Convenient Synthesis and Physiological Activities of Flavonoids in Coreopsis lanceolata L. Petals and Their Related Compounds

Chalcones, flavanones, and flavonols, including 8-methoxybutin isolated from Coreopsis lanceolata L. petals, were successfully synthesized with total yields of 2–59% from O-methylpyrogallols using the Horner–Wadsworth–Emmons reaction as a key reaction. Aurones, including leptosidin, were also successfully synthesized with 5–36% total yields using the Aldol condensation reaction as a key reaction. Each chalcone, flavanone, flavonol, and aurone with the 3,4-dihydroxy groups in the B-ring showed high antioxidant activity. Additionally, each of the chalcones, flavanones, flavonols, and aurones with the 2,4-dihydroxy groups in the B-ring showed an excellent whitening ability.


Flavonoids Synthesis
The process used to synthesize the chalcones, flavanones, and flavonols is shown in Scheme 1. The protection of 1a,b with chloromethyl methyl ether (MOMCl) produced compound 2a,b. The

Antioxidant and Tyrosinase Inhibitory Activity of the Synthesized Flavonoids
Next, the physiological activities of these synthesized compounds were investigated. The antioxidant activity and whitening effect were assessed based on the 2,2-diphenyl-1-picrylhydrazyl   the basis of their 1 H-NMR and 13 C-NMR spectral data. The 1 H-NMR spectrum of 14a showed a signal for an olefinic proton at δ 6.84 (s). The olefinic carbon was observed at δ 112.16. According to the 13 C-NMR study of the aurones, a signal for the olefinic carbon of the Z-isomer was observed at about 110 ppm, whereas that of E-isomer was observed at about 120 ppm [7]. Therefore, the structures of 14a-l were classified as the (Z)-form. Finally, the deprotection of the MOM group of 14a-k with 3 M HCl produced compounds 15a-k with 51-97% yields. Scheme 1. Synthesis of chalcones 7a-l, flavanones 8a-l, and flavonols 11a-l. Scheme 2. Synthesis of aurones 15a-k.

Antioxidant and Tyrosinase Inhibitory Activity of the Synthesized Flavonoids
Next, the physiological activities of these synthesized compounds were investigated. The antioxidant activity and whitening effect were assessed based on the 2,2-diphenyl-1-picrylhydrazyl

Antioxidant and Tyrosinase Inhibitory Activity of the Synthesized Flavonoids
Next, the physiological activities of these synthesized compounds were investigated. The antioxidant activity and whitening effect were assessed based on the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay [8] and tyrosinase inhibition assay [9], respectively. The results are summarized in Tables 1 and 3-4. The antioxidant activity was evaluated based on the scavenging rate of the DPPH radical under the condition where the final concentration of the samples and DPPH radical were prepared at 0.040 mM and 0.040 mM, respectively. A correlation was found between the physiological activity and

The General Procedure for the Protection of 2-O-Methylpyrogallol 1a,b with Chloromethyl Methyl Ether
A solution of 1a,b (100.0 mmol) in N,N-dimethylformamide (DMF) (50 mL) was added to a suspension of sodium hydride (60% in mineral oil, 9.60 g, 240.0 mmol or 4.80 g, 120.0 mmol) in DMF (150 mL) at 0 • C. After being stirred at room temperature for 30 min, chloromethyl methyl ether (15.2 mL, 200.0 mmol or 11.4 mL, 150.0 mmol) was added to the mixture at 0 • C. After being stirred at room temperature for 6 h, 100 mL Et 2 O was added to the mixture. The reaction mixture was poured into ice water (400 mL). The mixture was extracted with Et 2 O. The organic layer was washed with water and brine and dried over anhydrous MgSO 4 . The solvent was evaporated in vacuo and the residue was chromatographed on silica gel with CHCl 3 -Et 2 O (9:1) to produce 2a,b.

-Methoxymethyl Group
A solution of 6a-k (1.0 mmol) and 1.5 M hydrochloric acid aqueous solution (5 mL) in THF (5 mL) was stirred at room temperature for 45 min. The mixture was extracted with Et 2 O. The organic layer was washed with water and brine and dried over anhydrous MgSO 4 . The solvent was evaporated in vacuo and the residue was chromatographed on a preparative thin layer chromatography (hexane:EtOAc = 3:2) to produce chalcones 9a-k.  13  at room temperature. A solution of 1a,b (30.0 mmol) in 1,2-dichloroethane (30 mL) was added to the mixture and the reaction mixture was stirred at room temperature for 12 h. The mixture was poured into ice and a 2 M HCl solution and extracted with CHCl3. The organic layer was washed with water and dried over anhydrous MgSO 4 . The solvent was evaporated in vacuo and the residue was chromatographed on silica gel with CHCl3-Et 2 O (9:1) to produce 12a,b.

