Synthesis, spectroscopic and structural characterisation, DFT study and in vitro biological evaluation of a benzimidazole–azo functionalised 1,3-benzoxazepine-1,5-dione

Abstract

A novel seven-membered oxygen/nitrogen heterocycle containing benzimidazole, azo, and phenolic pharmacophores was synthesized in four steps and evaluated in vitro. Condensation of o-phenylenediamine with salicylaldehyde afforded 2-(1H-benzimidazol-2-yl)phenol, followed by azo coupling with diazotized 4-aminoacetophenone, condensation with benzocaine, and [2+5] cycloaddition of the resulting ketimine with phthalic anhydride. The target compound, ethyl 4-[3-(4-{[3-(1H-benzimidazol-2-yl)-4-hydroxyphenyl]diazenyl}phenyl)-3-methyl-1,5-dioxo-1,5-dihydro-4H-benzo[e][1,3]oxazepin-4-yl]benzoate (13), was obtained as a red solid in 79% yield (m.p. 280–282 °C). FT-IR and ¹³C NMR data confirmed ring formation, with lactone and lactam carbonyl bands at 1712.67 and 1689.53 cm⁻¹ and three carbonyl resonances at 167.03, 166.36, and 165.78 ppm. Powder X-ray diffraction indicated a predominantly amorphous structure with residual crystalline sodium chloride. B3LYP/6-31G(d)//GFN2-xTB calculations showed 228 real vibrational modes, a HOMO–LUMO gap of 3.592 eV, chemical hardness of 1.796 eV, and electrophilicity of 3.867 eV. The azo bridge contributed substantially to LUMO localization and electronic conjugation, while an intramolecular O–H···N hydrogen bond (1.690 Å) stabilized the structure. In vitro evaluation against HepG2 cells showed 53.4% viability at 400 µg mL⁻¹, whereas WRL-68 cells retained 80.8% viability, indicating preferential cytotoxicity toward hepatocellular carcinoma cells.