Does the combination of optimal substitutions at the C²-, N⁵- and N⁸-positions of the pyrazolo-triazolo-pyrimidine scaffold guarantee selective modulation of the human A₃ adenosine receptors?

In an attempt to study the optimal combination of a phenyl ring at the C²-position and different substituents at the N⁵- and N⁸-positions towards the selective modulation of human A₃ adenosine receptors (hA₃AR), we synthesized a new series of 2-para-(un)substituted-phenyl-pyrazolo-triazolo-pyrimidines bearing either a methyl or phenylethyl at N⁸ and chains of variable length at N⁵. Through biological evaluation, it was found that the majority of the compounds had good affinities towards the hA₃AR in the low nanomolar range. Compound 16 possessed the best hA₃AR affinity and selectivity profile (KᵢhA₃=1.33nM; hA₁/hA₃=4880; hA₂A/hA₃=1100) in the present series of 2-(substituted)phenyl-pyrazolo-triazolo-pyrimidine derivatives. In addition to pharmacological characterization, a molecular modeling investigation on these compounds further elucidated the effect of different substituents at the pyrazolo-triazolo-pyrimidine scaffold on affinity and selectivity to hA₃AR.