Design, Synthesis, In Vitro and In Vivo Characterization of Selective NKCC1 Inhibitors for the Treatment of Core Symptoms in Down Syndrome

Abstract

Intracellular chloride concentration [Cl^-]_i is defective in several neurological disorders. In neurons, [Cl^-]_i is mainly regulated by the action of the Na⁺-K⁺-Cl^- importer NKCC1 and the K⁺-Cl^- exporter KCC2. Recently, we have reported the discovery of ARN23746 as the lead candidate of a novel class of selective inhibitors of NKCC1. Importantly, ARN23746 is able to rescue core symptoms of Down syndrome (DS) and autism in mouse models. Here, we describe the discovery and extensive characterization of this chemical class of selective NKCC1 inhibitors, with focus on ARN23746 and other promising derivatives. In particular, we present compound 40 (ARN24092) as a backup/follow-up lead with in vivo efficacy in a mouse model of DS. These results further strengthen the potential of this new class of compounds for the treatment of core symptoms of brain disorders characterized by the defective NKCC1/KCC2 expression ratio.

Figure 1

Structures of ARN23746 and bumetanide.

Figure 2

Representation of the points subject to chemical manipulation of 3 (ARN22393) and 4 (ARN22394).

Figure 3

Pharmacophore model generation and fitting. (A) Identification of the low energy conformations of bumetanide (white sticks) and generation of the pharmacophore hypothesis (transparent spheres). (B–E) Overlap of bumetanide, furosemide, 1 (ARN23746), and 33 (containing a bulky pyrrolidine group, which is useful to appreciate spatial rotation of the sulfonamide) with the pharmacophore features. Red arrows highlight features’ mismatches between the compounds and the pharmacophore model. Furosemide is the less potent NKCC1 inhibitor, and it shows a different fit onto the model in comparison to other bumetanide derivatives. In the middle circle, the overlap of bumetanide (transparent sticks) and the main scaffold of our selective inhibitors (outlined sticks) is represented. Due to a slight rotation upon the aromatic plane, the fitting of our inhibitors differs from that of bumetanide derivatives.

Figure 4

In vitro activity and selectivity of compound 40 in cell-based assays. (A) Left, example traces obtained in the Ca²⁺ influx assay on 3 days in vitro (DIV) neuronal cultures for each compound (100 μM). The arrows indicate the addition of GABA (100 μM) and KCl (90 mM). Right, quantification of the effect of the indicated compounds (10 and 100 μM) in experiments as those on the left. Data are represented as a percentage of the respective control DMSO. Data represent mean ± SEM from three independent experiments. 10 μM: Kruskal–Wallis one-way ANOVA on ranks, H = 24.747, DF = 2, P < 0.001, Dunn’s post hoc test, *P < 0.05, ***P < 0.001; 100 μM: Kruskal–Wallis one-way ANOVA on ranks, H = 23.646, DF = 2, P < 0.001, Dunn’s post hoc test, ***P < 0.001. (B) Left, example traces obtained in the Cl^– influx assay on NKCC2-transfected HEK293 cells for each compound (10 μM). The arrow indicates the addition of NaCl (74 mM) to initiate the NKCC1-mediated Cl^– influx. Right, quantification of the NKCC2 inhibitory activity in experiments as those on the left. Data are presented as a percentage of the respective control DMSO. Data represent mean ± SEM from four independent experiments (one-way ANOVA, F(2, 48) = 21.161, P < 0.001, Dunnett’s post hoc test, ***P < 0.001). (C) Left, example traces obtained in the Tl influx assay on KCC2-transfected HEK293 cells for each compound (10 μM). The arrows indicate the addition of Tl₂SO₄ (2 mM) and NaCl (74 mM). Right, quantification of the KCC2 inhibitory activity in experiments as those on the left. Data are presented as a percentage of the respective control DMSO. Data represent mean ± SEM from three independent experiments (one-way ANOVA, F(2, 37) = 19.194, P < 0.001, Dunnett’s post hoc test, ***P < 0.001).

