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In recent years, special attention has been paid to the A 3 adenosine receptor (A 3 AR) as a possible pharmacological target to treat intestinal inflammation. In this work, it was set up a novel method to quantify the concentration of a promising anti-inflammatory agent inside and outside of intestinal barrier using the everted gut sac technique. The compound chosen for the present study is one of the most potent and selective A 3 AR agonist reported so far, named AR 170 (N 6 -methyl-2-phenylethynyl-5'-N-methylcarboxamidoadenosine). In order to evaluate the intestinal absorption of AR 170 the radioligand binding assay in comparison with HPLC-DAD was used. Results showed that the compound is absorbed via passive diffusion by paracellular pathway. The concentrations determined in the serosal (inside the sac) fluid by radioligand binding assay are in good agreement with those obtained through the widely used HPLC/MS protocol, demonstrating the reliability of the method. It is worthwhile to note that the radioligand binding assay allows detecting very low concentrations of analyte, thus offering an excellent tool to measure the intestinal absorption of receptor ligands. Moreover, the AR 170 quantity outside the gut sac and the interaction with A 3 AR could presuppose good topical anti-inflammatory effects of this compound., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A3 Adenosine receptors are promising drug targets for a number of diseases and intense efforts are dedicated to develop selective agonists and antagonists of these receptors. A series of adenosine derivatives with 2-(ar)-alkynyl chains, with high affinity and different degrees of selectivity for human A3 adenosine receptors was tested for the ability to inhibit forskolin-stimulated adenylyl cyclase. All these derivatives are partial agonists at A3 adenosine receptors; their efficacy is not significantly modified by the introduction of small alkyl substituents in the N(6)-position. In contrast, the adenosine-5'-N-ethyluronamide (NECA) analogs of 2-(ar)-alkynyladenosine derivatives are full A3 agonists. Molecular modeling analyses were performed considering both the conformational behavior of the ligands and the impact of 2- and 5'-substituents on ligand-target interaction. The results suggest an explanation for the different agonistic behavior of adenosine and NECA derivatives, respectively. A sub-pocket of the binding site was analyzed as a crucial interaction domain for receptor activation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The present study examined the effect of two A(2A) adenosine receptor (AR) agonists, CGS 21680 and VT 7, on high-palatability food (HPF) intake in a model of binge eating in sated rats and on low-palatability food (LPF) intake in food-deprived rats. Binge eating was induced in female rats by three 8-day cycles of food restriction/refeeding, followed by acute stress. Two groups of rats were used: NR+NS rats normally fed and not stressed and R+S rats exposed to cycles of food restriction/refeeding and then stressed. R+S rats had higher intake of HPF than the NR+NS controls. The two A(2A)AR agonists were tested at doses of 0.1 and 0.05 mg/kg intraperitoneally; VT 7 did not modify locomotor activity at either dose, whereas CGS 21680 only slightly reduced it at the higher dose tested. The injection of 0.1 mg/kg of both agonists markedly reduced HPF intake both in R+S and in NR+NS rats. The dose of 0.05 mg/kg was inactive. CGS 21680 and VT 7, 0.1 mg/kg, also reduced the standard LPF intake in 24 h food-deprived rats; however, they did not reduce water intake, indicating that their effect on food intake is selective. The dose of 0.05 mg/kg was inactive. Thus, A(2A)AR agonists exert a rather general effect on food intake, inhibiting both HPF intake in sated rats and LPF intake in food-deprived rats. They may potentially be useful pharmacological agents to control binge-related eating disorders and to reduce food overconsumption associated with obesity.

