Discovery and Profiling of New Multimodal Phenylglycinamide Derivatives as Potent Antiseizure and Antinociceptive Drug Candidates

Abstract

We developed a focused series of original phenyl-glycinamide derivatives which showed potent activity across in vivo mouse seizure models, namely, maximal electroshock (MES) and 6 Hz (using both 32 and 44 mA current intensities) seizure models. Following intraperitoneal (i.p.) administration, compound (R)-32, which was identified as a lead molecule, demonstrated potent protection against all seizure models with ED₅₀ values of 73.9 mg/kg (MES test), 18.8 mg/kg (6 Hz, 32 mA test), and 26.5 mg/kg (6 Hz, 44 mA test). Furthermore, (R)-32 demonstrated efficacy in both the PTZ-induced kindling paradigm and the ivPTZ seizure threshold test. The expression of neurotrophic factors, such as mature brain-derived neurotrophic factor (mBDNF) and nerve growth factor (NGF), in the hippocampus and/or cortex of mice, and the levels of glutamate and GABA were normalized after PTZ-induced kindling by (R)-32. Importantly, besides antiseizure activity, (R)-32 demonstrated potent antinociceptive efficacy in formalin-induced pain, capsaicin-induced pain, as well as oxaliplatin- and streptozotocin-induced peripheral neuropathy in mice (i.p.). No influence on muscular strength and body temperature in mice was observed. Pharmacokinetic studies and in vitro ADME-Tox data (i.e., high metabolic stability in human liver microsomes, a weak influence on CYPs, no hepatotoxicity, satisfactory passive transport, etc.) proved favorable drug-like properties of (R)-32. Thermal stability of (R)-32 shown in thermogravimetry and differential scanning calorimetry gives the opportunity to develop innovative oral solid dosage forms loaded with this compound. The in vitro binding and functional assays indicated its multimodal mechanism of action. (R)-32, beyond TRPV1 antagonism, inhibited calcium and sodium currents at a concentration of 10 μM. Therefore, the data obtained in the current studies justify a more detailed preclinical development of (R)-32 for epilepsy and pain indications.

Keywords: antinociceptive activity; antiseizure activity; hybrid molecules; in vitro ADME-Tox studies; in vitro functional studies; multimechanistic compounds.

Figure 1

Development process yielding a chemical prototype compound KJ-5 (*disclosed as compound 53 in Jakubiec et al.(38)) and general structures of new hybrid molecules reported herein.

Scheme 1. Synthesis of Intermediates and the Final Compounds 3–12 (Series A) and 21–24 (Series B)

Scheme 2. Synthesis of Intermediates and the Final Enantiomers (R)-31–(R)-33 and (S)-31–(S)-33 (Series C)

Figure 2

Effect of eutomers (R)-31, (R)-32, and (R)-33 on the thresholds for the onset of (A) myoclonic twitch, (B) generalized clonus, and (C) forelimb tonic extension in the timed ivPTZ test in mice. (R)-31, (R)-32, and (R)-33 were administered, i.p., 30 min before the seizure test. Control animals received vehicle. Data are presented as means + SD in mg/kg of PTZ necessary to induce each of the three end points (n = 7–14 animals). Statistical significance was evaluated using Student’s t-test: **p < 0.01, ****p < 0.0001 vs the vehicle-treated group (GraphPad Prism 8.4.3).

Figure 3

Effect of repeated treatment with (R)-32 in PTZ-induced kindling model in mice. (R)-32, VPA, or vehicle were administered i.p. every 24 h. PTZ at a subconvulsive dose of 40 mg/kg was given i.p. three times a week, 30 min after administration of compound (R)-32, VPA, or vehicle. Data are presented as means of seizure severity (n = 10–15 animals). Statistical significance was evaluated by a mixed effects model for repeated measures followed by Tukey’s post hoc test: **p < 0.01, ****p < 0.001 vs control group (GraphPad Prism 8.4.3).

