Synthesis, In Vitro, and In Silico Anti-Inflammatory Screening of Orthocetamol Azo Derivatives

Abstract Chronic inflammation drives various non-communicable diseases, necessitating the search for novel therapeutic agents. Integrating heteroatoms in the inhibitor drug design enhances potency by enabling multifaceted interactions with the target cell receptor. Herein, a new series of orthocetamol azo derivatives, featuring a nitrogen-based moiety (–N=N–), was synthesized via the diazotization of orthocetamol with substituted aniline derivatives, yielded 31–95%. The in vitro anti-inflammatory evaluation on orthocetamol azo derivatives showed a minimum 49.4±1.3% NO inhibition against LPS-induced RAW 264.7 macrophages at 50 μM. Notably, para-bromo substituted compound exhibited the most potent NO inhibition of 92.8±0.3%, with an IC50 value of 16.0±0.6 µM, comparable to the positive control, Dexamethasone (16.9±2.2 µM). In silico molecular docking analysis of the p-Br orthocetamol azo against the COX-2 enzyme (PDB ID: 5IKR), a key regulator often associated with NO production pathways, revealed a binding affinity of –7.2 kcal/mol driven by a stable hydrogen bond with TYR355 and a predicted isoform-selective π-alkyl interaction with VAL523. While these computational insights suggest a potential preference for COX-2, further pharmacokinetic profiles of p-Br orthocetamol azo confirmed its druglikeness through adherence to the Lipinski rule, in addition to an optimal balance of ADMET properties. The finding suggested p-Br orthocetamol azo could serve as a promising therapeutic candidate, warranting further in vitro enzymatic assays to experimentally validate its COX-1/COX-2 selectivity profile in anti-inflammatory drug development.