Affinity probes for the GABA-gated chloride channel: Selection of 5e-tert-Butyl-2e-[4-(substituted-ethynyl)phenyl]-1,3-dithianes and optimization of linker moiety

The noncompetitive blocker (NCB) site of the γ-aminobutyric acid (GABA)-gated chloride channel is the target for many important insecticides and potent convulsants. This site is specifically blocked by ³H ethynylbicycloorthobenzoate (³H EBOB) and other trioxabicyclooctane radioligands and might be suitable for affinity probes with an appropriate heterocyclic substituent and linker moiety. Optimal potency at the NCB site is achieved with 5e-tert-butyl-2e-4-(substituted-ethynyl)phenyl]-1,3-dithianes compared with analogs in which the butyldithiane portion is replaced with butyldithiane-sulfoxide or -sulfone, n-propyltrioxabicyclooctane or dioxatricyclododecene. Three positions were examined for coupling the linker and dithiane: C-2 of the dithiane; a branched substituent within the alkynyl moiety; the terminus of a straight chain extension from the ethynyl group, which proved to be the best. Optimized linkers for addition to the ethynylphenyldithiane to achieve appropriate length and fit within the active site, i.e. receptor potency, are CH₂OCH₂C(O)SCH₂CH₂(SH or NH₂) and the corresponding thiolates and amides. Several compounds with these spacers block the chloride channel, measured as inhibition of ³H EBOB binding, at 4–50 nM.