Nucleoside analogues may represent good candidates for the discovery of new antibacterial agents, therefore, a library of adenosine analogues was assessed for their antibacterial activity, and the relationship between the structure and activity of these molecules was outlined. Antibacterial activity was evaluated against that of reference strains of Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Escherichia coli and Pseudomonas aeruginosa. We tested 54 adenosine analogues, modified both at ribose and base moieties, including adenine and 1/3-deazaadenine derivatives substituted in the 2- and/or N(6)-positions and bearing N-9 sugar moieties, such as ribose, 2'-deoxyribose, 3'-deoxyribose, 2',3'-dideoxyribose or cycloalkyl groups like cyclopentane. The data obtained, MIC and minimal bactericidal concentrations demonstrated that the presence of bulky substituents such as cycloheptyl and cyclooctyl rings on the N(6)-amino, together with a chlorine atom in the 2-position, conferred antibacterial activity against the Gram-positive group with MIC values ranging from 16 to 128 mg l(-1). The intact sugar moiety seemed to be not essential for antimicrobial activity and nucleosides bearing deoxyribose or cyclopentyl groups associated with bulky substituents in N(6)-position showed good antimicrobial properties. Furthermore, N-1 proved to be non-crucial and the 2-chloro-N(6)-cyclooctyl-1-deaza-3'-deoxyadenosine and 2',3'-dideoxyadenosine compounds were among the more active in the series with an MIC of 32 mg l(-1) against Staph. aureus and Strep. pneumoniae. None of the analogues was active against the two gram-negative species tested. Hence, adenosine derivatives bearing bulky substituents in the N(6)-position may represent good lead compounds for the future discovery of a novel series of antibacterial agents.

A new series of 2-aralkynyl-N(6)-methyl-MECAs 10-13 were synthesized and evaluated in radioligand binding studies and in a new Eu-GTP functional assay that provides a powerful alternative to radioisotope use. The new compounds possess high affinity and selectivity for the AA(3)R with N(6)-methyl-2-phenylethynylMECA (10) showing a subnanomolar affinity and about 100000-fold selectivity vs AA(1)R and AA(2A)R. Furthermore, the new nucleosides showed to be full agonists, the N(6)-methyl-2-(2-pyridinyl)ethynylMECA (13) being the most potent in the series.

A new series of N6-methoxy-2-(ar)alkynyladenosine derivatives has been synthesized and tested at the human recombinant adenosine receptors. Binding studies demonstrated that the new compounds possess high affinity and selectivity for the A3 subtype. Among them, compounds bearing an N-methylcarboxamido substituent in the 4'-position showed the highest A3 affinity and selectivity. In particular, the N6-methoxy-2-p-acetylphenylethynylMECA (40; Ki A3 = 2.5 nM, A3 selectivity versus A1 = 21 500 and A2A = 4200) results in one of the most potent and selective agonists at the human A3 adenosine receptor reported so far. Furthermore, functional assay, performed with selected new compounds, revealed that the presence of an alkylcarboxamido group in the 4'-position seems to be essential to obtain full agonists at the A3 subtype. Finally, results of molecular docking analysis were in agreement with binding and functional data and could explain the high affinity and potency of the new compounds.

Although significant progress has been made in the past few decades demonstrating that adenosine modulates a variety of physiological and pathophysiological processes through the interaction with four subtypes of a family of cell-surface G-protein-coupled receptors, clinical evaluation of some adenosine receptor ligands has been discontinued. Major problems include side effects due to the wide distribution of adenosine receptors, low brain penetration (which is important for the targeting of CNS diseases), short half-life of compounds, or a lack of effects, in some cases perhaps due to receptor desensitization or to low receptor density in the targeted tissue. Currently, three A(2A) adenosine receptor agonists have begun phase III studies. Two of them are therapeutically evaluated as pharmacologic stress agents and the third proved to be effective in the treatment of acute spinal cord injury (SCI), while avoiding the adverse effects of steroid agents. On the other hand, the great interest in the field of A(2A) adenosine receptor antagonists is related to their application in neurodegenerative disorders, in particular, Parkinson's disease, and some of them are currently in various stages of evaluation. This review presents an update of medicinal chemistry and molecular recognition of A(2A) adenosine receptor agonists and antagonists, and stresses the strong need for more selective ligands at the A(2A) human subtype.

