The Synthesis and Biological activity of a Highly Selective Adenosine A2a. Receptor Agonist

Abstract

Three novel nucleosides 1, 2, and 3 were prepared that contained side chains at the 2-position of adenosine. Compound 1 was shown to be the most selective A_2a receptor agonist reported to date having an A₁/A₂ ratio of 2400. In addition, compound 1 was shown to reduce blood pressure in rats and dogs with only minimal effects on heart rate.     

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.     

New 2,6,9-trisubstituted adenines as adenosine receptor antagonists: a preliminary SAR profile

A new series of 2,6,9-trisubstituted adenines (5–14) have been prepared and evaluated in radioligand binding studies for their affinity at the human A₁, A_2A and A₃ adenosine receptors and in adenylyl cyclase experiments for their potency at the human A_2B subtype. From this preliminary study the conclusion can be drawn that introduction of bulky chains at the N ⁶ position of 9-propyladenine significantly increased binding affinity at the human A₁ and A₃ adenosine receptors, while the presence of a chlorine atom at the 2 position resulted in a not univocal effect, depending on the receptor subtype and/or on the substituent present in the N ⁶ position. However, in all cases, the presence in the 2 position of a chlorine atom favoured the interaction with the A_2A subtype. These results demonstrated that, although the synthesized compounds were found to be quite inactive at the human A_2B subtype, adenine is a useful template for further development of simplified adenosine receptor antagonists with distinct receptor selectivity profiles.     

Design,synthesis and evaluation of 2-aryl benzoxazoles as promising hit for the A2A receptor

The development of adenosine A_2A receptor antagonists has received much interest in recent years for the treatment of neurodegenerative diseases. Based on docking studies, a new series of 2-arylbenzoxazoles has been identified as potential A_2AR antagonists. Structure-affinity relationship was investigated in position 2, 5 and 6 of the benzoxazole heterocycle leading to compounds with a micromolar affinity towards the A_2A receptor. Compound F1, with an affinity of 1?μm, presented good absorption, distribution, metabolism and excretion properties with an excellent aqueous solubility (184?μm) without being cytotoxic at 100?μm. This compound, along with low-molecular weight compound D1 (K_i?=?10?μm), can be easily modulated and thus considered as relevant starting points for further hit-to-lead optimisation.     

Synthesis and Receptor Affinity of Polysubstituted Adenosines

Abstract

In a search for potent and selective adenosine agonists it has been found that 2-hexynyladenosine-5′-N-ethyluronamide (HENECA) displays high affinity at rat A_2A receptor combined with a good A_2A vs A₁ selectivity. The finding that HENECA shows good affinity also for A₃ receptors prompted us to investigate the effect of various substituents in different positions of this molecule.     

Characteristics of Adenosine-Mediated Inhibition of Tachykinin Release from Perifused Myenteric Plexus Synaptosomes

Abstract

Adenosine receptor agonists were shown to inhibit evoked release of the tachykinins Substance P and Neurokinin-A from perifused myenteric synaptosomes. The potencies of selective A₁ and A₂ agonists are consistent with activity at adenosine A₁ receptors located on the nerve endings.     

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.     

Development of Stable Cell Lines Expressing Different Subtypes of GabaA Receptors

Abstract

The experiments reported here were motivated by our interest to express in stably-transfected cells large amounts of recombinant rat GABA_A receptors. For this, we developed an original two step selection strategy, in which the first step consisted of transfecting HEK 293 cells with rat GABA_A receptor α and β subunits. G 418 resistant colonies isolated at this step were screened for [³H] muscimol binding to select for those that coexpressed α- and β-subunits. The best α and β subunit expressing colony was then supertransfected with a plasmid coding for the γ rat GABA_A receptor subunit and a mutant DHFR gene. After a second round of selection, this time in presence of methotrexate, those colonies that coexpressed ternary αβγ GABA_A receptor combinations were distinguished using [³H] flumazenil as a probe. This strategy was applied to the isolation of 3 GABA_A receptor clones, α₁β₂γ_2S, α₁β₂γ_2S and α₁β₂γ_2S, that expressed relatively high levels of these proteins. These 3 cell lines exhibited pharmacological and functional properties similar to cells transiently-transfected with equivalent subunit combinations. These cell lines therefore provide attractive models with which to evaluate the intrinsic activity and potency of compounds at recombinant GABA_A receptor subtypes.     

2-Phenylhydroxypropynyladenosine Derivatives as High Potent Agonists at A2B Adenosine Receptor Subtype

Abstract

Adenosine derivatives bearing in 2-position the (R,S)- phenylhydroxypropynyl chain were evaluated for their potency at human A_2B adenosine receptor, stably transfected on CHO cells, on the basis that (R,S)-2-phenylhydroxy-propynyl-5′-N-ethylcarboxyamidoadenosine [(R,S)-PHPNECA] was found to be a good agonist at the A_2B receptor subtype. Biological studies demonstrated that the presence of small alkyl groups in N ⁶-position of these molecules are well tolerated, whereas large groups abolished A_2B potency. On the other hand, the presence of an ethyl group in the 4′-carboxamido function seems to be optimal, the (S)-PHPNECA resulting the most potent agonist at A_2B receptor reported so far.     

