2-Amino-3-aroyl-4,5-alkylthiophenes: Agonist allosteric enhancers at human A1 adenosine receptors

2-Amino-3-benzoylthiophenes are allosteric enhancers (AE) of agonist activity at the A₁ adenosine receptor. The present report describes syntheses and assays of the AE activity at the human A₁AR (hA₁AR) of a panel of compounds consisting of nine 2-amino-3-aroylthiophenes (<b>3a-i</b>), eight 2-amino-3-benzoyl-4,5-dimethylthiophenes (<b>12a-h</b>), three 3-aroyl-2-carboxy-4,5- dimethylthiophenes (<b>15a-c</b>), 10 2-amino-3-benzoyl-5,6-dihydro 4<i>H</i>-cyclopenta[b]thiophenes (<b>17a-j</b>), 14 2-amino-3-benzoyl-4,5,6,7-tetrahydrobenzo[<i>b</i>]thiophenes (<b>18a-n</b>), and 15 2-amino- 3-benzoyl-5,6,7,8-tetrahydro-4<i>H</i>-cyclohepta[<i>b</i>]thiophenes (<b>19a-o</b>). An in vitro assay employing the A₁AR agonist [¹²⁵I]ABA and membranes from CHO-K1 cells stably expressing the hA¹AR measured, as an index of AE activity, the ability of a candidate AE to stabilize the agonist- A₁AR-G protein ternary complex. Compounds <b>3a-i</b> had little or no AE activity, and compounds<b> 12a-h</b> 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 <b>17a-c</b> lacked AE activity, suggesting the 2-amino group is essential. Polymethylene bridges linked thiophene C-4 and C-5 of compounds <b>17a-j</b>, <b>18a-n</b>, and <b>19a-o</b>. AE activity increased with the size of the -(CH₂)_n- bridge, <i>n</i> ) 3 < <i>n</i> ) 4 < <i>n</i> ) 5. The 3-carbethoxy substituents of <b>17a</b>, <b>18a</b>, and <b>19a</b> 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 hA₁AR 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.<br />