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The binding affinities at rat A1, A2a, and A3 adenosine receptors of a wide range of derivatives of adenosine have been determined. Sites of modification include the purine moiety (1-, 3-, and 7-deaza; halo, alkyne, and amino substitutions at the 2- and 8-positions; and N6-CH2-ring, -hydrazino, and -hydroxylamino) and the ribose moiety (2'-, 3'-, and 5'-deoxy; 2'- and 3'- O-methyl; 2'-deoxy 2'-fluoro; 6'-thio; 5'-uronamide; carbocyclic; 4'- or 3'-methyl; and inversion of configuration). (-)- and (+)-5'-Noraristeromycin were 48- and 21-fold selective, respectively, for A2a vs A1 receptors. 2-Chloro-6'-thioadenosine displayed a Ki value of 20 nM at A2a receptors (15-fold selective vs A1). 2-Chloroadenin-9-yl(beta-L-2'-deoxy-6'- thiolyxofuranoside) displayed a Ki value of 8 microM at A1 receptors and appeared to be an antagonist, on the basis of the absence of a GTP-induced shift in binding vs a radiolabeled antagonist (8-cyclopentyl-1,3-dipropyl-xanthine). 2-Chloro-2'-deoxyadenosine and 2-chloroadenin-9-yl(beta-D-6'-thioarabinoside) were putative partial agonists at A1 receptors, with Ki values of 7.4 and 5.4 microM, respectively. The A2a selective agonist 2-(1-hexynyl)-5'-(N-ethylcarbamoyl)adenosine displayed a Ki value of 26 nM at A3 receptors. The 4'-methyl substitution of adenosine was poorly tolerated, yet when combined with other favorable modifications, potency was restored. Thus, N6-benzyl-4'-methyladenosine-5'-(N-methyluronamide) displayed a Ki value of 604 nM at A3 receptors and was 103- and 88-fold selective vs A1 and A2a receptors, respectively. This compound was a full agonist in the A3-mediated inhibition of adenylate cyclase in transfected CHO cells. The carbocyclic analogue of N6-(3-iodobenzyl)adenosine-5'-(N-methyluronamide) was 2-fold selective for A3 vs A1 receptors and was nearly inactive at A2a receptors.

In the search for more selective A2-receptor agonists and on the basis that appropriate substitution at C2 is known to impart selectivity for A2 receptors, 2-alkynyladenosines 2a-d were resynthesized and evaluated in radioligand binding, adenylate cyclase, and platelet aggregation studies. Binding of [3H]NECA to A2 receptors of rat striatal membranes was inhibited by compounds 2a-d with Ki values ranging from 2.8 to 16.4 nM. 2-Alkynyladenosines also exhibited high-affinity binding at solubilized A2 receptors from human platelet membranes. Competition of 2-alkynyladenosines 2a-d for the antagonist radioligand [3H]DPCPX and for the agonist [3H]CCPA gave Ki values in the nanomolar range, and the compounds showed moderate A2 selectivity. In order to improve this selectivity, the corresponding 2-alkynyl derivatives of adenosine-5'-N-ethyluronamide 8a-d were synthesized and tested. As expected, the 5'-N-ethyluronamide derivatives retained the A2 affinity whereas the A1 affinity was attenuated, resulting in an up to 10-fold increase in A2 selectivity. A similar pattern was observed in adenylate cyclase assays and in platelet aggregation studies. A 30- to 45-fold selectivity for platelet A2 receptors compared to A1 receptors was found for compounds 8a-c in adenylate cyclase studies.

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.

