2-Amino-3-benzoylthiophenes are allosteric enhancers (AE) of agonist activity at the A(1) adenosine receptor. The present report describes syntheses and assays of the AE activity at the human A(1)AR (hA(1)AR) of a panel of compounds consisting of nine 2-amino-3-aroylthiophenes (3a-i), eight 2-amino-3-benzoyl-4,5-dimethylthiophenes (12a-h), three 3-aroyl-2-carboxy-4,5-dimethylthiophenes (15a-c), 10 2-amino-3-benzoyl-5,6-dihydro-4H-cyclopenta[b]thiophenes (17a-j), 14 2-amino-3-benzoyl-4,5,6,7-tetrahydrobenzo[b]thiophenes (18a-n), and 15 2-amino-3-benzoyl-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophenes (19a-o). An in vitro assay employing the A(1)AR agonist [(125)I]ABA and membranes from CHO-K1 cells stably expressing the hA(1)AR measured, as an index of AE activity, the ability of a candidate AE to stabilize the agonist-A(1)AR-G protein ternary complex. Compounds 3a-i had little or no AE activity, and compounds 12a-h had only modest activity, evidence that AE activity depended absolutely on the presence of at least a methyl group at C-4 and C-5. Compounds 17a-c lacked AE activity, suggesting the 2-amino group is essential. Polymethylene bridges linked thiophene C-4 and C-5 of compounds 17a-j, 18a-n, and 19a-o. AE activity increased with the size of the -(CH(2))(n)()- bridge, n = 3 < n = 4 < n = 5. The 3-carbethoxy substituents of 17a, 18a, and 19a did not support AE activity, but a 3-aroyl group did. Bulky (or hydrophobic) substituents at the meta and para positions of the 3-benzoyl group and also 3-naphthoyl groups greatly enhanced activity. Thus, the hA(1)AR contains an allosteric binding site able to accommodate 3-aroyl substituents that are bulky and/or hydrophobic but not necessarily planar. A second region in the allosteric binding site interacts constructively with alkyl substituents at thiophene C-4 and/or C-5.
2-Amino-3-aroylthiophenes are agonist allosteric enhancers (AE) at the A(1) adenosine receptor (A(1)AR). Here we report the syntheses of three kinds of novel 2-aminothiophenes and assays of their AE activity at the human A(1)AR (hA(1)AR), namely, (1) 2-amino-4,5-diphenylthiophene-3-carboxylates, 3a-h, (2) 2-amino-3-benzoyl-4,5-diphenylthiophenes, 7a-p, and (3) 2-amino-5-bromo-3-benzoyl-4-phenylthiophenes, 10a-h. An in vitro assay employing the A(1)AR agonist [(125)I]ABA and membranes from CHO-K1 cells stably expressing the hA(1)AR measured an index of AE activity, the ability of a candidate AE to stabilize the agonist-A(1)AR-G protein ternary complex, scored as the percentage of ternary complex remaining after 10 min of dissociation initiated by CPX and GTPgammaS. The AE activity score of 2-amino-4,5-dimethyl-3-(3-trifluoromethylbenzoyl)thiophene (PD 81,723), which was 19%, served as a standard for comparison. Two 3-carboxythiophene 3-trifluoromethylbenzyl esters, 3d (49%) and 3f (63%), had substantial AE activity. The 3-(1-naphthoyl) substituent of 7e (52%) also supported AE activity. Compounds in series 3 tended to be more potent, 10a and 10c having scores of 91 and 80%, respectively. The activity of 2-amino-5-bromo-3-ethoxycarbonyl-4-(3-nitrophenyl)thiophene, 10h (26%), is an exception to the rule that a 3-ethoxycarbonyl substituent cannot support AE activity.
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