A1 and A2 Adenosine Receptors Involvement in Controlling Purine and Acetylcholine Release From Rat Hippocampal Slices

Abstract

The different role of presynaptic A₁ and A₂ adenosine receptor subtypes on a possible autoregulation of endogenous purine outflow as well as on the ACh release, simultaneously assayed, was evaluated in rat hippocampal slices, at rest and under a field electrical stimulation.     

Synthesis of 3′-Acetamidoadenosine Derivatives as Potential A3 Adenosine Receptor Agonists

On the basis of high binding affinity of 3′-aminoadenosine derivatives 2b at the human A₃ adenosine receptor (AR), 3′-acetamidoadenosine derivatives 3a–e were synthesized from 1,2:5,6-di-O-isopropylidene-D-glucose via stereoselective hydroboration as a key step. Although all synthesized compounds were totally devoid of binding affinity at the human A₃AR, our results revealed that 3′-position of adenosine can only be tolerated with small size of a hydrogen bonding donor like hydroxyl or amino group in the binding site of human A₃AR.     

A1 receptors mediate adenosine inhibitory effects in mouse ileum via activation of potassium channels

AimsWe investigated the effects induced by exogenous adenosine on the spontaneous contractile activity of the longitudinal muscle of a mouse ileum, the receptor subtypes activated, the involvement of enteric nerves and whether opening of K⁺ channels was a downstream event leading to the observed effects.Main methodsMechanical responses of the mouse ileal longitudinal muscle to adenosine were examined in vitro as changes in isometric tension.Key findingsAdenosine caused a concentration-dependent reduction of the spontaneous contraction amplitude of the ileal longitudinal muscle up to its complete disappearance. This effect induced was markedly reduced by an A₁ receptor antagonist, but not by A₂ and A₃ receptor antagonists and mimicked only by the A₁ receptor agonist. Adenosine uptake inhibitors did not change adenosine potency. A₁ receptor expression was detected at the smooth muscle level. Adenosine responses were insensitive to tetrodotoxin, atropine or nitric oxide synthase inhibitor. Tetraethylammonium and iberiotoxin, BK_Ca channel blockers, significantly reduced adenosine effects, whilst 4-aminopyridine, a K_v blocker, apamin, a small conductance Ca²⁺-activated K⁺ (SK_Ca) channel blocker, charybdotoxin, an intermediate conductance Ca²⁺-activated K⁺ (IK_Ca) and BK_Ca channel blocker, or glibenclamide, an ATP-sensitive K⁺ channel blocker, had no effects. The combination of apamin plus iberiotoxin caused a reduction of the purinergic effects greater than iberiotoxin alone.SignificanceAdenosine acts as an inhibitory modulator of the contractility of mouse ileal longitudinal muscle through postjunctional A₁ receptors, which in turn would induce opening of BK_Ca and SK_Ca potassium channels. This study would provide new insight in the pharmacology of purinergic receptors involved in the modulation of the gastrointestinal contractility.     

More Than one Alpha Variant May Exist in a GabaA/benzodiazepine Receptor Complex

Abstract

G A B A_A/Benzodiazepine receptors are formed by the assembly of presumably five polypeptides with unknown stoichiometry. Six α, three β, two λ, and one δ subunit have been characterized on the molecular level. In analogy to the nicotinic acetylcholine receptor, and supported by functional analysis of recombinantly expressed GABA_A receptor subunits, a structure containing at least three different polypeptides has been proposed for the functional GABA_A and benzodiazepine regulated Cl^?-channel. Using an α₁ subunit specific antiserum we could show that additional α variants are present in α₁ subunit containing GABA_A/Benzodiazepine receptor complexes. This suggests that the diversity of GABA_A/Benzodiazepine receptors may be larger than previously thought.     

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.     

Minireview: Differential Targeting and Retention of G Protein-Coupled Receptors in Polarized Epithelial Cells

Abstract

Localization of receptors in discrete cellular microdomains undoubtedly contributes to their interaction with particular effectors and receptor targets. For G protein-coupled receptors, virtually nothing is known about the mechanisms and structural features responsible for their targeting to and retention in varying surface domains. We have shown that the G_j/G_o-coupled α_2A-adrenergic receptor (α_2AAR) is directly targeted to the lateral subdomain of MDCK II cells. Mutational analysis has revealed that regions in or near the bilayer are likely critical for α_2AAR targeting, whereas endofacial domains contribute to α_2AAR retention on the lateral surface. Although the α_2BAR also is enriched on the lateral subdomain at steady-state, its polarization occurs after initial random delivery to both apical and basolateral surfaces followed by a selective accumulation on the lateral subdomain. The α_2CAR also is expressed on the lateral subdomain and achieves its localization via direct delivery to the basolateral surface; however, the α_2CAR also exists in an as yet not fully characterized intracellular compartment. Interestingly, another G_j/G_o-coupled receptor, the A₁ adenosine receptor, is enriched on the apical surface of MDCK II calls and achieves this localization by direct apical delivery. These findings indicate that receptor delivery to polarized surfaces is not determined by receptor coupling to a specific subpopulation of G proteins.     

