Beyond Retigabine: Design, Synthesis, and Pharmacological Characterization of a Potent and Chemically Stable Neuronal Kv7 Channel Activator with Anticonvulsant Activity

Figures

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  Scheme 1. Synthesis of 1,4-Phenylenediamine (2), Benzene-1,2,4-triamines (13−20), 2-(pyrrolidin-1-yl)benzene-1,4-diamine (23), 2-(Piperidin-1-yl)benzene-1,4-diamine (24), N4-Alkylbenzene-1,2,4-triamine (25,26,27), and N-(3,4-Diaminophenyl)acetamide (28) Derivatives

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  Scheme 2. Synthesis of 3,4-Diaminophenol Derivative 31

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  Scheme 3. Synthesis of Benzene-1,2,4-triamine Derivatives 41–43, 47, 51, 52, and 57

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  Scheme 4. Synthesis of N-((2-Amino-3-fluoro-4-(benzyl)amino)phenyl)amide (59 and 60), N-(2-Amino-4-(4-(trifluoromethyl)phenethyl)phenyl)amide (67 and 68) Derivatives, and 2-Amino-N-hexyl-4-((4-(trifluoromethyl)benzyl)amino)benzamide (71)

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  Figure 1. Functional characterization of a first series of retigabine derivatives (compounds 13–20, 71, 2, 23, 24, 41–43, 47, 51, 52, 57). (A) Retigabine compounds 23a and 24a structures with the three different areas investigated by structure-based approach. Highlighted: area 1 in blue, area 2 in orange, and area 3 in red. (B) Retigabine binding pocket in Kv7.2 channels. The two Kv7.2 subunits shown are colored in cyan and salmon. Bound conformations of 23a (yellow) and 24a (green) are shown in thin solid sticks. For each ligand, three bound conformations (sampled at 0, 60, and 120 ns from 120 ns-long MD simulations of the ligand/Kv7.2 complex) are shown. Experimentally solved bound conformation of retigabine (PDB ID: 7CR2) is shown in magenta thick transparent sticks. (C) FluxOR fluorescence signals generated in Kv7.2/Kv7.3-transfected cells by the following: vehicle DMSO 0.1% (gray curve), retigabine (RET, purple curves), and retigabine + XE991 co-administrated (black curve). Non-transfected cell (NT) signal is also shown. (D,E) Average value of maximal fluorescence (FMAX/F0; D) and initial slope (E) of the FluxOR fluorescence signal calculated between 5 and 15 s. (F) Dose–response…

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  Figure 2. Molecular dynamics (MD) simulations of retigabine analogues. (A) Predicted bound conformations of 13 (orange), 14 (green), 17 (pink), 18 (light gray), and 19 (dark gray) at 60 ns of MD simulations are depicted in sticks. (B) Bound conformations of 15 (yellow) and 16 (magenta) at 60 ns of MD simulations are depicted in sticks. (C) Predicted bound conformation of 17 (magenta sticks) at 60 ns of MD simulations. (D) Predicted bound conformations of 23 (yellow sticks) and 24 (magenta sticks). In all panels, the two different Kv7.2 monomers are depicted in sticks and cartoons and colored in cyan and salmon. H-bonds are represented by yellow dashed lines.

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  Figure 3. Functional characterization of a second series of retigabine derivatives with improved photostability (compound 25–28, 31, 59, 60, 67, 68). (A) Proposed mechanism for retigabine photo-oxidation. (B) Average FluxOR fluorescence signals obtained in Kv7.2/Kv7.3-transfected cells upon exposure to the indicated photostable compounds, each used at a concentration of 10 μM in comparison with retigabine (RET 10 μM; purple bar). * indicates values significantly different (p < 0.05) from respective control. (C) HPLC traces of retigabine (RET) and compound 60 at 550 nm after 3 h exposure to UV–visible light.

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  Figure 4. Effect of retigabine (RET), RL-81, and compound 60 on Kv7.2/Kv7.3 currents. (A) Representative macroscopic current traces recorded from CHO cell expressing Kv7.2/Kv7.3 channels in response to the indicated voltage protocol before (left) and after (right) application of 1 μM RET, RL-81 and compound 60, as indicated. Current scale, 200 pA; time scale, 200 ms. (B,C) Dose–response curves reporting the effects of the 3 indicated compounds on the V1/2 shift (ΔV1/2) in mV (B) and on maximal current density (Idrug/Icontrol; C) calculated at +20 mV for Kv7.2/Kv7.3 channels. (D,E) Current density of the indicated homomeric Kv7.2 channels (D) and the effect of 10 μM RET or 1 μM 60 on the indicated homomeric Kv7.2 mutant channels. * indicates values significantly different (p < 0.05) from respective controls.

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  Figure 5. Anticonvulsant efficacy of retigabine and compound 60 (c60) in pentylenetetrazol (PTZ)-induced acute seizures in mice. (A,B) Average values for the seizure score (A) and latency to onset of a maximal seizure (B) as a function of retigabine (RET) or compound 60 doses. Individual scores calculated in each animal are indicated by dots. (C,D) Effect of retigabine (RET) and compound 60, with (hatched bars) or without (empty bars) pretreatment with XE991 (XE 3 mg/kg) on seizure score (C) and latency to onset of a maximal seizure (D). (E) Effect of RET (1, 3 mg/kg), c60 (0.3–1 mg/kg) or XE991 (3 mg/kg) on mice survival rate after PTZ exposure. The number of animals used in each group was: 19 for controls, 4 for XE (3 mg/kg), 8 for RET (1 mg/kg), 10 for RET (3 mg/kg), 8 for XE + RET (3 + 1 mg/kg); 3 for c60 (0.1 mg/kg), 15 for c60 (0.3 mg/kg), 8 for c60 (1 mg/kg), and 8 for XE + c60 (3 + 0.1 mg/kg). The asterisk (*) indicates values significantly different (p < 0.05) from respective controls.