Integrated ADME, metabolomics, and multi-target docking of Novel methoxy- and thiophene-substituted benzodioxane...

Benzodioxane carboxylic acid-based hydrazones are emerging pharmacophores with diverse bioactivities, yet their pharmacokinetic and metabolic characteristics remain underexplored. This study integrates experimental and computational approaches to evaluate two novel derivatives as (Z)-N-(3-methoxybenzylidene)-2,3-dihydrobenzo[b] [1,4] dioxine-6-carbohydrazide (MBDH) and (Z)-N'-(thiophen-2-ylmethylene)-2,3-dihydrobenzo[b] [1,4] dioxine-6-carbohydrazide (TBDH). Pharmacokinetic analysis (10 mg/kg, oral, rats) demonstrated that TBDH showed longer systemic retention (t₁⁄₂ = 12.81 ± 0.64 h; MRT = 12.30 ± 0.49 h; C_max = 98.94 ± 2.97 µg/mL; AUC_0-24 = 1038.94 ± 0.02 µg h/mL) than MBDH (t₁⁄₂ = 9.39 ± 0.47 h; MRT = 10.16 ± 0.40 h; C_max = 112.7 ± 3.38 µg/mL; AUC_0-24 = 900 ± 0.03 µg h/mL). LC-MS/MS metabolomic profiling revealed extensive Phase I and Phase II transformations. MBDH primarily underwent hydroxylation and demethylation followed by glucuronidation and glutathione conjugation, whereas TBDH additionally exhibited thiophene ring hydroxylation, sulfonation, and thiolation, contributing to its higher metabolic stability. Bio Transformer 3.0 predictions correlated strongly with experimental data, confirming the proposed biotransformation routes. Molecular docking indicated strong interactions of metabolites with pharmacologically relevant targets such as acetylcholinesterase, β-glucosidase, peroxidase, serine protease, and steroid receptors. TBDH metabolites exhibited notable multi-target binding, with the strongest affinity observed for β-glucosidase (- 8.8 kcal/mol; RMSD 1.8-0.9 Å) and serine protease (- 7.9 kcal/mol; RMSD 1.6-1.1 Å). The integration of LC-MS/MS metabolomics, pharmacokinetics, and in silico modeling provides a comprehensive understanding of structure-dependent metabolic behavior. Collectively, these findings highlight the influence of methoxy versus thiophene substitution on bioavailability and target affinity, identifying TBDH as a metabolically stable and pharmacologically promising benzodioxane hydrazone candidate.