Clerodane Diterpenes from Casearia corymbosa as Allosteric GABA(A) Receptor Modulators

[Image: see text] An EtOAc extract of Casearia corymbosa leaves led to an allosteric potentiation of the GABA signal in a fluorometric imaging plate reader (FLIPR) assay on Chinese hamster ovary (CHO) cells stably expressing GABA(A) receptors with an α(1)β(2)γ(2) subunit composition. The activity was tracked by HPLC-based activity profiling, and four known (2, 3, 4, and 8) and five new clerodane-type diterpenoids (1, 5–7, and 9) were isolated. Compounds 1–8 were obtained from the active time window. The absolute configuration of all compounds was established by ECD. Compounds 3, 7, and 8 exhibited EC(50) values of 0.5, 4.6, and 1.4 μM, respectively. To explore possible binding sites at the receptor, the most abundant diterpenoid 8 was tested in combination with diazepam, etazolate, and allopregnanolone. An additive potentiation of the GABA signal was observed with these compounds, while the effect of 8 was not inhibited by flumazenil, a negative allosteric modulator at the benzodiazepine binding site. Finally, the activity was validated in voltage clamp studies on Xenopus laevis oocytes transiently expressing GABA(A) receptors of the α(1)β(2)γ(2)S and α(1)β(2) subtypes. Compound 8 potentiated GABA-induced currents with both receptor subunit compositions [EC(50) (α(1)β(2)γ(2)S) = 43.6 μM; E(max) = 809% and EC(50) (α(1)β(2)) = 57.6 μM; E(max) = 534%]. The positive modulation of GABA-induced currents was not inhibited by flumazenil, thereby confirming an allosteric modulation independent of the benzodiazepine binding site.