2,8-Disubstituted adenosine derivatives as partial agonists for the adenosine A2A receptor.

Novel 2,8-disubstituted adenosine derivatives were synthesized in good overall yields starting from 2-iodoadenosine. Binding affinities were determined for rat adenosine A(1) and A(2A) receptors and human A(3) receptors. Some compounds displayed good adenosine A(2A) receptor affinities, with most of the 2-(1-hexynyl)- and 2-[(E)-1-hexenyl]-substituted derivatives having K(i) values in the nanomolar range. Although the introduction of an 8-alkylamino substituents decreased the affinity for the adenosine A(2A) receptor somewhat, the selectivity for this receptor compared to A(3) was improved significantly. The 8-methylamino (12) and 8-propylamino (14) derivatives of 2-(1-hexynyl)adenosine (3), showed reasonable A(2A) receptor affinities with K(i) values of 115 and 82nM, respectively, and were 49- and 26-fold selective for the adenosine A(2A) receptor compared to the A(3) receptor. The compounds were also evaluated for their ability to stimulate the cAMP production in CHO cells expressing the human adenosine A(2A) receptor. 2-(1-Hexynyl)adenosine (3) and 2-[(E)-1-hexenyl]adenosine (4) both showed submaximal levels of produced cAMP, compared to the reference full agonist CGS 21680, and thus behaved as partial agonists. Most 8-alkylamino-substituted derivatives of 3, displayed similar cAMP production as 3, and behaved as partial agonists as well. Introduction of alkylamino groups at the 8-position of 4, showed a slight reduction of the efficacy compared to 4, and these compounds were partial agonists also.

Inhibition of nucleoside transport by new analogues of nitrobenzylthioinosine.

Nitrobenzylthioinosine (NBTI, 1) was systematically modified by attachment of substituents at positions C6 and N9, and also by substitution of N1 with C. These modifications were chosen to reduce the polarity of the new compounds. Incorporation of the nitro functionality into a benzoxadiazole ring system was considered first. These new nucleosides showed high affinity (1.5-10nM) towards the nucleoside transport protein as present on human erythrocyte ghosts. Next, modification of this benzoxadiazole ring system with C, S and O in different positions produced a number of less polar nucleosides with affinity in the higher nanomolar range. Modification of N9 was achieved with different alkyl and alcohol substituents. An n-butyl substituent proved best, although all variations yielded substantial decreases in affinity. Replacement of N1 by a carbon atom in combination with a 2-Cl substituent also resulted in a relatively potent NBTI derivative (47 nM).

Modulation of agonist responses at the A(1) adenosine receptor by an irreversible antagonist, receptor-G protein uncoupling and by the G protein activation state.

Potency and intrinsic activity of agonists depend on ligand structure, but are also regulated by receptor-G protein stoichiometry. A potential functional reserve in adenosine A(1) receptor-mediated G protein activation was investigated by stimulation of guanosine-5'-(gamma-[35S]thio)-triphosphate ([35S]GTPgammaS) binding by the full agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA) and the partial agonist 5'-deoxy-5'-methylthioadenosine (MeSA). Pretreatment of rat brain membranes with the irreversible antagonist 1-propyl-3-[3-[[4-(fluorosulfonyl)benzoyl]oxy]-propyl]-8-cyclopentylxanthine revealed no classical receptor reserve for either agonist. The functional significance of the G protein coupling state of the receptor and occupancy of G proteins by guanine nucleotides was assessed after partial uncoupling of receptor-G protein complexes with N-ethylmaleimide and in the presence of increasing GDP concentrations. Agonist EC(50) values in G protein activation were increased after NEM pretreatment and at higher GDP concentrations, and a decrease in the relative intrinsic activity of MeSA was observed. The shift of agonist concentration-response curves to the right, the decrease in maximal effects and the decrease in relative intrinsic activity of the partial agonist point to a functional reserve which has to be attributed to GDP-free receptor-G protein complexes. The mechanisms of action of FSCPX, NEM and GDP were fully consistent with the two-state model of receptor activation. The apparent reserve revealed by GDP reflects a shift from spontaneously active GDP-free receptor-G protein complexes (RG)(*), which can bind [35S]GTPgammaS, to (RG) occupied by GDP. The abundance of (RG)(*) is favored by agonists and by the absence of GDP.

2,5'-Disubstituted adenosine derivatives: evaluation of selectivity and efficacy for the adenosine A(1), A(2A), and A(3) receptor.

Novel 2,5'-disubstituted adenosine derivatives were synthesized in good overall yields starting from commercially available guanosine. Binding affinities were determined for rat adenosine A(1) and A(2A) receptors and human A(3) receptors. E(max) values were determined for the stimulation or inhibition of cAMP production in CHO cells expressing human adenosine A(2A) (EC(50) values as well) or A(3) receptors, respectively. The compounds displayed affinities in the nanomolar range for both the adenosine A(2A) and A(3) receptor, without substantial preference for either receptor. The derivatives with a 2-(1-hexynyl) group had the highest affinities for both receptors; compound 4 (2-(1-hexynyl)adenosine) had the highest affinity for the adenosine A(2A) receptor with a K(i) value of 6 nM (A(3)/A(2A) selectivity ratio of approximately 3), whereas compound 37 (2-(1-hexynyl)-5'-S-methyl-5'-thioadenosine) had the highest affinity for the adenosine A(3) receptor with a K(i) value of 15 nM (A(2A)/A(3) selectivity ratio of 4). In general, compounds with a relatively small 5'-S-alkyl-5'-thio substituent (methyl-5'-thio) displayed the highest affinities for both the adenosine A(2A) and A(3) receptor; the larger ones (n- or i-propyl-5'-thio) increased the selectivity for the adenosine A(3) receptor. The novel compounds were also evaluated in cAMP assays for their (partial) agonistic behavior. Overall, the disubstituted derivatives behaved as partial agonists for both the adenosine A(2A) and A(3) receptor. The compounds showed somewhat higher intrinsic activities on the adenosine A(2A) receptor than on the A(3) receptor. Compounds 37, 40 and 45, 48, with either a 5'-S-methyl-5'-thio or a 5'-S-i-propyl-5'-thio substituent had the lowest intrinsic activities on the adenosine A(2A) receptor. For the A(3) receptor, compounds 34, 35, 38, 39, and 46, 47, with a 5'-S-ethyl-5'-thio or a 5'-S-n-propyl-5'-thio substituent had the lowest intrinsic activities.