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-1oxime (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-4naphthoquinone-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.

Tautomerism in o-hydroxyanilino-1,4-naphthoquinone derivatives: Structure, NMR, HPLC and density functional theoretic investigations

“…The retention time of peak-1 is 8.3 min and peak-2 is 9.1 min with peak area 1.47% and 98.53% respectively. Although the peak area is negligible the nature of chromatogram is similar to what is observed for tautomers 13 . Based on the peak areas it can be concluded that the tautomer stability at peak II is maximum.…”

“…The tautomers of o-hydroxyanilino-1,4-naphthoquinone derivatives have been investigated in this report with the help of 1 H, 13 C, DEPT, gDQCOSY, gHSQCAD NMR experiments, HPLC and cyclic voltammetry. All the compounds exists as 'ortho'and 'para' tautomers in DMSO-d 6 solvent and only 'para' tautomer is predominant in CD 3 CN solvent.…”

“…Structural parameters of these tautomers derived from the SCRF method within the framework of dispersion corrected DFT agree well with the single crystal X-ray data. The 1 H and 13 C chemical shifts were assigned to all compounds and it was observed that proton chemical shifts of 'ortho' tautomers showed upfield shift. The H(3) proton facilitating intramolecular hydrogen bonding in 'ortho' tautomers of 1A-3A reveals largest deshielding in the GIAO calculated 1 H NMR spectra in consonance with the experimental data.…”

“…Recently we studied the tautomeric equillibria of naphthoquinoneoximes, by reversed phase chromatographic technique [13,14]. The tautomeric equilibrium is shown to be dependent on pH of mobile phase.…”

“…The isolation of the tautomers carried out by preparative HPLC and characterized by chromatographic (HPLC) as well as spectroscopic techniques. Stability of tautomers depends on solvent polarity which is used for solubility of compound [13]. Tautomeric equillibria further provide facile path for biological activity.…”