Flavonoid derivatives have been optimized as relatively rigid antagonists of adenosine receptors with particular selectivity for the A3 receptor subtype. A quantitative study of the structure-activity relationships for binding of flavonoids to adenosine A1, A2A, and A3 receptors has been conducted using comparative molecular field analysis (CoMFA). Correlation coefficients (cross-validated r2) of 0.605, 0.595, and 0.583 were obtained for the three subtypes, respectively. All three CoMFA models have the same steric and electrostatic contributions, implying similar requirements inside the binding cavity. Similarities were seen in the topology of steric and electrostatic regions with the A1 and A3 receptors, but not the A2A. Substitutions on the phenyl ring at the C-2 position of the chromone moiety may be considered important for binding affinity at all adenosine receptors. In the A3 model a region of favorable bulk interaction is located around the 2'-position of the phenyl ring. The presence of a C-6 substituent in the chromone moiety is well tolerated and increases the A1/A3 selectivity. The CoMFA coefficient contour plots provide a self-consistent picture of the main chemical features responsible for the pKi variations and also result in predictions which agree with experimental values.

Ecto-nucleotidases are plasma membrane-bound enzymes that sequentially dephosphorylate extracellular nucleotides such as ATP. This breakdown of ATP and other nucleotides obscures the characterization and classification of P2 (nucleotide) receptors. We therefore studied suramin and several of its analogs, divalent cations and ATP gamma S for their ability to inhibit ecto-ATPase in human blood cells. Suramin itself and Ni2+ were the more potent, non-competitive inhibitors with micromolar affinity. ATP gamma S also displayed micromolar affinity and inhibited ecto-ATPase competitively. The data obtained with the divalent cations demonstrate that coordination of the phosphate chain but not the N7 of the adenine ring is required for the breakdown of ATP by ecto-ATPase. Divalent cations that coordinate both the phosphate chain and N7 inhibit ecto-ATPase in a non-competitive manner.

A novel class of non-nitrogen-containing heterocycles, the tetrahydrobenzothiophenones, was found to bind to adenosine receptors as antagonists in the micromolar range. Affinity was determined in radioligand-binding assays at rat brain A1 and A2a receptors. A structure-activity analysis indicated that a 3-thioether group is favored and affinity at A2a, but not at A1, receptors is highly dependent on this thioether substituent. A carboxylic acid-derived substituent is required at the 1-position of the thiophene ring, with esters being more potent in binding at A1 receptors than the corresponding carboxyl hydrazide or carboxylic acid derivatives. The methyl (15) and ethyl (16) esters are about equipotent at A1 but not at A2a receptors. A 4-keto group on the saturated ring is favored for receptor affinity. Dimethyl substitution at the 6-position of the saturated ring is allowed. One of the most potent derivatives was the nonselective compound ethyl 3-(benzylthio)-4-oxo-4,5,6,7-tetrahydrobenzo[c] thiophene-1-carboxylate (BTH4, 7; Figure 1), which antagonized adenosine agonist-induced inhibition of adenylyl cyclase in rat adipocyte membranes with a KB value of 1.62 +/- 0.73 microM and adenosine agonist-induced stimulation of adenylyl cyclase in pheochromocytoma cell membranes with a KB value of 9.19 +/- 0.98 microM. Displacement of radioligand binding by BTH4 (7) at cloned human A3 receptors was negligible but one slightly A3 selective compound (11, 3.9-fold over A1 and >7.5-fold over A2a) was found. A 1-methylpropyl thioether (17) was 29-fold selective for A1 and A2a receptors. BTH4 (7) alone, at 10 mg/kg, stimulated locomotor activity in mice but paradoxically acted, under certain circumstances, synergistically with an A1 selective agonist to depress locomotor activity. A pharmacophore model relating structural features of xanthine and non-xanthine adenosine antagonists to BTH4 (7) suggests a high degree of similarity in electrostatic surfaces, assuming that the thiophene ring superimposes the region of the uracil ring of xanthines.