2-Amino-3-aroyl-4,5-alkylthiophenes:  Agonist Allosteric Enhancers at Human A<INF>1</INF> Adenosine Receptors

2-Amino-3-benzoylthiophenes are allosteric enhancers (AE) of agonist activity at the A<INF>1</INF> adenosine receptor. The present report describes syntheses and assays of the AE activity at the human A<INF>1</INF>AR (hA<INF>1</INF>AR) of a panel of compounds consisting of nine 2-amino-3-aroylthiophenes (<BO>3a</BO>−<BO>i</BO>), eight 2-amino-3-benzoyl-4,5-dimethylthiophenes (<BO>12a</BO>−<BO>h</BO>), three 3-aroyl-2-carboxy-4,5-dimethylthiophenes (<BO>15a</BO>−<BO>c</BO>), 10 2-amino-3-benzoyl-5,6-dihydro-4H-cyclopenta[b]thiophenes (<BO>17a</BO>−<BO>j</BO>), 14 2-amino-3-benzoyl-4,5,6,7-tetrahydrobenzo[b]thiophenes (<BO>18a</BO>−<BO>n</BO>), and 15 2-amino-3-benzoyl-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophenes (<BO>19a</BO>−<BO>o</BO>). An in vitro assay employing the A<INF>1</INF>AR agonist [¹²⁵I]ABA and membranes from CHO-K1 cells stably expressing the hA<INF>1</INF>AR measured, as an index of AE activity, the ability of a candidate AE to stabilize the agonist-A<INF>1</INF>AR-G protein ternary complex. Compounds <BO>3a</BO>−<BO>i </BO>had little or no AE activity, and compounds <BO>12a</BO>−<BO>h </BO>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 <BO>17a</BO>−<BO>c</BO> lacked AE activity, suggesting the 2-amino group is essential. Polymethylene bridges linked thiophene C-4 and C-5 of compounds <BO>17a</BO>−<BO>j</BO>, <BO>18a</BO>−<BO>n</BO>, and <BO>19a</BO>−<BO>o</BO>. AE activity increased with the size of the -(CH<INF>2</INF>)<INF>n</INF><INF></INF>- bridge, n = 3 &lt; n = 4 &lt; n = 5. The 3-carbethoxy substituents of <BO>17a</BO>, <BO>18a</BO>, and <BO>19a</BO> 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<INF>1</INF>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.