Many investigators have noted structural similarities

Many investigators have noted structural similarities between certain flavonoids and benzodiazepines, such as diazepam, that are the most widely studied positive modulators of GABAA receptors. Benzodiazepines can act on these receptors via ‘two distinct and separable mechanisms’ (Walters, Hadley, Morris, & Amin, 2000). At nanomolar concentrations, benzodiazepines act in a classic flumazenil-sensitive manner to enhance the action of GABA, while at micromolar concentrations, benzodiazepines act in a flumazenil-insensitive manner. Flavonoids can act on GABAA receptors at low concentrations in either a flumazenil-sensitive or flumazenil-insensitive manner as modulators of these receptors (Hanrahan et al., 2011). Furthermore, many flavonoids act in a biphasic manner, potentiating GABA actions at low concentrations and inhibiting at high concentrations. In addition, some flavonoids have agonist actions on certain GABA receptors, directly gating the Ezatiostat hydrochloride in the absence of GABA. Clearly flavonoids can interact with a variety of specific active sites on ionotropic GABA receptors. Unraveling these active sites remains a major task.
6-Substituted Flavones Previous studies have shown the 6-position on flavones as being relevant to determining the effects on recombinant GABAA receptors (Hall et al., 2004, Ren et al., 2010). A study of flavones (Fig. 1), each varying only at position 6, were compared, including 6-fluoroflavone, 6-chloroflavone, 6-bromoflavone, 6-hydroxyflavone, and 6,2′-dihydroxyflavone demonstrated 6-bromoflavone to be a positive modulator at GABAA receptors acting through flumazenil-sensitive high-affinity benzodiazepine sites (Ren et al., 2011). In contrast, the other two 6-haloflavones and 2′-hydroxyflavone were neutralizing modulators, while 6,2′-dihydroxyflavone is a negative modulator of GABAA receptors. In contrast, 6-methylflavone is known to be a flumazenil-insensitive positive modulator of GABAA receptors (Hall et al., 2004). The fact that flavone analogues differing only at position 6 showed drastically different pharmacological properties clearly points to 6-substitution being an important determinant of efficacy and binding sites. 2′-Methoxy-6-methylflavone (2′MeO6MF, Fig. 2) acts as a positive modulator at α2β1γ2L and all α1-containing GABAA receptor subtypes (Karim et al., 2012). In contrast, at α2β2/3γ2L, it directly activates these receptors without potentiating GABA. This activation is attenuated by bicuculline and gabazine but not flumazenil indicating a novel site. Mutation studies show that position 265 in the β1/2 subunit was key to whether 2′MeO6MF acts as an activator or a potentiator. In hippocampal neurons, 2′MeO6MF directly activated single-channel currents that showed the hallmarks of GABAA chloride currents. In the continued presence of 2′MeO6MF, the single-channel conductance increased and these high-conductance channels were disrupted by the γ(381–403) MA peptide, indicating that such currents are mediated by α2/γ2-containing GABAA receptors. In mice, 2′MeO6MF displayed anxiolytic-like effects in two unconditioned models of anxiety: the elevated plus maze and light/dark tests. 2′MeO6MF induced sedative effects at higher doses in the hole board, actimeter, and barbiturate-induced sleep time tests. No myorelaxant effects were observed in the horizontal wire test (Karim et al., 2012). 3-Hydroxy-2′-methoxy-6-methylflavone (3-OH-2′MeO6MF, Fig. 2) was found to potentiate GABA-induced currents at recombinant α1/2β and α1/2/4/6β1–3γ2L but not α3/5β1–3γ2L receptors (Karim et al., 2011). 3-OH-2′MeO6MF preferentially activated β2/3- over β1-containing receptors, with the highest efficacy observed at α2β2/3γ2L. In addition, this flavone acted as a potent bicuculline-sensitive allosteric agonist at α4β2/3δ receptors, as a partial agonist at α4β1δ receptors, and was devoid of potentiation effects at extrasynaptic α4β2/3δ receptors. The affinity of 3-OH-2′MeO6MF for α4β2/3δ receptors is 10-fold higher than at α4β1δ GABAA receptors. In mice, 3-OH-2′MeO6MF also induced anxiolytic-like effects in the elevated plus maze and light/dark paradigms (Karim et al., 2011). This 6-substituted flavone thus exhibited a unique profile at GABAA receptor subtypes.