Valproic acid, lauric acid, acetaminophen, aspirin, ibuprofen, and resveratrol were purchased from TCI (Seoul, Korea). MeOD, CDCl3, and D2O were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO) was purchased from Duksan Pure Chemicals (Seoul, Korea). Dulbecco’s modified Eagle medium (DMEM), Roswell Park Memorial Institute Medium (RPMI), fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Capricorn Scientific GmbH (Ebsdorfergrund, Germany).
General procedures: All substrates and reagents were obtained commercially. 1H-NMR spectra were measured in ppm using an Agilent MR400 (400 MHz) FT-NMR spectrometer, with (CH3)4Si (TMS) as the internal standard and solvent. 13C-NMR spectra were recorded at 100 MHz. Chemical shifts are reported with respect to the solvent (DMSO = 2.5, 3.3, and 39.52 ppm for 1H- and 13C-NMR). A Büchi 510 melting point apparatus, electrothermal digital melting point apparatus, and general V4 were used to determine the melting points. 1C DuPont 2200. Mass spectrometry was performed using a Hewlett Packard (HP) 5890 GC/MSD [Column: HP-1 (crosslinked methyl silicone gum, 30 m × 0.25 mm × 0.3 µm); detector: 5972 A mass detector 70 eV, EI].
Reaction of resveratrol and lauric acid (Compounds 1, 2, and 3): Lauric acid (0.03 mol) and resveratrol (0.01 mol) were mixed with THF (solvent). The sieve was added, and the reaction mixture was refluxed at 60°C for 24 h. After confirming the progress and completion of the reaction using TLC, the sieve was removed using vacuum filtration. The remaining sieve was removed by washing twice with THF. After eliminating the sieve by filtration, the THF in the filtrate was removed using a rotary vacuum evaporator. The products were purified using flash chromatography (EtOAc:hexane at a ratio of 2:8) to yield corresponding compounds as ivory-colored solids.
( E )-4-(3,5-dihydroxystyryl)phenyl dodecanoate 1: Yield: 37.2%; mp: 108 ~ 109 ℃; Rf: 0.2 (TLC eluent; EtOAc: n-Hexane = 3: 7, v/v); MASS (70 eV), m/z (rel. intensity %): 410.2; 1H NMR (DMSO, 400 MHz): δ; 9.24 (s, 2H), 7.57 (d, J = 8.6 Hz, 2H), 7.01–7.08 (m, 4H), 6.41 (d, J = 2.1 Hz, 2H), 6.13 (t, J = 2.1 Hz, 2H), 1.23 (m, 18H), 0.83 (t, J = 6.8 Hz, 3H); 13C NMR (DMSO, 200MHz): δ; 172.257 (1C), 158.968 (2C), 150.213 (1C), 139.096 (1C), 135.145 (1C), 127.846 (2C), 127.339 (2C), 122.436 (2C), 105.132 (2C), 102.807 (1C), 33.921 (2C), 31.739 (1C), 29.425 (1C), 29.308 (1C), 29.155 (1C), 29.109 (1C), 28.822 (1C), 24.768 (1C), 22.542 (1C) 14.406 (1C); Anal. Calcd. for C26H34O4: C, 76.06; H, 8.35; O, 15.59; Found: C, 76.02; H, 8.32; O, 15.58