Figure 5

Assessment of in vivo diuresis and efficacy of compound 40 in Ts65Dn mice. (A) Schematic representation the experimental protocol for the treatment of adult WT and Ts65Dn mice and assessment of the diuretic effect. (B) Quantification of the mean ± SEM and single animal cases of the urine volume collected for 2 h after mice were treated with the indicated drugs (two-way ANOVA on Ranks, F_treatment(2, 66) = 54.315, P < 0.001, Tukey’s post hoc test, ***P < 0.001). (C) Schematic representing the experimental protocol for the treatment of adult WT and Ts65Dn mice with 40 for in vivo efficacy assessment of memory and hyperactivity in DS mice. (D) Top, schematic representation of the T-maze test. Bottom, quantification of the mean ± SEM and single animal cases of correct choices in mice treated with the indicated drugs (two-way ANOVA, F_interaction(1, 50) = 4.036, P = 0.050, Tukey’s post hoc test, *P < 0.05, **P < 0.01). (E) Top, schematic representation of the novel-object recognition test. Bottom, quantification of the mean ± SEM and single animal cases of the discrimination index in mice treated with the indicated drugs (two-way ANOVA, F_treatment(1, 46) = 7.640, P = 0.008, Tukey’s post hoc test, *P < 0.05, **P < 0.01). (F) Top, schematic representation of the CFC test. Bottom, quantification of the mean ± SEM and single animal cases of the freezing response in mice treated with the indicated drugs (two-way ANOVA, F_interaction(1, 42) = 6.209, P = 0.017, Tukey’s post hoc test, *P < 0.05, **P < 0.01). (G) Top, schematic representation of the open field test. Bottom left, quantification of the mean ± SEM and single animal cases of the distance traveled during the test in mice treated with the indicated drugs (two-way ANOVA, F_genotype(1, 46) = 6.206, P = 0.016, Tukey’s post hoc test, *P < 0.05, **P < 0.01). Bottom right, quantification of the mean ± SEM and single animal cases of the average walking speed in mice treated with the indicated drugs (two-way ANOVA, F_genotype(1, 46) = 6.206, P = 0.016, Tukey’s post hoc test, *P < 0.05, **P < 0.01). (H) Quantification of the body weight of WT and Ts65Dn mice across the 3 weeks of treatment with the indicated drugs.

Scheme 1

Reagents and conditions: (i) HSO₃Cl, 120 °C, 46%; (ii) NH₄OH, THF, 0 °C to rt, 48%; (iii) amine hydrochloride, TEA, DCM, 0 °C to rt, 58–74%; and (iii) amine, toluene, 100 °C, 72–97%.

Scheme 2

Reagents and conditions: (i) amine, 80–100 °C and (ii) HCOONH₄, Pd(OH)₂, MeOH, Ar, 80 °C.

Scheme 3

Reagents and conditions: (i) methylamine or dimethylamine (1 M in THF), THF 0 °C to rt and (ii) amine, TEA, 1,4-dioxane, 100 °C.

Scheme 4

Reagents and conditions: (i) potassium phtalimide, DMF, rt and (ii) hydrazine hydrate, EtOH, reflux.

Scheme 5

Reagents and conditions: (i) amine, THF, 0 °C to rt, (ii) amine hydrochloride, DIPEA, THF, 0 °C to rt; and (iii) amine 53a, TEA, 1,4-dioxane, 100 °C.

Scheme 6

Reagents and conditions: (i) dimethylamine, DIPEA, THF, 0 °C to rt, 41% and (ii) amine 53a, TEA, 1,4-dioxane, 100 °C, 88%.

Scheme 7

Reagents and conditions: (i) HOS₃Cl, 0 to 120 °C, 35%; (ii) dimethylamine, DIPEA, THF, 0 °C, 70%; (iii) TMS-CHN₂, DCM/MeOH 80:20, 0 °C to rt, 92%; (iv) amine 52a, TEA, 1,4-dioxane, 100 °C, 84%; (v) LiOH aq, THF, rt, 72–86%; (vi) BBr₃, DCM, 0 °C to rt, 73–83%; and (vii) alkyl halide, acetonitrile, K₂CO₃, 80 °C, 72–78%.