Separation and isolation of tautomers of 2-hydroxy-4-naphthoquinone-1-oxime derivatives by liquid chromatography: antiproliferative activity and DFT studies

Abstract

Reversed phase HPLC separation and isolation of isomers of 2-hydroxy-4-naphthoquinone-1-oxime (Lwox) and 3-methyl-2-hydroxy-4-naphthoquinone-1-oxime (Phox) have been investigated. Two distinct peaks are observed in the chromatogram of Lwox and are assigned to ‘para’ tautomer; 2-hydroxy-4-naphthoquinone-1-oxime (3) and ‘ortho’ tautomer; 4-hydroxy-2-naphthoquinone-1-oxime (4). The tautomeric equilibrium of 3 and 4 has been manipulated by incrementally increasing the pH of the mobile phase from 2.5 to 10.5, and altering the solvent polarity. At pH > 6.8 the tautomers are well-separated from each other. There is no separation of Phox isomers between pH 2.5 and 10.5. Isolation of the tautomers has been carried out by preparative HPLC, with 3 and 4 obtained as ammonium bicarbonate adducts and characterized by LC-MS, FT-IR, and UV-visible spectroscopy. Red-orange 3 is characterized by a paranaphthoquinone stretch at 1287 cm−1 and a charge transfer band at 420 nm; yellow 4 exhibits, a similar stretch at 1246 cm−1 and absorption band at 406 nm. Compounds 3 and 4 were screened for selective antiproliferative activity in three cancer cell lines of different tissue types (COLO 205 (human colorectal adenocarcinoma), U87 MG (glioblastoma astrocytoma) and MIAPaCa-2 (human pancreatic carcinoma). Geometry-optimized structures for tautomers 3 and 4 (3′ and 4′ in Phox) were computed using the B3LYP method. Structures, 3 and 3′ are 4.7 and 5.8 kcal mol−1 more stabilized than 4 and 4′, respectively, as a result of a hydrogen bond interaction between the 2-hydroxyl group and the nitrogen of the oxime