<i>In vivo</i> and <i>in silico</i> toxicity profiling of a novel 1,5-benzodiazepine derivative

Benzodiazepines (BZDs) are widely used but are often associated with significant side effects such as amnesia, dependence, and withdrawal syndrome, prompting the search for safer alternatives. The current study aimed to assess the possible toxic effects of a novel 1,5-BZD derivative, which we named MAL. <i>In silico</i> analysis showed that MAL binds to the GABA-A receptor with a binding energy of -8.9 kcal/mol, and to its α1, β2, and γ2 subunits with binding energies of -7.6, -7.5, and -7.6 kcal/mol, respectively, indicating strong receptor affinity. To explore the safety of MAL, toxicological testing was conducted in laboratory animals in accordance with internationally accepted guidelines. Acute toxicity was evaluated by a single oral administration of 2 g/kg body weight to male mice, monitored over 14 days. Since BZDs are typically prescribed for up to two weeks in clinical practice, a 28-day repeated-dose toxicity study was also conducted. Mice received MAL (5, 25, 50, and 100 mg/kg) daily for 28 days to evaluate potential toxic effects. No adverse effects were observed on body weight gain, relative organ weights, or food and water consumption. Histological examination of major organs revealed no abnormalities, and biochemical analyses confirmed the absence of hepatotoxicity or nephrotoxicity. Additionally, we present ProTox, a comprehensive, open-source webserver designed for <i>in silico</i> toxicity prediction. In this study, ProTox predicted an oral LD50 of MAL at 3 g/kg. Based on the Globally Harmonized System (GHS) of classification, this places MAL in Category V. These findings support the potential safety of MAL for pharmaceutical formulations.