Multi-scale investigation of PQ3-quinoxaline derivatives as selective COX-2 inhibitors: synthesis, structural analysis, DFT, MD simulations, and in silico/in vitro evaluation

Quinoxaline has emerged as a promising scaffold in drug discovery, particularly in the development of anti-inflammatory agents, due to its structural versatility and broad pharmacological potential. Its flexible chemical framework permits diverse modifications, enabling the design of derivatives that can target critical molecular pathways implicated in cancer progression. The title compounds of 3-phenylquinoxalin-2(1H)-one (PQ3a-f), were synthesized in good yields from 3-phenylquinoxalin-2(1H)-one (1) in DMF, with 1-bromoalkanes (2a-f), and tetrabutylammonium bromide (BTBA). The crude products obtained have been recrystallized and elucidated by spectroscopic techniques (¹HNMR, ¹³CNMR and LC-MS), the 3D of PQ3a crystal structure was confirmed by single crystal X-ray diffraction (XRD) studies. Hirshfeld surface analysis confirmed the C-H…O intermolecular interactions. 2D fingerprint plot of PQ3a shows that the major contribution to the overall Hirshfeld surface area is from HH (72.7 %) contacts. 3D energy-frameworks calculations shows that the majority of dispersion energy over the other energies. DFT calculations are performed to know the PQ3a molecular properties at B3LYP/6–31 + G(d,p) basis sets. The energy gap between the HOMO-LUMO is found to be 3.81 eV, indicating the nature of potent biologically active molecule. Among the six synthesized PQ3a-f derivatives, PQ3a exhibited the most potent COX-2 inhibition (IC₅₀ = 10.24 μM) with moderate COX-1 inhibition (IC₅₀ = 31.67 μM), yielding a selectivity index (SI) of 3.09, indicative of COX-2 selectivity. PQ3f showed moderate COX-2 inhibition (IC₅₀ = 25.54 μM) and COX-1 inhibition (IC₅₀ = 46.45 μM), with an SI of 1.82. In contrast, the remaining derivatives (PQ3b-d) demonstrated weak or non-selective COX inhibition compared to the reference drug celecoxib (COX-2 IC₅₀ = 0.54 μM; SI = 21.15). Docking results revealed strong binding affinity, particularly with COX-2, supported by key interactions with Ser530 and Leu531. Molecular dynamics simulations (MDs) confirmed the stability of the PQ3a-COX-2 complex over 500 ns, highlighting sustained hydrophobic interactions and a stable hydrogen bond with Ser530. These findings suggest PQ3a as a promising selective COX-2 inhibitor for anti-inflammatory therapy.