Evaluation of Novel Cloxyquin Analogs for K 2P 18.1 Channel Modulation

Figures

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  Scheme 1. Synthesis of 5‐chloroquinoline derivatives 2b–2e. Reagents and reaction conditions: (a) 1a, glycerol, H2SO4 conc., KI, 140°C, 2 h, 16%. (b) 1b, glycerol, H2SO4 80%, KI, 140°C, 6.5 h, 45%. (c) 2b, H2SO4 conc., methanol, 80°C, overnight, 33%. (d) 2b, CH3NH2, EDC─HCl, NEt3, CH2Cl2, rt, 2 h, 15%. (e) 2b, 1. oxalyl chloride, DMF, CH2Cl2, 1 h 0°C, 2 h rt. 2. LiAlH4, diethyl ether, −60°C, 1.5 h, 15%.

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  Scheme 2. Synthesis of 5‐bromoquinolines 3b–3g. Reagents and reaction conditions: (a) CH3I (1 equivalent), K2CO3, DMF, rt, 2 d, 35%. (b) DMF, KOtBu, rt, 1 d, 6%. (c) salicylaldehyde, EtOH, rt, 10 min, 64%. (d) KOCN, CH3COOH/H2O (70:30 V/V), rt, 1.5 h, 54%. (e) CH3COCl, NEt3, CH2Cl2, rt, 2.5 h, 65%. (f) NaNO2, HCl, H2O, 0°C, 30 min, then KI, 0°C, 2.5 h, 53%.

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  Scheme 3. Synthesis of 5‐bromoquinolines 4a–4c. Reagents and reaction conditions: (a) n‐BuLi, THF, −78°C, 10 min, then DMF, −78°C, 10 min, 55%. (b) o‐phenylenediamine, THF/H2O (1:1), reflux, 24 h, 30%. (c) n‐BuLi, −78°C, THF, 1 h, then B(OCH3)3, rt, 1.5 h, 30%.

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  Figure 1. Pharmacological evaluation of 5‐chloro‐/5‐bromo‐quinolines 2–4. (A) Schematic representation of utilized pulse protocol (top) and resulting current traces recorded via TEVC in hK2P18.1 channel expressing Xenopus laevis oocytes. (B) Ic values determined for 100 µM 2–4. Values are given as mean ± SEM. Columns for different compound classes are colored in red (Class 1), blue (Class 2), purple (Class 3), and green (Class 4). Significance of log2 (Ic) ≠ 0 was determined by a general linear regression model (see 4.2–1) and is indicated by **p < 0.01 and ***p < 0.001. (C) Sample trace showing the effect of 100 µM 3d. SEM, standard error of mean; TEVC, two‐electrode voltage clamp.

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  Figure 2. Time‐dependent hydrolysis of 3d. (A) Reaction scheme of 3d hydrolysis in aqueous solutions forming 3a and salicylaldehyde 5. (B) Overlay of 88 UV‐Vis spectra from 60 s (violet) to 89 min (red) after solution preparation. (C) Overlay of 1H‐NMR recorded after 12 min of solution preparation every 10 min. (D, E) Linear regression of UV‐Vis absorbance (ln (A421 nm(t)/A0 421 nm), 2D) and 1H‐NMR data (ln (I 3d /IH2O). (E) Fitting procedures and parameters are given in Supporting Information: Table 1 and Section 4.

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  Figure 3. Docking poses for 3d and 4b in comparison to cloxyquin (clx). (A) Overlay of newly generated docking pose for 3d compared to previously generated docking pose of cloxyquin. Most important interacting amino acids (F145, E349, F352, and Q359) are shown from both complexes (hK2P18.1/clx, hK2P18.1/3 d). Major conformational changes of amino acid side chains can only be observed for E349 (black circle). (B, C) Close up depiction of 3d (B) and 4b (C) docking poses at the cloxyquin binding site. H bond interactions are indicated by yellow dotted lines.