Syntheses of Resveratrol Analogues and Evaluation of Their Antioxidant Activity

Experimental

Chemicals and Measurements. All chemicals used were purchased from Aldrich Chemical Co., Junsei or other com-mercial companies, and used without further purification unless otherwise stated. NMR spectra were recorded at Varian Mercury TM 300 MHz FT-NMR for 1H and 75 MHz for 13C, with the chemical shifts (δ) reported in parts per million (ppm) relative to TMS and the coupling constants (J) quoted in Hz. CDCl3, acetone-d6 and methanol-d4 were used as solvents with TMS as an internal standard. Bio-Sun UV irradiation system (300W) from Vilber Lourmat was used for the isomerization of trans resveratrol to cis resveratrol. Flash chromatography was carried out using Silica gel Merck 60 (230-400 mesh). Thin-layer chromatography (TLC) was performed on TLC Silica gel 60 F254 (Merck, layer thickness 0.2 mm), glass-backed Silica gel plates, with visualization by UV light (254 nm) or by treatment with p-anisaldehyde. Melting points were measured on a MEL-TEMP II apparatus and were uncorrected. UV absorptions were measured on Shimadzu UV-1800 spectrometer and Origin 8 program was used to calculate the IC50 values of the compounds.

5-[(4-Hydroxyphenylimino)methyl]benzene-1,3-diol (2). The reaction mixture of 3,5-dihydroxybenzaldehyde 7 (500 mg, 3.62 mmol), 4-hydroxyanline 8 (395 mg, 3.62 mmol) and Na2SO4 (500 mg) in methylene chloride (20 mL) was stirred for 3 hr at room temperature under argon. After the reaction, the solvent was evaporated. Then the residue was dissolved in acetonitrile and filtered with fritted glass. The filtrate was dried in vacuo. The desired product 2 was obtained as a yellow solid (788 mg, 93%). Physical properties and NMR spectra of the compound 2 were matched to those in the previous report by Albada et al.18

(Z)-3,4',5-Trihydroxystilbene (4). The solution of (E)-3,4',5-trihydroxystilbene (100 mg, 0.438 mmol) in methanol-d4 (5 mL) in a petri dish was irradiated at 365 nm and 11 J (joule) for 0.5 h at rt under darkness. After the reaction, 1H NMR showed that the reaction mixture was composed of cis and trans resveratrol (cis:trans = 84:16). Because this mixture was converted to trans resveratrol during the further purification such as column chromatography or recrystal-lization, this mixture was used without further purification. 1H NMR (300 MHz, CD3OD) cis δ 7.08 (2H, d, J = 8.4 Hz), 6.62 (2H, d, J = 8.4 Hz), 6.43 (2H, d, J = 12.3 Hz), 6.31 (2H, d, J = 12.3 Hz), 6.20 (2H, d, J = 2.1 Hz), 6.11 (1H, t, J = 2.1 Hz); trans δ 7.35 (2H, d, J = 8.7 Hz,), 6.97 (1H, d, J = 16.2 Hz), 6.80 (1H, d, J = 16.2 Hz), 6.76 (2H, d, J = 8.7 Hz), 6.45 (2H, d, J = 2.1 Hz), 6.15 (1H, t, J = 2.1 Hz).

3,5-Bis(tetrahydro-2H-pyran-2-yloxy)ethynylbenzene (10). To the solution of (trimethylsilyl)diazomethane (2 M solution in diethyl ether, 6.21 mL, 12.42 mmol) in THF (20 mL) was added n-butyl lithium (2 M solution in cyclo-hexane, 5.59 mL, 11.18 mmol) at −78 °C under nitrogen atmosphere. Then to the above mixture was added 3,5-bis[(tetrahydro-2H-pyran-2-yl)oxy]benzaldehyde 920 (1.905 g, 6.218 mmol) in THF (40 mL) dropwise and the resulting mixture was stirred for 1 hour at −78 °C. The reaction was quenched with saturated ammonium chloride solution (25 mL). The organic layer was extracted with diethyl ether, washed with saturated sodium bicarbonate solution and brine, dried over Na2SO4, filtered and dried in vacuo. The resulting residue was applied to silicagel column chromato-graphy (EtOAc:hexanes = 1:1). The desired product 10 was obtained as an orange liquid (839 mg, 48%). Rf 0.62 (EtOAc: hexanes = 1:3); 1H NMR (300 MHz, CDCl3) δ 6.84 (2H, d, J = 1.8 Hz), 6.77 (1H, t, J = 1.8 Hz), 5.38 (2H, d, J = 3.6 Hz), 3.88 (2H, t, J = 9.6 Hz), 3.61 (2H, d, J = 11.4 Hz), 3.02 (1H, s), 2.00-1.57 (12H, m); 13C NMR (75 MHz, CDCl3) δ 160.19, 125.46, 114.42, 100.13, 96.50, 79.78, 79.07, 61.73, 30.81, 25.70, 19.32.

