Development of Riluzole Analogs with Improved Use-Dependent Inhibition of Skeletal Muscle Sodium Channels

Abstract

Several commercially available and newly synthesized riluzole analogs were evaluated in vitro as voltage-gated skeletal muscle sodium-channel blockers. Data obtained from the patch-clamp technique demonstrated that potency is well correlated with lipophilicity and the introduction of a protonatable amino function in the benzothiazole 2-position enhances the use-dependent behavior. The most interesting compound, the 2-piperazine analog of riluzole (14), although slightly less potent than the parent compound in the patch-clamp assay as well as in an in vitro model of myotonia, showed greater use-dependent Nav1.4 blocking activity. Docking studies allowed the identification of the key interactions that 14 makes with the amino acids of the local anesthetic binding site within the pore of the channel. The reported results pave the way for the identification of novel compounds useful in the treatment of cell excitability disorders.

Figure 1

Riluzole (1) and riluzole analogs 2–11.

Scheme 1

Reagents and conditions: i) hydrazine, ethylene glycol, 12 M HCl, 140 °C, 2.5 h; ii) CuCl₂, tert-butyl nitrite, acetonitrile, rt for 2 h, then 70 °C for 1.5 h; iii) suitable amines, acetonitrile, TEA, 70 °C, overnight; iv) 70% formic acid, 40% paraformaldehyde, 80 °C, 8 h.

Scheme 2

Reagents and conditions: i) CuCl₂, tert-butyl nitrite, acetonitrile, rt for 2 h, then 70 °C for 1.5 h; ii) piperazine, acetonitrile, TEA, 70 °C, overnight; iii) aq HCl.

Figure 2

Effects of 6-substituted riluzole analogs at 100 μM concentration on Nav1.4 sodium current amplitude. (A) Representative Nav1.4 sodium currents recorded in HEK293T cells in control conditions (CTRL) and during application of 100 μM compounds at 0.1 or 10 Hz stimulation frequency. (B) Mean effects of 100 μM compounds on the Nav1.4 sodium current amplitude. (C) Concentration–response curves of riluzole (1), 3, and 5, at 0.1 or 10 Hz stimulation frequency. Results are reported as mean values ± SEM from 3 to 8 cells.

Figure 3

Effects of the synthesized riluzole analogs (12, 14–19, 22) at 100 μM concentration on Nav1.4 sodium current amplitude. (A) Representative Nav1.4 sodium currents recorded in HEK293T cells in control conditions (CTRL) and during application of 100 μM riluzole (1), 14 or 22 at 0.1 or 10 Hz stimulation frequency. (B) Mean effects of 100 μM 12, 14–19, and 22 on the Nav1.4 sodium current amplitude. (C) Concentration–response curves of riluzole (1), 14, or 22, at 0.1 or 10 Hz stimulation frequency. Results are reported as mean values ± SEM from 3 to 8 cells.

Figure 4

Effects of riluzole (1) and compounds 14 and 22 on rat skeletal muscle fiber excitability in a myotonia-like condition induced by 50 μM 9-AC. The maximal number of elicitable action potentials (N-spikes) were measured in isolated rat extensor digitorum longus muscle fibers, in control saline solution (CTRL, not shown), in the presence of 50 μM 9-AC (A), and in the presence of 50 μM 9-AC and 1 μM riluzole (B). Similar experiments were repeated with various concentrations of riluzole and compounds 14 and 22, and averaged results are shown in (C), where each bar is the mean ± SEM from 5 to 21 fibers. Statistical analysis was performed using unpaired Student’s t-test: *at least p < 0.05 versus CTRL; °at least p < 0.05 versus 9-AC.

Figure 5

Identification of Nav1.4 ligand-binding cavities through SiteMap. The detected pockets are shown as red (C1), gray (C2), cyan (C3), orange (C4), and blue (C5) surfaces.

Figure 6

Top-scored docking pose of 14 within the identified cavity C1.

Figure 7

Effects of the synthesized riluzole analogs (12 and 22) at 100 μM concentration on Nav1.7 sodium current amplitude. (A) Representative Nav1.7 sodium currents recorded in HEK293T cells in control conditions (CTRL) and during application of 100 μM riluzole (1), 14, and 22, at 0.1 or 10 Hz stimulation frequency. (B) Concentration–response curves of riluzole (1), 14, and 22, at 0.1 or 10 Hz stimulation frequency. Results are reported as mean values ± SEM from 3 to 12 cells.