Carba Analogues of Flupirtine and Retigabine with Improved Oxidation Resistance and Reduced Risk of Quinoid Metabolite Formation

Abstract

The K_V 7 potassium channel openers flupirtine and retigabine have been valuable options in the therapy of pain and epilepsy. However, as a result of adverse reactions, both drugs are currently no longer in therapeutic use. The flupirtine-induced liver injury and the retigabine linked tissue discolouration do not appear related at first glance; nevertheless, both events can be attributed to the triaminoaryl scaffold, which is affected by oxidation leading to elusive reactive quinone diimine or azaquinone diimine metabolites. Since the mechanism of action, i. e. K_V 7 channel opening, seems not to be involved in toxicity, this study aimed to further develop safer replacements for flupirtine and retigabine. In a ligand-based design strategy, replacing amino substituents of the triaminoaryl core with alkyl substituents led to carba analogues with improved oxidation resistance and negligible risk of quinoid metabolite formation. In addition to these improved safety features, some of the novel analogues exhibited significantly improved K_V 7.2/3 channel opening activity, indicated by an up to 13-fold increase in potency and an efficacy of up to 176 % compared to flupirtine, thus being attractive candidates for further development.

Keywords: KV7; drug design; flupirtine; ion channels; retigabine.

Figure 1

Structures of flupirtine (1) and retigabine (2), the proposed toxification pathway to phenazinium salt 4 in the case of retigabine, and hapten‐protein adducts 5 in the case of flupirtine via azaquinone diimines or quinone diimines 3, and selected structural modifications carried out in previous work (6) and this work (7, 8).

Scheme 1

Synthesis of N‐1 monocarba analogues 7 and 21: a) trimethylsilylacetylene, CuI, Pd(PPh₃)₄, TEA, 60 °C, 16 h, 99 %; b) K₂CO₃, MeOH, RT, 5 h, 85 %; c) NH₃, EtOH, 0 °C–RT, 21 h, 83 %; d) HBr, AcOH, 100 °C, 8 h, 93 %; e) CuI, Pd(PPh₃)₄, TEA, DMF, 60 °C, 6 h, 80 %; f) phenylacetylene, CuI, Pd(PPh₃)₄, TEA, DMF, 60 °C, 1 h, 38 %; g) trimethylsilylacetylene, CuI, Pd(PPh₃)₄, TEA, DMF, 60 °C, 0.5 h, 74 %; h) K₂CO₃, MeOH, RT, 0.5 h, 75 %; i) 1‐bromo‐4‐fluorobenzene, CuI, Pd(PPh₃)₄, TEA, DMF, 60 °C; j) H₂, Pd/C, EtOAc, 40 °C, 40 h; k) ethyl chloroformate, THF, TEA, RT, 4 h, 25 %; l) Fe, AcOH, 50 °C, 1 h; m) ethyl chloroformate, THF, TEA, RT, 7 h, 30 %.

Scheme 2

Synthesis of N‐1/3 dicarba analogue 27: a) Zn(CN)₂, Pd(PPh₃)₄, DMF, 70 °C, 24 h, 96 %; b) Fe, AcOH, CaCl₂, EtOH, RT, 2 h, 71 %; c) isobutyl chloroformate, TEA, 4‐DMAP, DCM, RT, 4 d, 34 %; d) DIBAL−H, DCM, −85 °C, 4 h, 23 %; e) 1. 4‐fluoroaniline, DCM, RT, 5 h, 2. NaBH₄, MeOH, RT, 1 h, 82 %.

Scheme 3

Synthesis of N‐1/3 dicarba analogues 35 a‐f: a) Zn(CN)₂, Pd(OAc)₂, PPh₃, DMF, 100 °C, 1.5 h, 83 %; b) SnCl₂, EtOAc, 70 °C, 0.5 h, 99 %; c) Br₂, DCM, −78 °C–RT, 1 h, 96 %; d) CuCN, NMP, 170 °C, 7–8 h, 42–47 %; e) 1. acyl chloride, TEA, 4‐DMAP, THF, 0 °C, 0.5 h, 2. RT/70 °C, 16 h–7 d, 56–74 % (33 a,d); f) 1. acyl chloride, TEA, DCM, RT, 1 h, 2. RT/40 °C, 3–89 h, 67–88 % (33 b,c,e); g) Ni, HCOOH, 80 °C, 6–8 h, 37–88 %; h) 1. amine, molecular sieves, toluene, 120 °C, 4–8 h, 2. NaBH₄, MeOH, 1,4‐dioxane, 0 °C–RT, 17 h, 42–73 %.