Adenosine A(3) receptors are promising drug targets for a number of conditions like inflammatory diseases including asthma, ischemic injury or certain types of cancer. Consequently, intense efforts are dedicated to the development of selective A(3) agonists and antagonists. The only tritiated agonist that is available for radioligand binding is the nonselective [(3)H]5'-N-ethylcarboxamidoadenosine ([(3)H]NECA). Based on a recently characterized series of 2-substituted adenosine receptor agonists we developed a tritiated selective A(3) radioligand with high affinity. From this series 2-hexyn-1-yl-N(6)-methyladenosine (HEMADO) with a K(i)-value of 1.1 nM at the human A(3) subtype was chosen. HEMADO is 300-fold selective versus the A(1) subtype, and 1100-fold and more than 25,000-fold selective compared to the adenosine A(2A) and A(2B) receptors, respectively. The tritiated derivative [(3)H]HEMADO exhibited the same affinity as the unlabeled precursor. In concentrations up to 10 nM no specific binding to adenosine A(1), A(2A) or A(2B) receptors was observed confirming the high selectivity for adenosine A(3) receptors. Characterization of [(3)H]HEMADO in radioligand binding studies revealed reversible binding to the human adenosine A(3) subtype. In saturation binding studies for the A(3) subtype a K(D)-value of 1.1 nM was determined. Nonspecific binding at a radioligand concentration of 1 nM amounted to 1-2% of total binding. Competition binding with a panel of adenosine receptor ligands clearly confirmed the correct A(3) pharmacology of the binding site labeled by [(3)H]HEMADO. With [(3)H]HEMADO we present a tritiated agonist with high affinity and A(3)-selectivity and very low nonspecific binding. [(3)H]HEMADO is a useful tool for specific screening for A(3) receptor agonists and antagonists in improved radioligand binding assays with the human subtype.

In this article, we report on the synthesis of 2',3',5'-tri-O-acetyl-2-amino-1-deazaadenosine and of 2',3',5'-tri-O-acetyl-2-amino-N(6)-cyclopentyl-1-deazaadenosine, which are very versatile intermediates for the preparation of 2-substituted 1-deazaadenosine derivatives. The two synthesized compounds showed to be quite unstable, with the N(6)-substituted derivatives being less stable than the N(6)-unsubstituted counterpart, according to the calculated HOMO-LUMO energy gap. Stability studies were performed through HPLC-MS analysis.

Newly synthesized mono-, di-, and triphosphate of 2-alkynyl adenosines showed very different behavior in human platelet P2Y receptor models, according to the different alkynyl chains. In fact, 2-hexynyladenosine di- (5) and triphosphate (7) induced platelet shape change and aggregation and inhibited PGE(1)-induced increase in platelet cyclic AMP. On the contrary, the corresponding 2-phenylethynyladenosine di- (6) and triphosphate (8) did not induce platelet shape change or aggregation, but inhibited platelet aggregation induced by ADP.

In the search for agonists for the elusive A2B adenosine receptor subtypes, 2-phenylhydroxypropynyl-5'-N-methylcarboxamido adenosine (PHPMECA, 14), 2-phenylhydroxypropynyl-5'-N-propylcarboxamido adenosine (PHPPECA, 15), and N6-ethyl-2-phenylhydroxypropynyl-5'-N-ethylcarboxamidoadenosine (19) were synthesized on the basis that introduction of alkynyl chains in 2-position of adenosine derivatives resulted in reasonably good A2B potency compared to NECA [see N6-ethyl-2-phenylhydroxypropynyl adenosine (5) EC50 = 1,700 nM and 2-phenylhydroxypropynyl-5'-N-ethylcarboxamido adenosine (PHPNECA, 8) EC50 = 1,100 nM, respectively]. Radioligand binding studies and adenylyl cyclase assays, performed with recently cloned human A1, A2A, A2B, and A3 adenosine receptors, showed that these modifications produced a decrease in potency at A2B receptor, as well as a general reduction in affinity at the other receptor subtypes. On the other hand, the contemporary presence of an ethyl substituent in N6-position and of a 4'-ethylcarboxamido group in the same compounds led to (R,S)-N6-ethyl-2-phenylhydroxypropynyl-5'-N-ethylcarboxamidoadenosine and (S)-N6-ethyl-2-phenylhydroxypropynyl-5'-N-ethylcarboxamidoadenosine, which did not show the expected increase in potency at A2B subtype. Hence, (S)-2-phenylhydroxypropynyl-5'-N-ethylcarboxamidoadenosine [(S)-PHPNECA] with EC50 A2B = 220 nM remains the most potent agonist at A2B receptor reported so far.