( E )-5-(4-hydroxystyryl)-1,3-phenylene didodecanoate 2: Yield: 27.0%; mp: 119 ~ 120 ℃; Rf: 0.3 (TLC eluent; EtOAc: n-Hexane = 3: 7, v/v); MASS (70 eV), m/z (rel. intensity %): 592.4; 1H NMR (MeOD, 400 MHz): δ; 9.60 (s, 1H), 7.35 (d, J = 8.6 Hz, 2H), 6.99 (m, 1H), 6.88 (m, 1H), 6.80 (m, 1H), 6.77 (d, J = 8.6 Hz, 2H), 6.69 (s, 1H), 2.55 (m, 2H), 2.26 (m, 2H), 1.72 (m, 2H), 1.59 (m, 2H), 1.28 (m, 32H), 0.89 (t, J = 6.8 Hz, 6H); 13C NMR (MeOD, 100 MHz): δ; 177.474 (1C), 177.329 (1C), 158.344 (1C), 153.096 (2C), 141.267 (1C), 130.250 (2C), 128.705 (1C), 125.635 (2C), 116.198 (2C), 111.191 (2C), 108.332 (1C), 34.799 (1C), 34.704 (1C) 32.777 (1C), 32.768 (1C), 30.450 (1C), 30.427 (1C), 30.313 (1C), 30.164 (1C), 30.313 (1C), 30.097 (1C), 29.948 (2C), 29.861 (2C), 25.820 (2C), 25.706 (2C), 23.436 (2C), 14.133 (2C); Anal. Calcd. for C38H56O5: C, 76.99; H, 9.52; O, 13.49; Found: C, 77.02; H, 9.52; O, 13.48
( E )-5-(4-(dodecanoyloxy)styryl)-1,3-phenylene didodecanoate 3: Yield: 50.7%; m.p: 132–133 ℃; Rf: 0.4 (TLC eluent; EtOAc: n-Hexane = 3: 7, v/v); MASS (70 eV), m/z (rel. intensity %): 774.6; 1H NMR (400 MHz, CDCl3): δ; 7.09 (t, J = 8.6 Hz, 2H), 6.88 (m, 2H), 6.77 (d, J = 8.6 Hz, 3H), 6.67 (d, J = 8.2 Hz, 2H), 2.25 (m, 6H), 1.54 (m, 6H), 1.18 (m, 48H), 1.80 (t, J = 6.8 Hz, 9H); 13C NMR (CDCl3, 100 MHz): δ; 180.363 (3C), 156.451 (1C), 156.400 (1C), 153.653 (1C), 133.457 (2C), 129.432 (2C), 115.295 (7C), 34.150 (1C), 31.942 (1C), 29.637 (24C), 24.725(1C), 22.718 (1C), 14.132 (3C); Anal. Calcd. for C50H78O6: C, 77.47; H, 10.14; O, 12.38; Found: C, 77.44; H, 10.12; O, 12.37
Reaction of resveratrol and valproic acid (Compounds 4 and 5): Valproic acid (0.03 mol) and resveratrol (0.01 mol) were mixed with THF (solvent). The sieve was added and the reaction mixture was refluxed at 60°C for 24 h. After confirming the progress and completion of the reaction by TLC, the sieve was removed using a vacuum filtration apparatus. The remaining sieve was removed by washing twice with THF. After eliminating the sieve by filtration, the THF in the filtrate was removed under vacuum. The products were purified using flash chromatography (EtOAc:hexane at a ratio of 2:8) to yield the corresponding compounds as brown solids.
