Experimental and theoretical investigations for novel 6-nitroquinoxaline-2,3‑dione derivatives: Synthesis, characterization, DFT calculations, ADME analysis, hirshfeld surface calculations, molecular docking studies, and antiproliferation evaluation

This work describes the synthesis of eight different 6-nitro-1,4-disubstituted-quinoxaline-2,3‑dione derivatives (4a-h). The starting material, 6-nitroquinoxaline-2,3‑dione 3 was prepared by reacting 4-nitro-o-phenylenediamine 1 with oxalic acid 2 at reflux in hydrochloric acid. Compound 3 was alkylated with various alkyl halides under solid-liquid phase transfer catalysis (PTC) to give the dialkyl quinoxalines (4a-h). The structures of all compounds obtained were elucidated based on spectroscopic measurements: ¹H, ¹³C NMR, MS and UV spectroscopy and confirmed by single-crystal X-ray diffraction studies in the case of 4a, 4b, 4f, and 4 h. The geometrical parameters and spectral data were also compared with those of a DFT geometry optimization and molecular orbital calculation at the B3LYP/6–311++G (d, p) level of theory. The closest contacts between the molecules of the four structures were investigated by analyzing their Hirshfeld surfaces and docking investigations in the protein EGFR (pdbid: 4HJO). Moreover, the experimental results are correlated to the calculated ones and showed great compatibility. Cell proliferation was used to measure the immunomodulatory activity of 4a-h compared to a control consisting only of normal cells cultured alone to determine their antiproliferative screening which concluded that the quinoxaline derivatives 4c, 4d, 4f and 4 g showed immunomodulatory potential.