Synthesis, biological evaluation, and in silico studies of paracetamol-derived 4-hydroxyphenylamino-pyridine conjugates as analgesic agents.

Abstract

A new series of paracetamol-derived 4-hydroxyphenylamino-pyridine-3-carbonitrile hybrids (3a-o) was designed and synthesized in good yields (35.3-76.7%) through the reaction of N-(4-hydroxyphenyl)acetamide (1) with various arylidenemalononitriles (2) in the presence of freshly prepared sodium alkoxide solutions. The chemical structures and purity of the target compounds were fully verified via 1H & 13C NMR, IR, and microanalysis. The synthesized compounds were evaluated for their analgesic activity using the hot plate, acetic acid-induced writhing, and formalin-induced licking and biting assays. Several derivatives, particularly 3h, 3i, 3l, and 3o, demonstrated significant central and peripheral analgesic effects, comparable to or greater than paracetamol in selected experimental endpoints. To further explore their possible mode of action, selected compounds were investigated for their effects on serum COX-2 protein expression, prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), and nitric oxide (NO). The selected compounds were administered at paracetamol-equivalent doses (150 mg kg-1), and serum samples were collected at the end of the experiment. NO was assessed using an ELISA-based assay. The tested derivatives reduced these inflammatory and nitrosative stress-associated biomarkers, suggesting that modulation of inflammatory pathways may contribute, at least in part, to their analgesic effects. Correlation analysis further revealed an association between analgesic activity and biomarker changes, although this association does not establish a causal relationship. Molecular docking and molecular dynamics simulations were performed to investigate the interaction of the most active compound (3l) with the COX-2 binding site. The predicted binding mode involved hydrogen bonding with Tyr323 and several hydrophobic interactions. Analyses of root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), radius of gyration (Rg), and solvent-accessible surface area (SASA) supported the structural stability of the simulated ligand-protein complex throughout the simulation period. Overall, these findings identify 3h, 3i, 3l, and 3o as promising analgesic candidates warranting further pharmacological and mechanistic investigation.

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