A high-affinity fluorenone-based beta(2)-adrenergic receptor antagonist with a photoactivatable pharmacophore

To develop molecules capable of directly probing the catechol binding regio n of the beta (2)-adrenergic receptor (beta (2)AR), novel benzophenone- and fluorenone-based beta (2)AR antagonists were prepared as potential photoaf finity probes. While the benzophenone-containing ligands bound with relativ ely modest affinity, one of the fluorenone-based compounds, 4-(2-hydroxy-3- isopropylaminoprspoxy)-7-amino-6-iodofluorenone (iodoaminoflisopolol, IAmF) , showed very high affinity for the beta (2)AR, inhibiting [I-125]-ICYP bin ding with an apparent K-i of approximately 1 x 10(-9) M. In comparison to t he benzophenone ligands, the fluorenone ligands have one additional carbon- carbon bond that creates a planar unsaturated ring system and leads to a la rge increase in receptor binding affinity. Unlike previous beta (2)AR photo affinity ligands, an attractive and unique feature of the fluorenone deriva tive IAmF is that the large planar unsaturated ring (believed to correspond to the catechol end of other beta (2)AR ligands) serves as both the bindin g pharmacophore and the photoreaction center for this molecule. With this p otential for directly probing the catechol binding region of the beta (2)AR , we synthesized and tested IAmF in carrier-free radioiodinated form ([I-12 5]IAmF). When photoreduction was conducted at 350 nm for 20 min, [I-125]IAm F was able to produce cross-linked products in both triethylamine and metha nol, with a reactivity pattern similar to that found in benzophenone photoc hemistry. As a final test of suitability as a photoaffinity label, specific labeling of the beta (2)AR in membranes (protectable by 10 muM alprenolol) was demonstrated. [I-125]IAmF represents a new class of beta (2)AR photoaf finity labels that can directly probe the catecholanalogous antagonist phar macophore binding site in the beta (2)AR ligand binding pocket.