Synthesis of Dimeric Cinnamoylamide Derivatives and Evaluation of Their Depigmenting Activities

Experimental Section

Synthesis of N,N'-Dicoumaroyl-putrescine (2a).

Acetoxy Coumaric Acid (4): To a solution containing coumaric acid (5.2 g, 0.03 mol), triethyl amine (7.7 g, 0.076 mol), and 4-(dimethylamino)pyridine (cat) in tetrahydrofuran (60 mL) was added acetic anhydride (7.8 g, 0.076 mol). The reaction mixture was refluxed for 3 h, and the solvent was removed at reduced pressure. The residue was dissolved in dichloromethane and washed with HCl (1 M) solution. The organic layer was dried with anhydrous MgSO4 and concentrated to produce a crude product. The resultant was purified by crystallization using dichloromethane and hexane to produce acetoxy coumaric acid 4 (5.5 g) at 85% yields. 1H NMR (300MHz, DMSO-d6) δ 7.75 (d, 2H, J = 8.7 Hz), 7.60 (d, 1H, J = 15.6 Hz), 7.19 (d, 1H, J = 8.7 Hz), 6.55 (d, 1H, J = 15.6 Hz), 2.28 (s, 3H). 13C NMR (125 MHz, DMSO-d6) δ 168.4, 166.9, 151.1, 142.2, 131.3, 128.8, 121.7, 118.7, 20.2. Ms-FAB (m/e) 207 (M++1).

N,N'-Diacetoxycoumaroyl-putrescine (5): Acetoxy coumaric acid (3.9 g, 0.019 mol) was dissolved in 15 mL of SOCl2 at 0 ℃ and refluxed for 1 h. SOCl2 was removed in vacuo, and crude acid chloride was dissolved in CH2Cl2 (10 mL). Then, the prepared acid chloride solution was added to a solution of 1,4-diaminobutane (0.8 g, 0.0095 mol) and triethyl amine (1.9 g, 0.019 mol) in methylene chloride (50 mL). The reaction mixture was stirred for 2 h at room temperature, concentrated in vacuo and the residue was extracted with ethyl acetate (300 mL) and washed with water. The organic layer was dried with anhydrous MgSO4 and concentrated to produce a crude product. The resultant was purified by column chromatography using ethyl acetate and hexane to afford N,N'-diacetoxycoumaroyl-putrescine 5 (2.8 g) at 65% yields. 1H NMR (300 MHz, DMSO-d6) δ 8.14 (s, 1H), 7.60 (d, 2H, J = 8.7 Hz), 7.42 (d, 1H, J = 15.6 Hz), 7.17 (d, 1H, J = 8.7 Hz), 6.59 (d, 1H, J = 15.6 Hz). 3.20 (m, 2H), 2.29 (s, 3H), 1.49 (m, 2H). 13C NMR (125 MHz, DMSO-d6) δ 168.4, 164.1, 150.5, 136.8, 132.0, 127.9, 121,79, 121.75, 37.7, 26.1, 20.2. Ms-FAB (m/e) 465 (M++1).

N,N'-Dicoumaroyl-putrescine (2a). N,N'-Diacetoxycoumaroyl-putrescine (2.8 g, 0.006 mol) was dissolved in KOH (0.5 M) solution and heated to 50 ℃ for 30 min. The reaction mixture was concentrated in vacuo, and ethylacetate (100 mL) was added, followed by HCl (1 M) solution. The organic layer was dried with anhydrous MgSO4 and concentrated to produce a crude product. The resultant was purified by column chromatography using ethyl acetate and hexane to afford N,N'-dicoumaroyl-putrescine 2a (1.6 g) at 70% yields. 1H NMR (300 MHz, DMSO-d6) δ 9.81 (bs, 1H), 7.97 (s, 1H), 7.38 (d, 2H, J = 8.7 Hz), 7.30 (d, 1H, J = 15.6 Hz), 6.78 (d, 1H, J = 8.7 Hz), 6.40 (d, 1H, J = 15.6 Hz), 3.20 (m, 2H), 1.46 (m, 2H). 13C NMR (125 MHz, DMSO-d6) δ 164.6, 158.1, 137.8, 128.5, 125.3, 118.1, 115.1, 37.7, 26.2. Ms-FAB (m/e) 381 (M++1).

Mushroom Tyrosinase Assay. Mushroom tyrosinase and ʟ-tyrosine were purchased from Sigma Chemical (Saint Louis, Missouri, USA). The reaction mixture for mushroom tyrosinase activity consisted of 150 μL of 0.1 M phosphate buffer (pH 6.5), 3 μL of sample solution, 8 μL of mushroom tyrosinase (2,100 unit/mL, 0.05 M phosphate buffer at pH 6.5), and 36 μL of 1.5 mM ʟ-tyrosine. Tyrosinase activity was determined by reading the optical density at 490 nm using a microplate reader (Bio-Rad 3550, Richmond, CA, USA) after incubation for 20 min at 37 ℃. The inhibitory activity of each sample is expressed as the concentration that inhibits enzyme activity by 50% (IC50).

DPPH Radical Scavenging Assay. DPPH radical-scavenging assay was carried out using the following procedure: a reaction mixture containing various concentrations of the test samples and DPPH methanolic solution (0.2 mM) was incubated at room temperature for 30 min and the absorbance was measured at 517 nm. Scavenging activity was expressed as a percent of the control DPPH solution (100%). The synthetic antioxidant trolox and ʟ-ascorbic acid were included as positive controls.

Cell Culture. The B16/F1 melanoma cells line was obtained from the Korean cell Line Bank (Seoul, Korea). Cells were cultured in Dulbecco’s Modified Eagle medium (DMEM) containing Fetal Bovine Serum (FBS, 10%), penicillin (100 U/mL), and streptomycin (0.1 mg/mL) at 37 ℃ in a humidified atmosphere of 5% CO2.

Measurements of Cell Viability. Cell viability was measured using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Cells were plated in 96-well plates and cultured for 24 h. After treatment with kojic acid and dimeric cinnamoylamide derivatives (2a-2l), 100 μL MTT (5 mg/mL in PBS) was added to each well. Cells were incubated at 37 ℃ for 30 min, dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals, and the absorbance was measured at 560 nm using a microplate reader a (Molecular Devices Co., Sunnyvale, CA, USA).

Measurements of Melanin Content. Cells (2 × 104 cells/mL) were seeded into 24-well plates and dicinnamoylamide derivatives were added in triplicate. The medium was changed daily and after 4 d of culture, the cells were lysed with 0.1 mL of 1 N NaOH. Then 100 μL of each crude cell extract was transferred to a 96-well plate. Relative melanin content was measured at 400 nm with a microplate reader (Molecular Devices).