Arylpiperazinylalkyl derivatives of 8-amino-1,3-dimethylpurine-2,6-dione as novel multitarget 5-HT/D receptor agents with potential antipsychotic activity

A series of new 7-arylpiperazinylalkyl-1,3-dimethyl-purine-2,6-dione derivatives with diversified 8-amino substituent in 8 position was synthesized and their 5-HT_1A, 5-HT_2A, 5-HT₆, 5-HT₇, and D₂ receptor affinities were determined. The binding study allowed identifying some potent 5-HT_1A/5-HT_2A/5-HT₇/D₂ ligands. The most interesting because of their multireceptor profile were 8-piperidine (30–35) and 8-dipropylamine (45–47) analogs with four and five carbon aliphatic linkers. The selected compounds 24, 31, 34, 39, 41, 43, 45, and 46 in the functional in vitro evaluation for all targeted receptors showed significant partial D₂ agonist, partial 5-HT_1A agonist, and 5-HT_2A antagonist properties. The advantageous in vitro affinity of compound 34 for 5-HT_1A and D₂ receptors has been explained by means of molecular modeling, taking into consideration its partial agonist activity towards the latter one. In behavioral studies, compounds 32 and 34 revealed antipsychotic-like properties, significantly decreasing d-amphetamine-induced hyperactivity in mice.     

The effects of zolpidem treatment on GABA(A) receptors in cultured cerebellar granule cells: changes in functional coupling

AimsHypnotic zolpidem is a positive allosteric modulator of γ-aminobutyric acid (GABA) action, with preferential although not exclusive binding for α1 subunit-containing GABA_A receptors. The pharmacological profile of this drug is different from that of classical benzodiazepines, although it acts through benzodiazepine binding sites at GABA_A receptors. The aim of this study was to further explore the molecular mechanisms of GABA_A receptor induction by zolpidem.Main methodsIn the present study, we explored the effects of two-day zolpidem (10 μM) treatment on GABA_A receptors on the membranes of rat cerebellar granule cells (CGCs) using [³H]flunitrazepam binding and semi-quantitative PCR analysis.Key findingsTwo-day zolpidem treatment of CGCs did not significantly affect the maximum number (B_max) of [³H]flunitrazepam binding sites or the expression of α1 subunit mRNA. However, as shown by decreased GABA [³H]flunitrazepam binding, two-day exposure of CGCs to zolpidem caused functional uncoupling of GABA and benzodiazepine binding sites at GABA_A receptor complexes.SignificanceIf functional uncoupling of GABA and benzodiazepine binding sites at GABA_A receptors is the mechanism responsible for the development of tolerance following long-term administration of classical benzodiazepines, chronic zolpidem treatment may induce tolerance.     

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.     

Imidazo[1,2-α]pyridines possess adenosine A1 receptor affinity for the potential treatment of cognition in neurological disorders

Previous research has shown that bicyclic 6:5-fused heteroaromatic compounds with two N-atoms have variable degrees of adenosine A₁ receptor antagonistic activity. Prompted by this imidazo[1,2-α]pyridine analogues were synthesized and evaluated for their adenosine A₁ and A_2A receptor affinity via radioligand binding studies and subjected to a GTP shift assay to determine its adenosine A₁ receptor agonistic or antagonistic functionality. Imidazo[1,2-α]pyridine, the parent scaffold, was found devoid of affinity for the adenosine A₁ and A_2A receptors. The influence of substitution on position C2 showed no improvement for either adenosine A₁ or A_2A receptor affinity. The addition of an amino or a cyclohexylamino group to position C3 also showed no improvement of adenosine A₁ or A_2A receptor affinity. Surprisingly para-substitution on the phenyl ring at position C2 in combination with a cyclohexylamino group at position C3 led to adenosine A₁ receptor affinity in the low micromolar range with compound 4d showing: (1) the highest affinity for the adenosine A₁ receptor with a K_i value of 2.06 µM and (2) adenosine A₁ receptor antagonistic properties. This pilot study concludes that para-substituted 3-cyclohexylamino-2-phenyl-imidazo[1,2-α]pyridine analogues represent an interesting scaffold to investigate further structure-activity relationships in the design of novel imidazo[1,2-α]pyridine-based adenosine A₁ receptor antagonists for the treatment of neurodegenerative disorders.     

3′-Amidated 3′-Deoxyxylofuranose Analogues of N 6-−Cyclopentyladenosines: a New Class of Non-Xanthine Antagonists at the Adenosine A1 Receptor.

Abstract

Full adenosine A₁ receptor agonists like CPA and other N ⁶-^?substituted adenosine analogs have previously been shown to become partial agonists upon deletion of the 3′-hydroxyl moiety. The present study further explored the C-3′ site for modification. The modest affinity at A₁ and A_2a receptors found in the 3′-amido-3′-deoxyxyIofuranosyladenine series prompted us to synthesize the corresponding N ⁶-^?cyclopentyl derivatives, which proved to exhibit potent antagonistic behaviour at the A₁ receptors. This represents a new perspective in the purinergic field.