Scheme 4

Synthesis of N‐1/3 dicarba analogues 43 a‐c: a) EtOH, SOCl₂, 90 °C, 18 h, 88 %; b) NBS, THF, 0 °C–RT, 17 h, 77 %; c) LiAlH₄, THF, RT, 5 h, 54 %; d) MnO₂, toluene, 80 °C, 2 h, 41 %; e) acyl chloride, DIPEA, DCM, 0 °C–RT, 16.5–24.5 h 48–50 %; f) 1. 4‐fluoroaniline, toluene, molecular sieves, 120 °C, 5 h, 2. NaBH₄, MeOH, 1,4‐dioxane, 0 °C, 17 h, 57–74 %; g) trimethylboroxine or cyclopropylboronic acid, Pd(PPh₃)₄, Na₂CO₃, 1,4‐dioxane, H₂O, 140 °C, μW irradiation, 75 min, 15–43 %.

Scheme 5

Synthesis of N‐1/3 dicarba analogues 48 and 53: a) Phthalic anhydride, AcOH, 130 °C, 5 h, 83 %; b) Ni, HCOOH, 80 °C, 6 h, 81 %; c) 4‐fluoroaniline, molecular sieves, toluene, 120 °C, 6 h; d) NaBH₄, MeOH, 1,4‐dioxane, 0 °C–RT, 17 h, 68 % (c+d); e) H₂, Pd/C, EtOAc, RT, 6 h, 67 % (c+e); f) Cbz‐Cl, DIPEA, DCM, RT, 4 h, 84 %; g) N₂H₄, H₂O, THF, RT, 16 h, 98 %; h) DIC, HOBt, DMF, RT, 16 h, 69 %; i) HBr, AcOH, RT, 2 h, 89 %; j) H₂, Pd/C, EtOAc, RT, 20 min, 61 %; k) DBU, MeCN, RT, 16 h, 76 %; l) KOH, H₂O, MeOH, RT, 16 h, 74 %; m) acyl chloride, DIPEA, DCM, 0 °C, 2.5 h, 94 %; n) pyrrolidine, EtOH, 60 °C, 7 h, 87 %; o) H₂, Pd/C, MeOH, RT, 2 h, 50 %.

Figure 2

Left: Crystal structures of 1,2‐diphenylethane (A) and N‐benzylaniline (C) in comparison to the conformation of retigabine in the bound state (B) obtained from a cryo‐EM structure of K_V7.2 in complex with retigabine. Right: Helmholtz free energy landscapes for the conformational space analysis of N‐benzylaniline and 1,2‐diphenylethane in octan‐1‐ol, as representative scaffolds for the investigated ligand structures. The star denotes the corresponding ring angle and ring distance in retigabine bound to K_V7.2.

Figure 3

Predicted induced‐fit docking poses of retigabine (A), 27 (B), 35 d (C), and 43 c (D) in the binding pocket of the heterotetrameric K_V7.2/3 potassium channel. The hydrogen bonds (yellow dashed lines) of the carbamate group are mostly preserved for all compounds, while the alkyl chain binds to a larger hydrophobic pocket. Inverting the secondary amine nitrogen atom position results in a hydrogen bond interaction with the backbone carbonyl oxygen atom of W236. The hydrogen bond interactions to S342 and F305, formed by flupirtine and retigabine, are not mandatory for activity. The primary amino group can therefore be replaced by hydrophobic substituents to form interactions with a small pocket between I339 and L338. An inversion of the pyridine ring realized in 43 c enables the pyridine nitrogen atom to act as a hydrogen bond acceptor for S342.

Figure 4

Comparison of the concentration‐activity curves of 35 a and flupirtine.

Figure 5

Cyclic voltammograms of retigabine (green) and the N‐1/3 dicarba analogue 35 a (red) in 0.1 M Tris‐HCl buffer (pH 7.4) with anodic peak potential values (E_p.a.) indicated by dashed lines.