A High-Affinity Fluorenone-Based β2-Adrenergic Receptor Antagonist with a Photoactivatable Pharmacophore

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Abstract
To develop molecules capable of directly probing the catechol binding region of the β2-adrenergic receptor (β2AR), novel benzophenone- and fluorenone-based β2AR antagonists were prepared as potential photoaffinity probes. While the benzophenone-containing ligands bound with relatively modest affinity, one of the fluorenone-based compounds, 4-(2-hydroxy-3-isopropylaminopropoxy)-7-amino-6-iodofluorenone (iodoaminoflisopolol, IAmF), showed very high affinity for the β2AR, inhibiting [125I]ICYP binding with an apparent Ki of approximately 1 × 10-9 M. In comparison to the benzophenone ligands, the fluorenone ligands have one additional carbon−carbon bond that creates a planar unsaturated ring system and leads to a large increase in receptor binding affinity. Unlike previous β2AR photoaffinity ligands, an attractive and unique feature of the fluorenone derivative IAmF is that the large planar unsaturated ring (believed to correspond to the catechol end of other β2AR ligands) serves as both the binding pharmacophore and the photoreaction center for this molecule. With this potential for directly probing the catechol binding region of the β2AR, we synthesized and tested IAmF in carrier-free radioiodinated form ([125I]IAmF). When photoreduction was conducted at 350 nm for 20 min, [125I]IAmF was able to produce cross-linked products in both triethylamine and methanol, with a reactivity pattern similar to that found in benzophenone photochemistry. As a final test of suitability as a photoaffinity label, specific labeling of the β2AR in membranes (protectable by 10 μM alprenolol) was demonstrated. [125I]IAmF represents a new class of β2AR photoaffinity labels that can directly probe the catechol-analogous antagonist pharmacophore binding site in the β2AR ligand binding pocket.