Synthesis and integrative multimodal evaluation of cholic acid-based hydrazone conjugates: in vitro, in silico, and in vivo studies.

Cholic acid-derived hydrazones represent a promising scaffold for multifunctional drug discovery. We synthesized and characterized two novel cholic acid-based hydrazone derivatives: DFBCH (2,4-difluorobenzylidene conjugate) and MEBCH (3-methoxybenzylidene conjugate) through various spectroscopic techniques, confirming the structural integrity of the synthesized compounds, followed by their comprehensive biological evaluation. In vitro screening showed a greater antibacterial zone of inhibition of MEBCH compound as compared to DFBCH against the tested strains. MEBCH demonstrated potent antioxidant activity (IC50 1.87 ± 0.20 mg/mL) relative to ascorbic acid (IC50 7.3 ± 1.40 mg/mL) by DPPH assay illustrating superior antioxidant activity. Both the compounds exhibited greater % inhibition against Hela cell line as compared to doxorubicin, suggesting greater inhibitory activity. MEBCH was the superior inhibitor of acetylcholinesterase (IC50 1.67 ± 0.61 µM), surpassing the standard donepezil (IC50 3.13 ± 0.1 µM), and demonstrated effective tyrosinase inhibition (IC50 1.87 ± 0.88 µM), comparable to kojic acid (IC50 2.38 ± 0.75 µM). Both of these compounds demonstrated effective β-glucosidase inhibition with an IC50 ≈ 2.23 µM, comparable to standard miglustat (IC50 2.02 ± 1.05 µM). Molecular docking (Glide) revealed MEBCH producing a favorable docking score with AChE (- 6.711; E-model - 57.480), indicating a stable predicted binding pose relative to DFBCH. BioTransformer 3.0 predicted CYP 450-mediated pathways for both analogues; docking of predicted metabolites showed retained or improved affinity (MEBCH metabolite to AChE - 6.3 kcal/mol) and lower RMSD bounds (AChE; lb/ub ≈ 5.8/6.2 Å; β-glucosidase lb/ub ≈ 5.25/5.73 Å). DFT calculations revealed small HOMO-LUMO gaps and solvent-sensitive dipole moments consistent with moderate chemical reactivity and solvent stabilization. Single-dose oral pharmacokinetics (10 mg/kg) in rats revealed MEBCH with higher Cmax, AUC, longer t1/2, and greater oral bioavailability than DFBCH. Collectively, these data nominate MEBCH as a lead cholic-hydrazone for further preclinical development against enzyme targets relevant to neurological and oxidative-stress pathways.