Adenosine derivatives bearing in 2-position the (R,S)-phenylhydroxypropynyl chain were evaluated for their potency at human A2B 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 A2B receptor subtype. Biological studies demonstrated that the presence of small alkyl groups in N6-position of these molecules are well tolerated, whereas large groups abolished A2B 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 A2B receptor reported so far.

Adenosine (Ado) derivatives substituted at the C2 position with an alkynyl chain are endowed with high affinity for A(1), A(2A) and A(3) human adenosine receptors, while being less active at the low affinity A(2B) subtype. On the other hand, the introduction of an alkynyl chain at the C8 position of adenosine is detrimental for the affinity and potency at A(1), A(2A), and A(2B) receptors, while is more tolerated by the A(3) receptor. The evaluation of the stimulation of [35S]GTPgammaS binding revealed that 2-alkynyladenosines behave as adenosine receptors agonists while, on the contrary, 8-alkynyladenosines behave as antagonists. With this work we demonstrated, by means of an NMR-based and a computational conformational analysis, that 8-alkynyladenosines, differently from 2-alkynyladenosines, cannot adopt the sugar-base anti conformation required for adenosine receptor activation.Furthermore, using the recently reported X-ray crystal structure of bovine rhodopsin as template, we built a 3D model of the seven transmembrane domains of the human adenosine A(3) receptor with the homology modeling. After identification of the binding site we carried out docking experiments, demonstrating that the two class of molecules have different binding modes that explain their different degree of affinity and the shift of their activity from agonism to antagonism.

The search for potent and selective A(2A) adenosine receptor agonists has been particularly fruitful in the early nineties. A series of 2-amino, 2-alkoxy, 2-alkythio-, 2-alkynyl-, and 2-alkenyl-derivatives of adenosine (Ado, 1) and N-ethylcarboxamidoadenosine (NECA, 30) have been synthesized and tested mainly on different model of rat A(1) and A(2A) receptor subtypes. From these studies some ligands, such as CGS 21680 (33), HENECA (42), and (S)-PHPNECA (46b), showed to possess high A(2A) affinity combined with good A(2A) vs A(1) selectivity. More detailed characterization of these ligands at the four cloned human adenosine receptor subtypes revealed that none of the prototypical adenosine receptor agonists exhibits at the same time high affinity and selectivity for the human A(2A)AR subtype. Both NECA and CGS 21680, which are avalaible as radioligands for this subtype, have lower affinity at human than at rat receptor. The 2-alkynylNECA derivatives HENECA an PHPNECA showed high affinity also at human A(3) receptors. In particular, (S)-PHPNECA displayed K(i)s in the low nanomolar range at A(1), A(2A), and A(3)subtypes and an EC(50) of 220 nM at human A(2B) receptor. On the other hand, it is now well known that the coronary vasodilation induced by Ado in different species is mediated by activation of A(2A)AR and a compound capable of producing coronary vasodilation through activation of A(2A)AR, but that is devoid of A(1)- and A(1)-agonist activity would have advantage over Ado for use in myocardial perfusion imaging studies. Other potential therapeutic applications of selective A(2A)AR agonists are as anti-aggregatory, anti-inflammatory, anti-psychotic, and anti-Huntington's disease agents. This review is aimed at presenting a complete overview of the medicinal chemistry development of A(2A) adenosine receptor agonists and at stressing the strong need for more selective ligands at A(2A) human subtype.

