Anxiety, a multidimensional behavioral disorder, has been widely studied in neuroscience to understand its causes. The medications available for treatment show variable efficacy and side effects. To discover new drugs, animal models such as zebrafish (Danio rerio) have been used due to their genetic homology with humans. This study aimed to synthesize, characterize, and evaluate the anxiolytic activity of a hydrazone derivative in zebrafish models as well as to investigate its mechanism of action. The hydrazones were synthesized from the condensation of isoniazid with hydroxylated aldehydes and characterized by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and NMR. For in vivo testing, six fish were treated with different doses of hydrazones (4, 20, 40 mg/kg), a negative control (3% DMSO), and a positive control (DZP 4 mg/kg), assessing locomotor activity and acute toxicity over 96 h. The light-dark test and neuromodulatory analysis of GABA and serotonin were also performed. The hydrazones (E)-N'-(2,4-dihydroxybenzylidene)-isonicotinohydrazide and (E)-N'-(2,3,4-dihydroxybenzylidene)-isonicotinohydrazide exhibited anxiolytic efficacy, reduced by flumazenil and granisetron. MPO analyses suggest that the compound resides in a physicochemical space formed by CNS-active drug candidates and exhibits ligand-receptor interactions, suggesting that the compounds may act similarly to the reference drug.
