Synthesis and characterization of a novel tritiated KATP channel opener with a benzopyran structure

The synthesis of a tritiated benzopyran-type opener of the ATP-dependent K+ channel (KATP channel), [3H]-PKF217 – 744 {(3S,4R)-N-[3,4-dihydro-2,2-dimethyl-3-hydroxy-6-(2-methyl-4-pyridinyl)-2H-1-benzopyran-4-yl]-3-[2,6-3H]pyridinecarboxamide} with a specific activity of 50 Ci mmol−1 is described. Binding of the ligand was studied in membranes from human embryonic kidney cells transfected with the sulphonylurea receptor isoforms, SUR2B and SUR2A, respectively. PKF217 – 744 was confirmed as being a KATP channel opener by its ability to open the Kir6.1/SUR2B channel, the recombinant form of the vascular KATP channel, and to inhibit binding of the pinacidil analogue, [3H]-P1075, to SUR2B (Ki=26 nM). The kinetics of [3H]-PKF217 – 744 binding to SUR2B was described by rate constants of association and dissociation of 6.9×106 M−1 min−1 and 0.09 min−1, respectively. Binding of [3H]-PKF217 – 744 to SUR2B/2A was activated by MgATP (EC50∼3 μM) and inhibited (SUR2B) or enhanced (SUR2A) by MgADP. Binding of [3H]-PKF217 – 744 to SUR2B was inhibited by representatives of the different structural classes of openers and sulphonylureas. Ki values were identical with those obtained using the opener [3H]-P1075 as the radioligand. Glibenclamide accelerated dissociation of the SUR2B-[3H]-PKF217 – 744 complex. The data show that the affinity of [3H]-PKF217 – 744 binding to SUR2B is ∼6 times lower than that of [3H]-P1075. This is due to a surprisingly slow association rate of the benzopyran-type ligand, suggesting a complex mechanism of opener binding to SUR. The other pharmacological properties of the two opener radioligands are identical. British Journal of Pharmacology (2001) 133, 275–285; doi:10.1038/sj.bjp.0704071