A series of N(6)-alkyl-2-alkynyl derivatives of adenosine (Ado) have been synthesized and evaluated for their affinity at human A(1), A(2A), and A(3) receptors and for their potency at A(2B) adenosine receptor subtypes. The corresponding 2-(1-alkynyl) derivatives of 5'-N-ethylcarboxamidoadenosine (NECA) and Ado are used as reference compounds. Binding studies demonstrated that the activities of 2-alkynylAdos were slightly increased for the adenosine A(1) receptor and slightly decreased for both A(3) and A(2B) subtypes compared to those of their corresponding NECA derivatives, whereas the A(2A) receptor affinities of the two series of nucleosides were similar. The presence of a methyl group on N(6) of the 2-alkynyladenosines, inducing an increase in affinity at the human A(3) receptor and a decrease at the other subtypes, resulted in an increase in A(3) selectivity. In particular, 2-phenylethynyl-N(6)-methylAdo (8b) showed an A(3) affinity in the low nanomolar range (K(i)(A(3)) = 3.4 nM), with a A(1)/A(3) and A(2A)/A(3) selectivity of about 500 and 2500, respectively. These findings motivated us to search for the preparation of new selective radioligands for the A(3) subtype; hence, a procedure to introduce a tritiated alkylamino group in these molecules was carried out. As far as the potency at the A(2B) receptor, the type of 2-alkynyl chain and the presence of the ethylcarboxamido group on the sugar seem to be very important; in fact, the (S)-2-phenylhydroxypropynylNECA [(S)-PHPNECA, 1e, EC(50)(A(2B)) = 0.22 microM] proved to be one of the most potent A(2B) agonist reported so far. On the other hand, the (S)-2-phenylhydroxypropynyl-N(6)-ethylAdo (9e, EC(50)(A(2B)) = 0.73 microM) showed a significantly increase of potency at the A(2B) subtype in comparison with the N(6)-methyl, N(6)-isopropyl, and the unsubstituted adenosine derivatives, although it resulted in being less potent than (S)-PHPNECA (1e, EC(50)(A(2B)) = 0.22 microM). These observations suggest that the introduction of an ethyl group in the N(6)-position and an ethylcarboxamido substituent in the 4'-position of (S)-2-phenylhydroxypropynyladenosine could lead to a compound endowed with high potency at the A(2B) receptor.

Some 8-alkynyladenosines were synthesized and evaluated for their adenosine receptor activity, utilizing radioligand binding studies (A(1), A(2A), A(3)) or adenylyl cyclase activity assays (A(2B)). Furthermore, the maximal induction of guanosine 5'-(gamma-thio)triphosphate ([35S]GTPgammaS) binding to G proteins and the inhibition of NECA-stimulated binding, in membranes of CHO cells which express the human A(3) receptor, were used to determine the intrinsic activity of these nucleosides at the A(3) adenosine receptor. The results showed that these new adenosine derivatives are very selective ligands for the A(3) receptor subtype and behave as adenosine antagonists, since they do not stimulate basal [35S]GTPgammaS binding, but inhibit NECA-stimulated binding. This is the first report that adenosine derivatives, with unmodified ribose moiety, are adenosine receptor antagonists.

Adenosine derivatives bearing different (ar)alkynyl chains at the 8-position were synthesized and tested at human adenosine receptors. Binding studies showed that all compounds possess affinity for the A3 subtype in the high nM range. Moreover, guanosine 5'-O-(3-[35S]thio)triphosphate binding assay indicated that the 8-alkynyl adenosines behaved as antagonists of NECA at A3 receptors.

2,6-Dichloro-1-deazapurine and 2,6-dichloro-3-deazapurine were coupled with 1,2-O-diacetyl-5-O-benzoyl-3-deoxy-beta-D-ribofuranose. Deprotection of the obtained compounds and reaction with liquid ammonia gave the desired 2-chloroadenine nucleosides, which were dechlorinated to afford the corresponding 1-deaza and 3-deazaadenosine derivatives. Biological studies performed on ADA from calf intestine showed that these new nucleosides are inhibitors of the enzyme.

2-Phenylethynyladenosine and its N6-methyl derivative were synthesized and evaluated in binding assays at human adenosine receptors stably transfected on CHO cells. Results showed that the N6-methyl-2-phenylethynyladenosine is endowed with very high affinity and selectivity at A3 receptor subtype. Hence, an alternative procedure for the synthesis of tritiated N6-methyl-2-phenylethynyladenosine was set up to introduce tritiated methylamine in the final step.