As shown in Fig. 3, the crystals of the compounds 13a and 2c used for X-ray diffraction analysis were obtained by slow evaporation
of acetone-ethanol (1:3) mixed solution at room temperature (13a: CCDC No. 1451159; 2c: CCDC No. 1451160).
2.2 Activity on tyrosinase
The biological activity of polyhydroxy chalcones against tyrosinase was evaluated (Table 1). All the compounds inhibit the
tyrosinase. Generally, the higher inhibitory activity can be observed with more hydroxyl in chalcone. For 2a and 3a, 5a and 6a, the
result showed that the most important factor for their efficacy was the location of the hydroxyl on benzene ring, with a significant
preference to a 4-substituted B ring rather than a substituted A ring. Moreover, a 2,4-substituted resorcinol with more hydroxyl on ring
A improved their activity, such as compounds 7a and 9a.
It was not hard to find that hydroxyl in chalcone skeleton seriously abolished the activator effect on tyrosinase according to the result
above. Therefore, some other groups were introduced to the chalcone to improve the activity. As shown in Table 2, chalcones
substituted with -X (14a, 15a) and -N(CH3)2 (13a) were more potent than ones with -H, -OH, -CH3 and -OCH3, suggesting that the
electron-withdrawing group (EWG) may be favorable to enhance the activity. In the view of best activity of compound 13a, further
modification was performed on this active molecule. Neither changing the location of the -OCH3 on A ring nor replacement of -OCH3
with -NO2, -NH2 or -NHAc enhanced the activator effect, which emphasizes the importance of the para-position of the substituted
group on A ring and the -N(CH3)2 on B ring.
With few exceptions, when A ring was replaced with naphthalene or pyridine ring, similar effect was found with compound 1b and
2b, proving that the benzene for the A ring was not necessary for this effect. However, the activity dramatically decreased once other
aromatic rings were introduced to B ring (compounds 1c-3c), which means that the benzene for B ring was essential for maintaining
the activity (Table 3). For the linker group between A and B ring, the α, β-unsaturated carbonyl was prolonged to give compound 1d-
2d, based on the active compounds synthesized above. Unfortunately, in Table 3, the extended distance between the two groups failed
to retain the biological activity.
2.3 Activity on tyrosinase and melanin synthesis in B16 cells
The viability of 13a on B16 melanoma cells was examined using the MTT assay. The cells were treated with various concentrations
of 13a (6.25-200 μg/mL). As shown in Fig. 4, there was no significant difference between the control and treated group at
concentrations of 6.25-200 μg/mL. 13a showed very small cytotoxic effects on B16 cells at concentrations of 200 μg/mL.
The effect of 13a on tyrosinase in cells was measured by L-DOPA oxidation (Fig. 5). Compared with treatment with medium only
(untreated condition), treatment with 13a at 0-40μg/mL resulted in a dose-dependent increase in tyrosinase activity in B16 cells. In the
melanin content assay, to exclude the possibility that a rise in melanin content may be induced by cell-proliferating effect of 13a, the
absorbance of the same number of cells across 13a concentrations (0-40 μg/mL) was measured as well. We found that melanin levels
increased in a dose-dependent manner by 13a treatment in B16 cells (Fig. 6). At 40 μg/mL of 13a, the melanin content only slightly
increased, so 20 μg/mL was chosen as an effective concentration of 13a for further experiments.
3. Conclusion
This work showed that good activator effect can be obtained with small chalcone molecules and the SAR was summed up for the
first time as follows. (1) The hydroxyl on either benzene of chalcone may cause an inhibitory activity, especially for the B ring. (2).The
A ring of the chalcone can be substituted with other conjugate aromatic rings to retain the activator effect while the B ring cannot. It
was speculated that the planar molecule was essential for the activity. (3) The linker should not be prolonged for maintaining the
activity. In addition, 13a increased both activity of the tyrosinase and the contents of the melanin in a dose-dependent manner in
murine B16 cells.
4. Experimental
4.1 Chemistry
Reagents and solvents were purchased from Sigma, Sodipro or VMR, and used without further purification. Dimethylsulfoxide
(DMSO), mushroom tyrosinase, L-3, 4-dihydroxyphenylalanine (L-DOPA), 3,4-dihydroxyphenylalanine and 3-(4,5-dimethylthiazol-2-
yl)-2, 5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma (St. Louis, MO, USA).
4.2 General method for preparation of chalcone and analogues (1a-21a, 1b-2b, 1c-3c, 1d-4d)
The corresponding aldehyde (1.0 equiv) and ketone (1.0 equiv) were dissolved in ethanol at 0 °C and 10% NaOH (1.0 equiv.)
solution was slowly added. After that, the mixture was allowed to warm up to room temperature and stirred for 48 h. The resulting
mixture was evaporated to dryness and the residue was purified by chromatography on silica gel eluted with petroleum ether-ethyl
acetate to afford the final compound. The characterization data of the synthesized compounds are in the Supporting information.