8‐(3‐Isothiocyanatostyryl)caffeine is a selective, irreversible inhibitor of striatal A2‐Adenosine receptors

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Abstract
8‐(3‐Isothiocyanatostyryl)caffeine (ISC) was synthesized and shown to inhibit selectively the binding of [3H]CGS 21680 (an A2a‐selective agonist) at adenosine receptors in striatal membranes. The Ki value at A2a‐receptors was found to be 110 nM (rat), with selectivity ratios for A2a versus A1‐receptors in rat, guinea pig, bovine, and rabbit striatum of >100‐fold. Preincubation of membranes with ISC caused a dose‐dependent, irreversible antagonism of the binding of [3H]CGS 21680, with an IC50 value of 3 μM. The irreversibility is likely due to the presence of the chemically reactive isothiocyanate group, since the binding of the corresponding analogue in which the isothiocyanate was replaced with a chloro group was completely reversible. The potency of ISC to irreversibly inhibit the binding of [3H]CGS 21680 in several species varied in the order rat ≈︁ guinea pig > bovine ≈︁ rabbit. In all four species, binding of the A1‐selective agonist [3H]R‐N6‐phenylisopropyladenosine was not diminished by pre‐treatment with 2 μM ISC. The kinetics of irreversible inhibition of rat A2a‐receptors by 2 μM ISC gave a t1/2 of approximately 3 min. Following partial inactivation, the remaining rat A2a‐binding sites retained the same Kd value as in control membranes for saturatin by [3H]CGS 21680. Thus, ISC appears to be a selective affinity label for A2a‐ versus A1‐receptors in the brain.