( E )-4-(3-hydroxy-5-((2-propylpentanoyl)oxy)styryl)phenyl 2-propylpenoate 4: Yield: 36.1%; mp: 165 ~ 166 ℃; Rf: 0.7 (TLC eluent; EtOAc: n-Hexane = 1: 9, v/v); MASS (70 eV), m/z (rel. intensity %): 480.3; 1H NMR (CDCl3, 400 MHz): δ; 9.20 (s, 1H), 7.38 (m, 1H), 7.36 (t, J = 6.3 Hz, 2H), 7.34 (m, 2H), 7.06 (t, J = 7.2 Hz, 4H), 1.73 (m, 4H), 1.54 (m, 4H), 1.43 (m, 8H), 1.25 (m, 2H), 0.96 (t, J = 7.2 Hz, 12H); 13C NMR (CDCl3, 100 MHz): δ; 174.878 (2C), 150.660 (1C), 129.208 (2C), 125.505 (2C), 121.460 (2C), 100.197 (4C), 94.554 (3C), 45.234 (2C), 34.652 (4C), 20.569 (4C), 13.898 (4C); Anal. Calcd. for C30H40O5: C, 74.97; H, 8.39; O, 16.64; Found: C, 75.00; H, 8.35; O, 16.63
( E )-5-(4-((2-propylpentanoyl)oxy)styryl)-1,3-phenylene bis(2-propylpentanoate) 5: Yield: 40.8%; mp: 184 ~ 185 ℃; Rf: 0.9 (TLC eluent; EtOAc: n-Hexane = 1: 9, v/v); MASS (70 eV), m/z (rel. intensity %): 606.3; 1H NMR (CDCl3, 400 MHz): δ; 7.26 (t, J = 6.3 Hz, 4H), 6.94 (t, J = 7.4 Hz, 2H), 6.87 (dd, J = 8.5 and 0.9 Hz, 3H), 2.41 (m, 3H), 1.46 (m, 24H), 0.93 (t, J = 7.2 Hz, 18H); 13C NMR (100 MHz, CDCl3): δ; 183.048 (3C), 155.476 (3C), 129.623 (3C), 120.706 (1C), 115.280 (3C), 45.115 (3C), 34.332 (6C), 20.549 (6C), 13.970 (6C); Anal. Calcd. for C38H54O6:C,75.21; H,8.97; O,15.82; Found: C,75.20; H,8.95; O,15.83
Synthesis of valproic acid esters using an anti-inflammatory analgesic drug (Compounds 6, 7, and 8): Valproic acid (0.01 mol) and acetaminophen (0.01 mol) were mixed with THF. The sieve was added and the reaction mixture was refluxed at 60°C for 24 h. After confirming the progress and completion of the reaction using TLC, the sieve was removed under vacuum. The remaining sieve was removed by washing twice with THF. After eliminating the sieve by filtration, the THF in the filtrate was removed using a vacuum rotary evaporator. The products were purified using flash chromatography (EtOAc:hexane at a ratio of 2:8) to yield the corresponding compounds, 4-acetamidophenyl 2-propylpentanoate, as brown solids.
2-acetoxybenzoic 2-propylpentanoic anhydride 6: Yield: 55.1%; mp: sticky oil, Rf: 0.6 (TLC eluent; EtOAc: n-Hexane = 1: 9, v/v); MASS (70 eV), m/z (rel. intensity %): 306.1; 1H NMR (MeOD, 400 MHz): δ; 7.14 (t, J = 6.7 Hz, 1H), 6.79 (t, J = 9.0 Hz, 3H), 2.31 (t, 3H), 1.97 (s, 1H), 1.38 (m, 8H), 0.89 (t, J = 7.2 Hz, 6H); 13C NMR (MeOD, 100 MHz): δ; 179.279 (1C), 171.787 (1C), 156.879 (1C), 129.048 (1C), 119.205 (4C), 114.877 (1C), 45.225 (1C), 34.525 (2C), 20.341 (2C), 13.114 (2C); Anal. Calcd. for C17H22O5: C, 66.65; H, 7.24; O, 26.11; Found: C, 66.64; H, 7.22; O, 26.12
4-acetamidophenyl 2-propylpentanoate 7: Yield: 57.1%; mp: 89 ~ 90 ℃; Rf: 0.5 (TLC eluent; EtOAc: n-Hexane = 5: 5, v/v); MASS (70 eV), m/z (rel. intensity %): 277.2; 1H NMR (CDCl3, 400 MHz): δ; 7.78 (s, 1H), 7.43 (d, J = 7.9 Hz, 2H), 6.96 (d, J = 7.8 Hz, 2H), 2.60 (m, 1H), 2.10 (s, 3H), 1.53 (m, 4H), 1.42 (m, 4H), 0.95 (t, J = 7.2 Hz, 6H); 13C NMR (CDCl3, 100 MHz): δ; 175.673 (1C), 168.718 (1C), 146.797 (1C), 135.527 (1C), 121.861 (2C), 120.920 (2C), 45.337 (1C), 34.652(2C), 22.755(1C), 20.705(2C), 14.023(2C); Anal. Calcd. for C16H23NO3: C, 69.23; H, 8.36; N, 5.05; O, 17.30; Found: C, 69.24; H, 8.35; N, 5.06; O, 17.31