5-[4-(Tetrahydro-2H-pyran-2-yloxy)phenyl]ethynyl-1,3-[bis(tetrahydro-2H-pyran-2-yloxy)]benzene (12). To a mixture of 3,5-bis(tetrahydro-2H-pyran-2-yloxy)ethynyl-benzene 10 (2.569 g, 8.447 mmol), PdCl2(PPh3)2 (0.296 g, 0.422 mmol), 4-[(tetrahydro-2H-pyran-2-yl)oxy]iodo-benz-ene 1121 (2.322 g, 9.29 mmol) and CuI (0.064 g, 0.42 mmol) in DMF (13 mL) was added trimethylamine (2.35 mL, 16.89 mmol) under nitrogen atmosphere. The resulting mixture was stirred for 15 h at room temperature under nitrogen atmosphere. The reaction was quenched with water, and the organic layer was extracted with methylene chloride. The organic layer was washed with water, dried over Na2SO4, filtered, and dried in vacuo. The residue was applied to column chromatography (Methylene chloride: hexanes = 1:1). The desired product 12 was obtained as a brown solid (2.304 g, 57%). Rf 0.55 (EtOAc:hexanes = 1:3); mp 204-206 °C; 1H NMR (300 MHz, CDCl3) δ 7.43 (2H, d, J = 8.7 Hz), 7.01 (2H, d, J = 8.7 Hz), 6.87 (2H, d, J = 1.8 Hz), 6.73 (1H, t, J = 1.8 Hz), 5.43 (3H, t, J = 3.9 Hz), 3.89 (3H, t, J = 9.6 Hz), 3.62 (3H, d, J = 11.4 Hz), 2.00-1.57 (18H, m); 13C NMR (75 MHz, CDCl3) δ 160.35, 158.06, 134.65, 125.09, 117.83, 114.84, 112.82, 99.86, 98.69, 96.50, 94.88, 61.90, 30.81, 25.70, 19.31.

5-[(4-Hydroxyphenyl)ethynyl]-1,3-benzenediol (5). To a solution of 5-[(4-tetrahydro-2H-pyran-2-yloxy)phenyl]ethynyl-1,3-[bis(tetrahydro-2H-pyran-2-yloxy)]benzene 12 (500 mg, 1.04 mmol) in MeOH was added Dowex 50X2-100 ion-exchange resin (excess) and the resulting mixture was stirred for 3 h at room temperature. The reaction mixture was filtered through Celite 545 and the filtrate was evaporated. The residue was applied to silicagel column chromatography (EtOAc:hexanes = 1:1). The desired pro-duct 5 was obtained as a brown solid (199 mg, 85%). Rf 0.23 (EtOAc:hexanes = 1:3); mp 235-239 °C; 1H NMR (300 MHz, CD3OD) δ 7.30 (2H, d, J = 8.7 Hz), 6.76 (2H, d, J = 8.7 Hz), 6.38 (2H, d, J = 1.8 Hz), 6.32 (1H, t, J = 1.8 Hz); 13C NMR (75 MHz, CD3OD) δ 161.62, 158.06, 134.65, 124.51, 117.83, 117.61, 113.01, 104.31, 98.69, 94.88.

Antioxidant Assay using ABTS. The radical cation was prepared by mixing an equal amount of 7 mM ABTS•+ (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) stock solution with 2.45 mM potassium persulfate stock solution. This mixture was stored at 0 °C for 12 h under darkness. The above ABTS solution was diluted with methanol appropri-ately to give UV absorption value of 1.000 at the 734 nm. The compounds 1-5 were dissolved in methanol to prepare 1000 μM stock solutions. Then these solutions were diluted with methanol to 500 μM, 250 μM, 125 μM, 62.5 μM, 31.25 μM, 15.63 μM, 7.81 μM, 3.90 μM, 1.95 μM, and 0.93 μM. In different 3 sets of test tubes, 0.9 mL of the ABTS solution and 0.1 mL of the compound solution were mixed under darkness. After 10 min of incubation, the UV absorption at 734 nm was measured. Control was used the mixture of 0.9 mL ABTS and 0.1 mL methanol. For the reference compound, ascorbic acid (Vitamin C) and quercetin were used. The radical scavenging rates were obtained from these UV absorption data and the resulting IC50 values were calculated using Origin 8.