The Search for New Anticonvulsants in a Group of (2,5-Dioxopyrrolidin-1-yl)(phenyl)Acetamides with Hybrid Structure-Synthesis and In Vivo/In Vitro Studies

Abstract

Epilepsy belongs to the most common and debilitating neurological disorders with multifactorial pathophysiology and a high level of drug resistance. Therefore, with the aim of searching for new, more effective, and/or safer therapeutics, we discovered a focused series of original hybrid pyrrolidine-2,5-dione derivatives with potent anticonvulsant properties. We applied an optimized coupling reaction yielding several hybrid compounds that showed broad-spectrum activity in widely accepted animal seizure models, namely, the maximal electroshock (MES) test and the psychomotor 6 Hz (32 mA) seizure model in mice. The most potent anticonvulsant activity and favorable safety profile was demonstrated for compound 30 (median effective dose (ED₅₀) MES = 45.6 mg/kg, ED₅₀ 6 Hz (32 mA) = 39.5 mg/kg, median toxic dose (TD₅₀) (rotarod test) = 162.4 mg/kg). Anticonvulsant drugs often show activity in pain models, and compound 30 was also proven effective in the formalin test of tonic pain, the capsaicin-induced pain model, and the oxaliplatin (OXPT)-induced neuropathic pain model in mice. Our studies showed that the most plausible mechanism of action of 30 involves inhibition of calcium currents mediated by Cav_1.2 (L-type) channels. Importantly, 30 revealed high metabolic stability on human liver microsomes, negligible hepatotoxicity, and relatively weak inhibition of CYP3A4, CYP2D6, and CYP2C9 isoforms of cytochrome P450, compared to reference compounds. The promising in vivo activity profile and drug-like properties of compound 30 make it an interesting candidate for further preclinical development.

Keywords: absorption, distribution, metabolism, excretion, toxicity (ADME-Tox) properties; anticonvulsant activity; antinociceptive activity; hybrid molecules; in vitro binding/functional studies; pyrrolidine-2,5-dione.

Figure 1

Design and general structure of hybrid 2-(2,5-dioxopyrrolidin-1-yl)-2-phenylacetamide derivatives (the main modification site is marked in green).

Scheme 1

Synthesis of intermediates 1, 2 and target compounds 3–31.

Figure 2

Antinociceptive activity of compounds 4 (A) and 30 (B) in the formalin test. Results are shown as time of licking in phase I (0–5 min after intraplantar injection of formalin) and in phase II (15–30 min after formalin injection). Each value represents the mean ± standard error of the mean (SEM) for 8–10 animals. Statistical analysis: one-way ANOVA followed by post hoc Dunnett’s test. Statistical significance compared to vehicle-treated animals (Tween): * p < 0.05, ** p < 0.01, **** p < 0.0001. C—control group.

Figure 3

Antinociceptive activity of compounds 4 (A) and 30 (B) in the capsaicin test. Results are shown as time of licking in the period of 5 min after intraplantar injection of capsaicin. Each value represents the mean ± SEM for 8–10 animals. Statistical analysis: one-way ANOVA followed by post hoc Dunnett’s test. Statistical significance compared to vehicle-treated animals (Tween): * p < 0.05, *** p < 0.00, **** p < 0.0001. C—control group.

Figure 4

Antiallodynic effects of compound 30 administered at the doses of 20 mg/kg, 40 mg/kg, and 80 mg/kg in the tactile allodynia evaluated in the von Frey test in the oxaliplatin (OXPT)-induced model of peripheral neuropathy. Results compared to vehicle-treated group (pre 30). Statistical analysis: repeated measures analysis of variance (ANOVA), followed by Dunnett’s post hoc comparison: * p < 0.05, *** p < 0.001, **** p < 0.0001.

Figure 5

The influence of 4, 24, 30, and reference inhibitor ketoconazole (KE) on CYP3A4 activity. Statistical significance (* p < 0.05, *** p < 0.001, **** p < 0.0001) was analyzed by Graph Pad Prism 8.0.1 software using one-way ANOVA and Bonferroni’s multiple comparison post test. The compounds were examined in triplicate.

Figure 6

The influence of 4, 24, 30, and reference inhibitor quinidin (QD) on CYP2D6 activity. Statistical significance (* p < 0.05,** p < 0.01, *** p < 0.001, **** p < 0.0001) was analyzed by Graph Pad Prism 8.0.1 software using one-way ANOVA and Bonferroni’s multiple comparison post test. The compounds were examined in triplicate.

Figure 7

The influence of 4, 24, 30 (10 µM) and the reference inhibitor sulfaphenazole (SE) on CYP2C9 activity. Statistical significance (** p < 0.01, *** p < 0.001, **** p < 0.0001) was analyzed by Graph Pad Prism 8.0.1 software using one-way ANOVA and Bonferroni’s multiple comparison post test. The compounds were examined in triplicate.

Figure 8

The effect of 4, 24, 30, and cytostatic drug doxorubicin (DX) and the mitochondrial toxin carbonyl cyanide 3-chlorophenyl-hydrazone (CCCP) on hepatoma HepG2 cell lines viability after 72 h of incubation at 37 °C, 5% CO₂. The statistical significance (GraphPad Prism 8.0.1) was evaluated by a one-way ANOVA, followed by Bonferroni’s comparison test (*** p < 0.001, **** p < 0.0001) compared with negative control dimethyl sulfoxide (DMSO) 1% in growth medium).