1
13
H NMR, C NMR, and HR-MS; when using triethylamine as the catalyst and acid-binding agent, and dichloromethane as
the solvent to control the temperature at 25ꢁC, the reaction was optimum. The results of antitumor activities of compounds on
HeLa cells indicated that compounds 3a and 5b displayed better antitumor activities against HeLa cells than resveratrol.
Compound 3a displayed the best antitumor activities with an IC value of 26.7 ꢀmol/L, which was higher than the value of
50
resveratrol (IC = 112 ꢀmol/L), and its inhibition ratio was 95.1% against HeLa cells at the concentration of 75 ꢀmol/L.
50
EXPERIMENTAL
All chemicals and reagents were purchased from commercial sources and were used without further purification.
All reactions were monitored by TLC (silica gel plates; Merck 60 F254). Column chromatography (CC): silica gel (Merck,
1
13
300–400 mesh). Mp: WPS-2A apparatus, uncorrected. H and C NMR Spectra: Bruker-Avance 500 (500 MHz) in DMSO-d ,
6
at 500 and 125 MHz, respectively, ꢂ relative to Me Si as internal standard, in ppm, J in Hz. IR spectra: Nicolet-IR-6700
4
–1
spectrometer (KBr); ꢃ in cm . MS: Shimadzu 2020 spectrometer. HR-MS: SolariX-70FT-MS Bruker spectrometer.
Measurement of the Antitumor Activities on Cervical Cancer HeLa Cells. The antitumor activities of the target
compounds on human cervical carcinoma cells (HeLa cells) in vitro were measured by the MTT assay, we collected the cells
in the log phase and adjusted the concentration of the cell suspension, then added the cell suspension (100 ꢀL) to each well and
adjusted the density of the cells under test to 1000–10000 holes by decking (the edge holes were filled with sterile PBS). The
cells were cultured at 37ꢁC for 24 h in a 5% CO incubator until the cell monolayers covered the bottom of the hole (96-well
2
flat-bottom plates).Then we added gradient concentration drugs in five gradients, 100 ꢀL per well, and cultured the cells for
24 h in a 5% CO incubator at 37ꢁC. Then we observed them with an inverted microscope, discarded the supernatant, added
2
20 ꢀL MTT solution (5 mg/mL, namely 0.5% MTT) to each well, continued to culture the cells for 4 h, discarded the
supernatant, added 150 ꢀL DMSO to each hole, and oscillated the cells for 10 min on a low-speed shaker until the crystals
were fully dissolved. Finally, the absorbance value of each well was measured by an automatic multimicroplate reader at
OD570 nm and 630 nm.
General Methods for the Preparation of the Compounds 2a–6b. A solution of 2-thiophenecarboxylic acid or 2-furoic
acid (2.8 g, 22.0 mmol) in thionyl chloride (5.2 g, 44.0 mmol) was stirred and refluxed for 3 h. Then the excess solvents were
removed under reduced pressure to give 2-thiophenecarbonyl chloride (a) or 2-furoyl chloride (b), which were then dissolved
in dichloromethane (5.0 mL). The resulting solution was slowly added to a mixture of resveratrol (1.0 g, 4.4 mmol) and
triethylamine (4.0 mL) in dichloromethane (20.0 mL) at 25ꢁC. Then the mixture was stirred for 4 h; after completion of the
reaction as monitored by thin-layer chromatography (TLC) and vaporizing the dichloromethane, the residue was extracted
with ethyl acetate and washed with brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated
in vacuo to give a brown crude product .The residue was purified by flash chromatography (silica gel, ethyl acetate–petroleum
ether, 8:1–1:1) to afford compounds 2a–6b as a white solid.
(E)-5-(4-((Thiophene-2-carbonyl)oxy)styryl)-1,3-phenylenebis(thiophene-2-carboxylate) (2a). Yield 30%,
–1
1
mp 104–106ꢁC. IR (ꢃ, cm ): 1724.7, 1251.2, 1058.6. H NMR (500 MHz, DMSO-d , ꢂ, ppm, J/Hz): 8.14 (2H, d, J = 4.9),
6
8.11 (1H, d, J = 4.9), 8.08 (2H, d, J = 2.7), 8.05 (1H, d, J = 2.6), 7.70 (2H, d, J = 8.5), 7.54 (2H, s), 7.48 (1H, d, J = 16.4), 7.34
13
(6H, s), 7.27 (1H, s). C NMR (125 MHz, DMSO-d , ꢂ, ppm): 160.5, 160.3, 151.5, 150.5, 140.2, 136.1, 135.8, 135.7, 135.1,
6
+
132.3, 132.0, 130.3, 129.3, 129.2, 128.3, 127.4, 122.8, 118.1, 115.6. ESI-MS m/z 559.0 [M + H] ; HR-MS calcd for C H O S ,
29 18 6 3
559.0338.
(E)-5-(4-Hydroxystyryl)-1,3-phenylenebis(thiophene-2-carboxylate) (3a). Yield 26%, mp 148.4–149.6ꢁC.
–1
1
IR (ꢃ, cm ): 3389.2, 1717.8, 1251.2, 1065.7. H NMR (500 MHz, DMSO-d , ꢂ, ppm, J/Hz): 9.90 (1H, s), 8.11 (2H, d, J = 4.8),
6
13
8.04 (2H, s), 7.70 (2H, d, J = 8.5), 7.36–7.25 (6H, m), 7.02 (1H, s), 6.93 (1H, s), 6.59 (1H, s). C NMR (125 MHz, DMSO-d ,
6
ꢂ, ppm): 159.0, 158.3, 151.9, 139.7, 135.6, 135.3, 132.4, 132.1, 128.7, 128.5, 128.2, 117.6, 116.2, 112.1. ESI-MS m/z 449.0
+
[M + H] ; HR-MS calcd for C H O S , 449.0512.
24 16 5 2
–1
(E)-3-Hydroxy-5-(4-hydroxystyryl)phenylthiophene-2-carboxylate (4a). Yield 10%, mp 207.6–208.1ꢁC. IR (ꢃ, cm ):
1
3384.5, 1704.1, 1251.2, 1058.6. H NMR (500 MHz, DMSO-d , ꢂ, ppm, J/Hz): 9.81 (1H, s), 9.62 (1H, s), 8.10 (1H, s), 8.02
6
(1H, t, J = 6.7), 7.43 (2H, d, J = 8.5), 7.35–7.30 (1H, m), 7.13 (1H, d, J = 16.3), 6.95 (2H, d, J = 16.7), 6.85 (1H, s), 6.77 (2H,
13
d, J = 8.5), 6.53 (1H, s). C NMR (125 MHz, DMSO-d , ꢂ, ppm): 158.9, 157.9, 151.8, 140.3, 135.6, 129.9, 129.2, 128.5,
6
+
128.3, 124.8, 116.0, 111.5, 110.2, 108.1. ESI-MS m/z 339.0 [M + H] ; HR-MS calcd for C H O S, 339.0686.
19 14
4
654