Anxiolytic effects of crystal and polycrystal of hydrazone derived from hydralazine in adult zebrafish.

Hydrazones are pharmacologically versatile compounds with emerging therapeutic potential. Here, we report the synthesis, structural characterization, and the anxiolytic potential of the hydrazone derived from hydralazine, (E)-1-(2-(pyridin-4-ylmethylene)hydrazineyl)phthalazine (HDZH4PIR). The molecular structure of the polycrystalline form of HDZH4PIR was confirmed by 1H and 13C NMR, and structural confirmation of its crystalline form was carried out by single-crystal X-ray diffraction. The anxiolytic potential in the adult zebrafish model of HDZH4PIR in its crystal and polycrystal forms was evaluated by vivo and in silico studies. Both forms were well-tolerated up to 40 mg kg-1 (LD50 > 40 mg kg-1), supporting safety for behavioral assays. In open-field tests, both forms reduced locomotion slightly without causing sedation. In the light/dark task, both forms showed diazepam-like anxiolytic activity, with distinct profiles: crystalline HDZH4PIR was most potent at 20-40 mg kg-1 and acted via serotonergic (5-HT1A) pathways. In contrast, polycrystalline HDZH4PIR showed a hormetic response and acted via GABAA receptors. This mechanistic divergence was confirmed through antagonist profiling and molecular docking studies that showed that polycrystals bound strongly to the benzodiazepine site of GABAA receptors (ΔG = -9.0 kcal mol-1), while crystals preferred the 5-HT1A orthosteric site (ΔG = -8.7 kcal mol-1). ADMET predictions suggest that crystallization enhances safety but reduces oral bioavailability and blood-brain barrier permeability. In conclusion, HDZH4PIR is a promising dual-pathway anxiolytic candidate, with its crystalline form enhancing serotonergic modulation and its polycrystalline form targeting GABAA receptors.