The General Procedure for the Protection of 13a with a Chloromethyl Methyl Ether
A solution of 13a (0.90 g, 5.0 mmol) in DMF (5 mL) was added to a suspension of sodium hydride (60% in mineral oil, 0.24 g, 6.0 mmol) in DMF (15 mL) at 0 • C. After being stirred at room temperature for 30 min, a chloromethyl methyl ether (0.57 mL, 7.5 mmol) was added to the mixture at 0 • C. After being stirred at room temperature for 6 h, Et 2 O (20 mL) was added to the mixture. The reaction mixture was poured into ice water (200 mL). The mixture was extracted with Et 2 O. The organic layer was washed with water and brine and dried over anhydrous MgSO 4 . The solvent was evaporated in vacuo and the residue was chromatographed on silica gel with CHCl 3 -Et 2 O (9:1) to produce 13c. 3.14. The General Procedure for the Synthesis of Aurones 14a-l Aluminum oxide (basic, 2.00 g, 19.6 mmol) was added to a solution of benzofuranones 13b,c (1.0 mmol) and benzaldehydes 5a-f (1.2 mmol) in dichloromethane (5 mL). The mixture was thoroughly stirred for 2 days at room temperature. The suspension was filtered off and the residue was washed with CHCl 3 . The filtrate was concentrated in vacuo and the residue was chromatographed on a preparative thin layer chromatography (CHCl 3 :Et 2 O = 9:1) to produce (Z)-aurones 14a-l.

The DPPH Radical Scavenging Assay
The measurement of the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging effect was performed according to the established procedure [8]. Sample compounds were dissolved in ethanol to obtain a 0.1 mM concentration. The DPPH free radical was dissolved in ethanol to obtain a concentration of 0.2 mM. The ethanol (100 µL) and DPPH solutions (50 µL) were added to a sample solution (100 µL) on a 96-well transparent microplate. The mix solution was mixed on a plate-mixer for 1 min. The mix solution was allowed to stand at 25 • C for 30 min in the dark, followed by measuring the absorbance with a microplate reader at 517 nm. The sample blank test (B) was performed with ethanol instead of the sample solution using a similar procedure. The blank test of the sample (C) was performed similarly, with ethanol instead of the DPPH solution. The blank test of the sample blank (D) was performed similarly, but with ethanol instead of the sample and DPPH solution. The DPPH radical scavenging rate was calculated as follows: where A is the absorbance of the sample, B is the absorbance of the sample blank, C is the absorbance of the blank of the sample, and D is the absorbance of the blank of the sample blank.

Tyrosinase Activity Inhibition Assay
The Tyrosinase activity was determined using the dopachrome method with L-3-(3,4-dihydroxyphenyl) alanine (L-DOPA) as the substrate [9]. Sample compounds were dissolved in DMSO to obtain a concentration of 3.0 mM. L-DOPA was dissolved in a 0.2 M phosphate buffer solution (PBS, pH 6.8) to obtain a concentration of 1.66 mM. The enzyme tyrosinase from mushrooms was dissolved in PBS to obtain a concentration of 600 units/mL. The sample solution (10 µL) was added to a L-DOPA solution (280 µL) on a 96-well transparent microplate. The mix solution was mixed on a plate-mixer for 1 min. The mix solution was left to stand at 25 • C for 5 min. The tyrosinase solution (10 µL) was added to the mixture and the mixture was incubated at 25 • C for 10 min, followed by measuring the absorbance with a microplate reader at 475 nm. The sample blank test (B) was performed with DMSO instead of the sample solution with similar procedure. The blank test of sample (C) was similarly performed with PBS instead of the enzyme solution. The blank test of the sample blank (D) was similarly performed with the DMSO and PBS instead of the sample and enzyme solutions, respectively. The percentage inhibition of tyrosinase activity was calculated as follows.
where A is the absorbance of the sample, B is the absorbance of the sample blank, C is the absorbance of the blank of the sample, and D is the absorbance of the blank of the sample blank.

Conclusions
In this study, chalcones, flavanones, and flavonols were easily synthesized, including 8-methoxybutin, which is a naturally occurring product from Coreopsis lanceolata L., using the HWE reaction as the key reaction in five to seven steps with overall yields of 18-59%, 13-53%, and 2-21% from O-methylpyrogallol 4a,b, respectively. The synthesis of aurones including leptosidin was achieved in four to five steps with overall yields of 5-36% from 4a,b using the aldol condensation reaction as a key reaction.
We found a correlation between the physiological activity and structures of the A-and B-rings of chalcone, flavanone, flavonol, and aurone. Each of chalcones 7b,h; flavanones 8b,h; flavonols 11b,h; and aurones 15b,h with the 3,4-dihydroxy groups on the B-ring had high antioxidant activity. The antioxidant activity in decreasing order was flavonol, chalcone, aurone, and flavanone.
The chalcones 7c,i and aurones 15c,i bearing the 2,4-dihydroxy groups on the B-ring had a high inhibitory activity potential. The whitening effect in decreasing order was chalcone, aurone, flavonol, and flavanone.