2-(4-isobutylphenyl)propanoic 2-propylpentanoic anhydride 8 : Yield: 67.3%; mp: sticky oil, Rf: 0.5 (TLC eluent EtOAc: n-Hexane = 1: 9, v/v); MASS (70 eV), m/z (rel. intensity %): 332.2; 1H NMR (DMSO, 400 MHz): δ; 7.35 (t, J = 8.1 Hz, 2H), 7.09 (t, J = 7.2 Hz, 2H). 3.79 (s, 1H), 2.57 (m, 2H), 2.19 (s, 1H), 1.66 (m, 1H), 1.38 (m, 7H), 1.24 (m, 4H), 0.89 (m, 12H); 13C NMR (MeOD, 100 MHz): δ; 176.254 (1C), 174.466 (1C), 140.168 (1C), 138.032 (1C), 129.946 (2C), 126.045 (2C), 63.750 (1C), 44.783 (1C), 34.598 (1C), 30.098 (2C), 24.868 (1C), 21.713 (2C), 20.440 (2C), 17.866 (2C), 13.347 (1C); Anal. Calcd. for C21H32O3: C, 75.86; H, 9.70; O, 14.44; Found: C, 75.84; H, 9.72; O, 14.42
Cell culture
5637 (human bladder cancer cells), Nuff (human newborn foreskin fibroblasts), and HDF [human dermal fibroblasts (HDFs)] cells were purchased from the American Type Culture Collection (ATCC). 5637 cells were cultured in RPMI supplemented with 10% FBS and 1% penicillin/streptomycin (100 U/mL). Nuff and HDF cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin (100 U/mL). All cells were maintained at 37°C in a humidified atmosphere containing 5% CO2.
MTT assay
Cytotoxicity of compound 2 was evaluated using an MTT assay.32 First, compound 2 was dissolved in DMSO. Then, it was dissolved in the free medium immediately before being used for cell treatment and was followed by 70% sonication and filtration of the material through a 0.45-µM filter (Millipore, Billerica, MA, USA). Cells were seeded into 96-well plates at a density of 1 × 104 cells/well prior to treatment. The next day, the medium was replaced with complement medium containing various concentrations of compound 2 at 37°C in a humidified CO2 incubator. After 24 h, the medium was replaced with 100 µL of 5 mg/mL MTT solution (Sigma Chemicals, St. Louis, MO, USA) in 1XPBS PBS, and the cells were incubated for 4 h at 37°C. MTT–formazan was dissolved in 100 µL of DMSO, and the absorbance at 550 nm was measured using a microplate reader. Cell viability (%) was calculated as follows
(O.D.550 of treated cells/O.D.550 of untreated cells) × 100
Annexin V and dead cell staining
Cell death and apoptosis of 5637 cells were measured using a MUSE™ Annexin V & Dead Cell kit, following the manufacturer’s protocol.33 Briefly, 5637 cells were seeded into 6-well plates at a density of 2×105 cells/well. After attachment, the cells were incubated with various concentrations of compound 2. After 24 h of incubation, the cells were harvested by centrifugation and resuspended in complete media. Then, 100 µL of MUSE Annexin V & Dead cell reagents were added to each tube and incubated for 20 min at room temperature in the dark. At the end of the incubation period, cells were analyzed using a MUSE Cell Analyzer (Millipore, Billerica, MA, USA).
Statistical analyses
Each experiment was performed at least three times in all cases. Data are presented as mean ± standard deviation (SD). Statistical differences among the groups were analyzed using GraphPad Prism 5. In all of the comparisons, a level of P < 0.05, was considered significant.