Nucleosides and Nucleotides. 200. Reinvestigation of 5‘-N-Ethylcarboxamidoadenosine Derivatives:  Structure−Activity Relationships for P₃ Purinoceptor-like Proteins

The non-P₁ and non-P₂ muscle relaxant effect of ATP in rabbit thoracic aorta has recently been attributed to a putative P₃ purinoceptor, which is activated by either adenosine or ATP. Since the physiological roles of this putative P₃ purinoceptor and of a new [³H]-5‘-N-ethylcarboxamidoadenosine (NECA)-binding protein from rat brain membranes called P₃ purinoceptor-like protein (P₃LP), due to its ligand specificity, have not been fully elucidated, we needed a specific ligand to obtain further information about the receptor. We examined the structure−activity relationship (SAR) of various 5‘-N-substituted-carboxamidoadenosine derivatives toward P₃LP and discovered a hydrophobic binding region near the 5‘-N-substituted-carboxamide group. From the linear alkyl N-substituted derivatives, the N-n-pentyl derivative 10 was found to be the most potent ligand with a K_i value of 12 nM. In the series of the N-cycloalkyl derivatives, the N-cyclohexyl derivative 27 was the strongest ligand with a K_i value of 18 nM. On the other hand, the N-substituents having branched alkyl side chains and bulky cycloalkyl groups did not show any potent affinities for P₃LP. Therefore, the hydrophobic pocket accommodates approximately a 10-carbon-atom-long linear alkyl side chain, while a considerably stronger hydrophobic binding region of about a 5-carbon-atom-long depth exists near the nitrogen atom of the amide group. This pocket also allows substitution with bulky hydrophobic groups since the 5‘-N-cycloalkyl derivatives have high affinities. We also examined the receptor selectivity for the selected nucleosides 10 and 27 with 1 [9-(6,7-dideoxy-β-d-allo-hept-5-ynofuranosyl)adenine, HAK2701] and NECA versus P₁ purinoceptor subtypes, such as adenosine A₁, A_2A, A_2B, and A₃ receptors, and found that 27 is the most selective ligand for P₃LP.