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

The synthesis of a tritiated benzopyran‐type opener of the ATP‐dependent K + channel (K ATP channel), [ 3 H]‐PKF217 – 744 {(3 S ,4 R )‐ N ‐[3,4‐dihydro‐2,2‐dimethyl‐3‐hydroxy‐6‐(2‐methyl‐4‐pyridinyl)‐2H‐1‐benzopyran‐4‐yl]‐3‐[2,6‐ 3 H]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 K ATP channel opener by its ability to open the Kir6.1/SUR2B channel, the recombinant form of the vascular K ATP channel, and to inhibit binding of the pinacidil analogue, [ 3 H]‐P1075, to SUR2B ( K i =26 n M ). The kinetics of [ 3 H]‐PKF217 – 744 binding to SUR2B was described by rate constants of association and dissociation of 6.9×10 6   M −1  min −1 and 0.09 min −1 , respectively. Binding of [ 3 H]‐PKF217 – 744 to SUR2B/2A was activated by MgATP (EC 50 ∼3 μ M ) and inhibited (SUR2B) or enhanced (SUR2A) by MgADP. Binding of [ 3 H]‐PKF217 – 744 to SUR2B was inhibited by representatives of the different structural classes of openers and sulphonylureas. K i values were identical with those obtained using the opener [ 3 H]‐P1075 as the radioligand. Glibenclamide accelerated dissociation of the SUR2B‐[ 3 H]‐PKF217 – 744 complex. The data show that the affinity of [ 3 H]‐PKF217 – 744 binding to SUR2B is ∼6 times lower than that of [ 3 H]‐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