AbstractBrain GABA_Α receptors are ionotropic receptors belonging to the class of Cys-loop receptors and are important drug targets for the treatment of anxiety and sleep disorders. By screening a compound library (2,112 compounds) at recombinant human α₄β₁δ GABA_Α receptors heterologously expressed in a HEK cell line, we identified a scaffold of spirocyclic compounds with nanomolar antagonist activity at GABA_Α receptors. The initial screening hit 2027 (IC₅₀ of 1.03 μM) was used for analogue search resulting in 018 (IC₅₀ of 0.088 μM). 018 was most potent at α_3,4,5-subunit containing receptors, thus showing preference for forebrain-expressed extrasynaptic receptors. Schild analysis of 018 at recombinant human α₄β₁δ receptors and displacement of [³H]muscimol binding in rat cortical homogenate independently confirmed a competitive profile. The antagonist profile of 018 was further validated by whole-cell patch-clamp electrophysiology, where kinetic studies revealed a slow dissociation rate and a shallow hill slope was observed. Membrane permeability studies showed that 2027 and 018 do not cross membranes, thus making the compounds less attractive for studying central GABA_Α receptors effects, but conversely more attractive as tool compounds in relation to emerging peripheral GABA_Α receptor-mediated effects of GABA e.g. in the immune system.