Novel Alaninamide Derivatives with Drug-like Potential for Development as Antiseizure and Antinociceptive Therapies─In Vitro and In Vivo Characterization

Abstract

In the present study, a series of original alaninamide derivatives have been designed applying a combinatorial chemistry approach, synthesized, and characterized in the in vivo and in vitro assays. The obtained molecules showed potent and broad-spectrum activity in basic seizure models, namely, the maximal electroshock (MES) test, the 6 Hz (32 mA) seizure model, and notably, the 6 Hz (44 mA) model of pharmacoresistant seizures. Most potent compounds 26 and 28 displayed the following pharmacological values: ED₅₀ = 64.3 mg/kg (MES), ED₅₀ = 15.6 mg/kg (6 Hz, 32 mA), ED₅₀ = 29.9 mg/kg (6 Hz, 44 mA), and ED₅₀ = 34.9 mg/kg (MES), ED₅₀ = 12.1 mg/kg (6 Hz, 32 mA), ED₅₀ = 29.5 mg/kg (6 Hz, 44 mA), respectively. Additionally, 26 and 28 were effective in the ivPTZ seizure threshold test and had no influence on the grip strength. Moreover, lead compound 28 was tested in the PTZ-induced kindling model, and then, its influence on glutamate and GABA levels in the hippocampus and cortex was evaluated by the high-performance liquid chromatography (HPLC) method. In addition, 28 revealed potent efficacy in formalin-induced tonic pain, capsaicin-induced pain, and oxaliplatin- and streptozotocin-induced peripheral neuropathy. Pharmacokinetic studies and in vitro ADME-Tox data proved favorable drug-like properties of 28. The patch-clamp recordings in rat cortical neurons showed that 28 at a concentration of 10 μM significantly inhibited fast sodium currents. Therefore, 28 seems to be an interesting candidate for future preclinical development in epilepsy and pain indications.

Keywords: ADME-Tox properties; antinociceptive activity; antiseizure activity; epilepsy; hybrid molecules; neuropathic pain.

Figure 1

Development process yielding in broad-spectrum antiseizure compounds KA-11, KA-104, and KJ-5 described previously, as well as a general structure of new hybrid molecules reported herein. * Data from ref (14) (see compound 11). ** Data from ref (16) (see compound 22). *** Data from ref (17). **** Data from ref (18) (see compound 53).

Scheme 1. Synthesis of Intermediates and Final Compounds of Series 1 (21–30), Series 2 (31–36), and Series 3 (45–48)

Figure 2

Acute effects of compounds 26 (A) and 28 (B) on seizure thresholds in the timed ivPTZ seizure test in mice. Both compounds were administered i.p. at a dose of 50 mg/kg, 30 min before the test. Control animals received vehicle. Data are presented as means (mg/kg PTZ) ± SEM (n = 9–13 animals). The statistical significance was evaluated by a Student’s t-test: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, as compared to the vehicle-treated group (GraphPad Prism 8).

Figure 3

Effect of compound 28 on the progression of PTZ-induced kindling in mice. Compound 28, VPA, or vehicle were administered i.p. every 24 h. PTZ (40 mg/kg, i.p.) was given three times a week, 30 min after administration of compound 28, VPA, or vehicle. Data are shown as means of seizure severity ± SEM (n = 13–15 animals). The statistical significance was evaluated by a mixed-effect model for repeated measures followed by Tukey’s post hoc test: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 as compared to the control group (GraphPad Prism 8).

Figure 4

Effect of treatments on glutamate and GABA concentrations in the hippocampus (A) and cortex (B). Data are shown as means of seizure severity ± SEM (n = 9–15 animals). The statistical significance was evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test: *p < 0.05, **p < 0.01, and ****p < 0.0001 (GraphPad Prism 8).

Figure 5

Effect of treatments on mature BDNF (mBDNF) expression in the hippocampus (A) and cortex (B) and on proNGF expression in the hippocampus (C) and cortex (D) with representative immunoblots, together with the total protein amount visualized by the stain-free technique. Data are shown as means of relative expressions ± SEM (n = 8 animals). The statistical significance was evaluated by one-way ANOVA followed by Tukey’s post hoc test: *p < 0.05 and **p < 0.01 (GraphPad Prism 8).

Figure 6

(A) Effect of compound 28 on the duration of licking/biting behavior in the acute phase (0–5 min after formalin injection and in the late phase and 15–30 min after formalin injection). The test compound or vehicle (1% Tween 80) was administered 30 min i.p. before the test. (B) The effect of compound 28 on the duration of the nociceptive response in capsaicin-induced pain. The test compound or vehicle (1% Tween 80) was administered 30 min (i.p.) before the capsaicin injection. The results are presented as bar plots showing the mean ± SEM. (C) Antiallodynic effects of compound 28 in the tactile allodynia in oxaliplatin (OXPT)-induced peripheral neuropathy. The compound was administered at the doses of 25, 50, and 75 mg/kg 30 min before the evaluation in the von Frey test carried out 3 h and 7 days after OXPT injection. (D) Antiallodynic effects of compound 28 in the tactile allodynia in streptozotocin (STZ)-induced peripheral neuropathy. The compound was administered at the doses of 12.5, 25, and 50 mg/kg 30 min before the evaluation in the von Frey test carried out 21 days after STZ injection. The statistical significance (A, B) was evaluated by one-way ANOVA followed by Dunnett’s post hoc test: **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 8–10 mice per group. The statistical significance (C, D) was evaluated by repeated measures analysis of variance (ANOVA), followed by Dunnett’s post hoc comparison: *p < 0.05, **p < 0.01, and ***p < 0.01 when results compared to the OXPT-treated group (Post Oxali/Pre 28) or STZ-treated group (Post 28) and ^∧p < 0.05, ^∧∧p < 0.01, and ^∧∧∧p < 0.001 when results compared to naive mice, n = 10 mice per group (GraphPad Prism 8).

Figure 7

Compound 28 inhibits fast voltage-gated sodium currents in prefrontal cortex pyramidal neurons. (A) Sodium current recordings in control (black trace), after application of 28 (blue trace), and after wash-out (red trace). Current traces were evoked by a rectangular voltage step. (B) Influence of 28 on sodium current is shown on an example neuron. Current traces were evoked once every 10 s. The vertical axis shows maximal current amplitudes (white circles) in control, in the presence of 28, and after wash-out. The horizontal axis shows the trace number. (C) Averaged normalized maximal sodium current amplitudes in control, in the presence of 28, and after wash-out. The statistical significance was evaluated by one-way ANOVA with Tukey’s post hoc test (n = 5): *p < 0.05 (GraphPad Prism 8).

Figure 8

Effect of 26, 28, and the reference drugs ketoconazole (KE) and quinidine (QD) on CYP3A4 (A) and CYP2D6 (B) activity. Statistical significance (*p < 0.05, **p < 0.01, ****p < 0.0001) was analyzed by GraphPad Prism 8.0.1 software using one-way ANOVA and Bonferroni’s multiple comparison post test.

Figure 9

Effect of 26, 28, and the reference drug doxorubicin (DOX) on the HepG2 cell viability. Statistical significance (**p < 0.01, ***p < 0.001, ****p < 0.0001) was analyzed by GraphPad Prism 8.0.1 software using one-way ANOVA and Bonferroni’s multiple comparison post test.