Figure 4

(R)-32 fails to affect REDs (recurrent epileptiform discharges) at any concentrations tested. (A) Representative traces of REDs recorded before (baseline), in the presence of 80 μM (R)-32, and 20 min after washout. (B) Time courses for 80 μM (R)-32’s effects of REDs duration (B1, green), frequency (B2, blue), and amplitude (B3, red). Dashed-line boxes from 20 to 40 min represent the duration of 80 μM (R)-32 exposure. Data points represent mean ± SD for REDs parameters in 1 min bins. (C) Concentration-dependent effects of (R)-32 on REDs duration (green), frequency (blue), and amplitude (red). Bars represent mean + SD of 13 slices (40 μM), 8 slices (80 μM), and 10 slices (120 μM).

Figure 5

Changes in glutamate and GABA concentrations in the hippocampus (A) and cortex (B). Data are shown as means + SD (n = 9–14 animals). Statistical significance was evaluated by one-way ANOVA followed by Tukey’s post hoc test: *p < 0.05, **p < 0.01, ***p < 0.001 (GraphPad Prism 8.4.3).

Figure 6

Changes in mature-BDNF (mBDNF) expression in hippocampus (A) and cortex (C) and in proNGF expression in hippocampus (D) and cortex (F) with representative immunoblots, together with total protein amount visualized by the stain-free technique (B,E). Data are shown as means of relative expressions + SD (n = 8). Statistical significance was evaluated by one-way ANOVA followed by Tukey’s post hoc test: **p < 0.01 (GraphPad Prism 8.4.3).

Figure 7

(A) Effect of compound (R)-31 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) Effect of compound (R)-31 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 (R)-31 in the tactile allodynia in oxaliplatin (OXPT)-induced peripheral neuropathy. The compound was administered at the doses of 50, 75, and 100 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 (R)-31 in the tactile allodynia in streptozotocin (STZ)-induced peripheral neuropathy. The compound was administered at the doses of 25, 50, and 100 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 < 0.0001, 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 < 0.0001 when results compared to the OXPT-treated group (Post Oxali/Pre (R)-31) or STZ-treated group (Post (R)-31) 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 8

(A) Effect of compound (R)-32 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) Effect of compound (R)-32 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 (R)-32 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 (R)-32 in the tactile allodynia in streptozotocin (STZ)-induced peripheral neuropathy. The compound was administered at the doses of 25, 50, and 100 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 < 0.05, **p < 0.01, ****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, ****p < 0.0001 when results compared to the OXPT-treated group (Post Oxali/Pre (R)-32) or STZ-treated group (Post (R)-32) 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 9

Compounds (R)-31 and (R)-32 inhibit fast voltage-gated sodium currents in prefrontal cortex pyramidal neurons. (A) Example sodium current recordings in control (black trace), after application of (R)-31 (blue trace) and after wash-out (red trace). Current traces were evoked by a rectangular voltage-step. (B) Influence of (R)-31 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 (R)-31 and after wash-out. The horizontal axis shows trace number. (C) Averaged, normalized maximal sodium current amplitudes in control, in the presence of (R)-31 and after wash-out. An asterisk shows statistical significance. (D) Example sodium current recordings in control (black trace), after application of (R)-32 (blue trace) and after wash-out (red trace). Current traces were evoked by a rectangular voltage-step. (E) Influence of (R)-32 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 (R)-32 and after wash-out. The horizontal axis shows trace number. (F) Averaged, normalized maximal sodium current amplitudes in control, in the presence of (R)-32 and after wash-out. An asterisk shows statistical significance.

Figure 10

Influence of (R)-31, (R)-32, and (R)-33 on: CYP3A4 activity (A) and the reference inhibitor ketoconazole (KE), CYP2D6 activity (B) and the reference inhibitor quinidine (QD), CYP2C9 activity (C) and the reference inhibitor sulfaphenazole (SE). The results are presented as means + SD. Statistical significance was evaluated by one-way ANOVA, followed by Bonferroni’s multiple comparison post hoc test (Graph Pad Prism 8.0.1 software) *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 11

Effect of (R)-31, (R)-32, and (R)-33 and cytostatic drug doxorubicin (DX) on the hepatoma HepG2 cell line viability after 72 h of incubation at 37 °C, 5% CO₂. The results are presented as means + SD. Statistical significance was evaluated by one-way ANOVA, followed by Bonferroni’s multiple comparison post hoc test (Graph Pad Prism 8.0.1 software, San Diego, CA, USA): *p < 0.05, ***p < 0.001, ****p < 0.0001 vs negative control (DMSO 1% in growth media).