Aniline analogues as synchronized Ca²⁺ oscillation modulators: structure-activity relationship and mechanism

Spontaneous synchronized Ca²⁺ oscillations (SCOs) are fundamental regulators of neuronal development, dendritic growth, and synaptogenesis. Modulation of SCOs is a promising strategy to discover neuromodulators for treating multiple neurological disorders. In the current study, an aniline compound (cpd) (1) was identified as a SCO modulator from the sponge Topsentia sp., suppressing SCO amplitude and frequency in primary cultured neocortical neurons with IC₅₀ values of 0.38 μM and 0.88 μM, respectively. Further synthesis led to the discovery of a series of aniline analogues. A subsequent structure-activity relationship (SAR) analysis revealed that a meta-substitution pattern and the hydrophobic C4-C5 chains are crucial for the SCO inhibition. Mechanistic investigation demonstrated that cpds 1 and 4 concentration-dependently suppressed veratridine-induced Ca²⁺ influx with IC₅₀ values of 1.89 μM and 1.35 μM, respectively in primary cultured neocortical neurons. In HEK-293 cells expressing Na_v1.2, cpds 1 and 4 shifted the voltage dependence of steady-state inactivation toward hyperpolarization by 10.99 and 12.67 mV, respectively, while producing no significant change in the steady-state activation. Cpds 1 and 4 preferentially bound to the inactivated state of Na_v1.2 with IC₅₀ values of 0.18 μM and 0.16 μM, respectively. Together these data suggest that suppression of SCOs by both cpds in cortical neurons is primary mediated through the inhibition of Na_v1.2. This work not only elucidates the SAR of aniline analogues as SCO inhibitors, but also advances the discovery of marine-derived small molecules as promising leads for neurological therapeutics.