The P2Y1 receptor is present in the heart, in skeletal and various smooth muscles, and in platelets, where its activation is linked to aggregation. Adenosine 3',5'- and 2',5'-bisphosphates have been identified as selective antagonists at the P2Y1 receptor (Boyer et al. Mol. Pharmacol. 1996, 50, 1323-1329) and have been modified structurally to increase receptor affinity (Camaioni et al. J. Med. Chem. 1998, 41, 183-190). We have extended the structure-activity relationships to a new series of deoxyadenosine bisphosphates with substitutions in the adenine base, ribose moiety, and phosphate groups. The activity of each analogue at P2Y1 receptors was determined by measuring its capacity to stimulate phospholipase C in turkey erythrocyte membranes (agonist effect) and to inhibit phospholipase C stimulation elicited by 10 nM 2-(methylthio)adenosine 5'-diphosphate (antagonist effect). 2'-Deoxyadenosine bisphosphate analogues containing halo, amino, and thioether groups at the 2-position of the adenine ring were more potent P2Y1 receptor antagonists than analogues containing various heteroatom substitutions at the 8-position. An N6-methyl-2-chloro analogue, 6, was a full antagonist and displayed an IC50 of 206 nM. Similarly, N6-methyl-2-alkylthio derivatives 10, 14, and 15 were nearly full antagonists of IC50 < 0.5 microM. On the ribose moiety, 2'-hydroxy, 4'-thio, carbocyclic, and six-membered anhydrohexitol ring modifications have been prepared and resulted in enhanced agonist properties. The 1,5-anhydrohexitol analogue 36 was a pure agonist with an EC50 of 3 microM, i.e., similar in potency to ATP. 5'-Phosphate groups have been modified in the form of triphosphate, methyl phosphate, and cyclic 3',5'-diphosphate derivatives. The carbocyclic analogue had enhanced agonist efficacy, and the 5'-O-phosphonylmethyl modification was tolerated, suggesting that deviations from the nucleotide structure may result in improved utility as pharmacological probes. The N6-methoxy modification eliminated receptor affinity. Pyrimidine nucleoside 3', 5'-bisphosphate derivatives were inactive as agonists or antagonists at P2Y receptor subtypes.

The tritiated analogue of 2-chloro-N6-cyclopentyladenosine (CCPA), an adenosine derivative with subnanomolar affinity and a 10,000-fold selectivity for A1 adenosine receptors, has been examined as a new agonist radioligand. [3H]CCPA was prepared with a specific radioactivity of 1.58 TBq/mmol (43 Ci/mmol) and bound in a reversible manner to A1 receptors from rat brain membranes with a high affinity KD-value of 0.2 nmol/l. In the presence of GTP a KD-value of 13 nmol/l was determined for the low affinity state for agonist binding. Competition of several adenosine receptor agonists and antagonists for [3H]CCPA binding to rat brain membranes confirmed binding to an A1 receptor. Solubilized A1 receptors bound [3H]CCPA with similar affinity for the high affinity state. At solubilized receptors a reduced association rate was observed in the presence of MgCl2, as has been shown for the agonist [3H]N6-phenylisopropyladenosine ([3H]PIA). [3H]CCPA was also used for detection of A1 receptors in rat cardio myocyte membranes, a tissue with a very low receptor density. A KD-value of 0.4 nmol/l and a Bmax-value of 16 fmol/mg protein was determined in these membranes. In human platelet membranes no specific binding of [3H]CCPA was measured at concentrations up to 400 nmol/l, indicating that A2 receptors did not bind [3H]CCPA. Based on the subnanomolar affinity and the high selectivity for A1 receptors [3H]CCPA proved to be a useful agonist radioligand for characterization of A1 adenosine receptors also in tissues with very low receptor density.

We have studied the ultrafast dynamics of NH-stretch vibrational excitations in Watson-Crick base pairs consisting of adenine and uracil derivatives. To estimate the influence of the A:U hydrogen bonding on the vibrational dynamics, we have also studied the uracil derivative in monomeric form. The vibrational relaxation of the NH-stretching mode is found to occur much faster in the Watson-Crick base pair than in monomeric uracil. From the delay dependence of the transient vibrational spectra, it can be concluded that both in base-paired and monomeric uracil, the energy relaxation takes place in two steps, the first step being a rapid transfer of energy from the NH-stretching mode to an accepting mode, the second step the relaxation of this accepting mode. The transient spectra show evidence that in the base pair the hydrogen bond between the nucleobases acts as the accepting mode, and that the hydrogen bonding between the bases is responsible for the extremely fast vibrational relaxation in this system.

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