Effects of Hydroxy and Methoxy Substituents on NMR Data in Flavonols

Kaempferide, kaempferol, and galangin belong to flavonol, one of flavonoid classes. Even they have the same moiety as flavonol, their binding affinities for a hexahistidine-tagged C-terminal nucleotide-binding domain are different with each other. The dissociation constants of flavonol, kaempferide, kaempferol, and galangin are 10.1, 4.5, 6.7, and 5.3 mM, respectively. Galangin contains two more hydroxyl groups than flavonol, and kaempferol has three more hydroxyl groups. Kaempferide differs with kaempferol in only one substituent: 4'-hydroxyl group of kaempferol is switched to 4'-methoxyl group in kaempferide. Likewise, galangin and kaempferide show different enzymatic kinetic parameters, Vmax/Km, for cytochrome P450 isomers CYP2C9 and CYP1A1 which take an important role in oxidative metabolism of galangin and kaempferide. The values of Vmax/Km of galangin for CYP2C9 and CYP1A1 were 59.0 and 10.2 μL/min/mg, respectively, and those of kaempferide were 5.1 and 1.9, respectively. While kaempferide has 4'methoxyl group, galangin does not have it. As a result, it can be considered that the substitution of hydroxyl or/and methoxyl groups on flavonols results in significant changes of the biological activities. Flavonol derivatives are being found still from natural sources. One of the best methods to identify them is NMR spectroscopy. Since the substitution of hydroxyl or/and methoxyl groups causes the chemical shift changes in the H and C NMR spectra, the elucidation of the effects of substituents on the chemical shifts in flavonol derivatives may help us predict the structures of the unknown compounds based on the simple one dimensional NMR experiments. In order to elucidate the substituent effects of chemical shifts on flavonol derivatives, the NMR experiments of flavonol (1) and nine flavonol derivatives (2-10) were carried out in this study. Of them, the C NMR data of three derivatives were already published. Because only the partial NMR data of others have been known, however, the complete assignments of their H and C NMR data are reported here. One of the C NMR data published previously showed the incorrect C NMR data, thus their corrected values were determined in this study. The structures and nomenclatures of flavonol derivatives 1-10 are shown in Figure 1. As mentioned above, the NMR data of three derivatives 2, 8, and 9 have been previously reported. We found that the NMR data of derivative 8 were partially incorrect. The C chemical shifts of the derivatives 8, 5,7,4'-trihydroxy-3'-methoxyflavonol, were reported by Kumari et al. Whereas C-3' and C-4' of 8 were assigned to the signals at 149.4 and 147.9 ppm in Kumari’s data, respectively, we found them to be at 147.4 and 148.9 ppm, respectively. That is, the order of the C chemical shifts of C-3' and C-4' was opposite in the Kumari’s data. In order to decide whether our assignments are correct or not, the interpretation of the NMR data was carried further out. The H peak at 3.84 ppm suggested the presence of 3'-methoxy proton. In the HMBC spectrum, 3'-methoxy proton was long-range coupled to the C peak at 147.4 ppm. Therefore, 147.4 ppm should be assigned to C-3'. The C chemical shifts of C-4' can be known through the HMBC spectrum of H-2' and H-6'. The H-2' and H-6' signals showed a longrange coupling with two C peaks at 146.7 and 148.9 ppm. Those peaks should be assigned to C-2 and/or C-4', respectively. Because 3-hydroxy proton peak at 9.41 ppm was long-range coupled to the C peak at 146.7 ppm in