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G.-Y. Liu et al. / European Journal of Medicinal Chemistry 112 (2016) 157e163
Scheme 1. 3,30-OH curcumin causes apoptosis in HepG2 cells through ROS-Mediated pathway.
4H), 6.90 (dd, J ¼ 8.0,1.6 Hz, 2H), 6.79 (d, J ¼ 8.0 Hz, 2H), 6.12 (s,1H);
4.3.9. 1,7-Bis(4-trifluoromethyphenyl)-heptane-3,5-dione (3c)
Yield: 51%, yellow powder; Rf ¼ 0.44 (petroleum ether/EtOAc
13C NMR 100 MHz (Acetone-d6),
d 184.6, 158.8, 141.4, 137.5, 131.0,
125.2, 120.7, 118.3, 115.5, 102.5; MS (EI) m/z 308 [M]þ.
(5:1)); mp 168e170 ꢁC; 1H NMR 400 MHz (CD3Cl),
d 7.66e7.71 (m,
10H), 6.68 (d, J ¼ 15.6 Hz, 2H), 5.90 (s, 1H); 13C NMR 100 MHz
(CD3Cl),
d 182.9, 139.1, 138.3, 131.8, 131.4, 128.2, 126.2, 125.9, 122.5,
4.3.3. 1,7-Bis(4-hydroxyphenyl)-heptane-3,5-dione (1c) [26]
102.5; HRMS (ESI) m/z 413.0964 [M þ H]þ, calcd for 412.0898,
error ¼ 1.6 ppm.
Yield: 51%; 1H NMR 300 MHz (Acetone-d6),
d
7.58 (d, J ¼ 15.9 Hz,
2H), 7.55 (d, J ¼ 8.7 Hz, 4H), 6.89 (d, J ¼ 8.7 Hz, 4H), 6.64 (d,
J ¼ 15.9 Hz, 2H), 5.98 (s, 1H); 13C NMR 75 MHz (DMSO-d6),
d 184.6,
160.6, 141.1, 131.0, 127.7, 122.1, 116.9, 101.8; MS (EI) m/z 308 [M]þ.
4.3.10. 1,7-Bis(phenyl)-heptane-3,5-dione (4a) [27]
Yield: 68%, yellow powder; 1H NMR 300 MHz (CD3Cl),
d 7.65 (d,
J ¼ 15.9 Hz, 2H), 7.55e7.58 (m, 4H), 7.38e7.42 (m, 6H), 6.61 (d,
4.3.4. 1,7-Bis(2-methoxyphenyl)-heptane-3,5-dione (2a)
J ¼ 15.6 Hz, 2H), 5.86 (s, 1H); 13C NMR 75 MHz (CD3Cl),
d 183.3,
Yield: 52%, yellow powder; Rf ¼ 0.35 (petroleum ether/EtOAc
140.6, 134.9, 130.1, 128.9, 128.1, 124.0, 101.8; MS (EI) m/z 276 [M]þ.
(4:1)); mp 118e120 ꢁC; 1H NMR 400 MHz (CD3Cl),
d 7.97 (d,
J ¼ 16.0 Hz, 2H), 7.55 (d, J ¼ 8.4 Hz, 2H), 7.32 (t, J ¼ 8.4 Hz, 2H), 6.95
(t, J ¼ 7.6 Hz, 2H), 6.91 (d, J ¼ 8.0 Hz, 2H), 6.70 (d, J ¼ 16.0 Hz, 2H),
4.4. Biology method
5.88 (s, 1H), 3.90 (s, 6H); 13C NMR 100 MHz (CD3Cl),
d 183.8, 158.3,
135.7, 131.2, 128.6, 124.7, 124.0, 120.7, 111.1, 101.5, 55.5; MS (EI) m/z
4.4.1. Cell culture
336 [M]þ.
Human liver hepatocellular carcinoma cells (HepG2) was pur-
chased from the Shanghai Institute of Biochemistry and Cell
Biology, Chinese Academy of Sciences and cultivated in RPMI 1640
supplemented with 2 mM glutamine, 10% (v/v) heat-inactivated
fetal calf serum, 100 kU/L penicillin and 100 kU/L streptomycin at
37 ꢁC in a humidified atmosphere with 95% air and 5% CO2.
4.3.5. 1,7-Bis(3-methoxyphenyl)-heptane-3,5-dione (2b)
Yield: 42%, yellow powder; Rf ¼ 0.34 (petroleum ether/EtOAc
(4:1)); mp 68e70 ꢁC; 1H NMR 400 MHz (CD3Cl),
d 7.61 (d,
J ¼ 15.6 Hz, 2H), 7.30 (t, J ¼ 8.0 Hz, 2H), 7.15 (d, J ¼ 7.6 Hz, 2H), 7.08
(s, 2H), 6.93 (d, J ¼ 8.0 Hz, 2H), 6.60 (d, J ¼ 15.6 Hz, 2H), 5.86 (s, 1H),
3.85 (s, 6H); 13C NMR 100 MHz (CD3Cl),
d 183.3, 160.0, 140.6, 136.5,
4.4.2. MTT assay
130.0, 124.4, 120.9, 116.0, 113.2, 101.9, 55.4; MS (EI) m/z 336 [M]þ.
The cell viability was assessed by MTT colorimetric assay as
described previously [24]. Cell viability was determined by a
colorimetric assay using MTT. HepG2 cells were seeded at a density
of 3 ꢂ 103/well in a complete growth medium in 96-well plates and
incubated for 24 h. The cells were incubated with the test com-
pounds for 48 h before the MTT assay. A fresh solution of MTT
(0.5 mg/mL) was added to each single well of the 96-well plate. The
plate was then incubated in a CO2 incubator for 4 h. The cells were
4.3.6. 1,7-Bis(4-methoxyphenyl)-heptane-3,5-dione (2c)
Yield: 41%, 1H NMR 300 MHz (CD3Cl),
d
7.59 (d, J ¼ 15.9 Hz, 2H),
7.50 (d, J ¼ 8.1 Hz, 4H), 6.53 (d, J ¼ 8.7 Hz, 4H), 6.47 (d, J ¼ 15.9 Hz,
2H), 5.78 (s, 1H), 3.85 (s, 6H); 13C NMR 75 MHz (CD3Cl),
183.3,
d
161.2, 140.1, 129.8, 127.7, 121.7, 114.3, 101.5, 55.4; MS (EI) m/z 336
[M]þ.
dissolved with 100 mL of DMSO and then analyzed in a multiwall-
plate reader (Bio-Rad M680) at 570 nm.
4.3.7. 1,7-Bis(2-trifluoromethyphenyl)-heptane-3,5-dione (3a)
Yield: 45%, yellow powder; Rf ¼ 0.45 (petroleum ether/EtOAc
(5:1)); mp 174e176 ꢁC; 1H NMR 400 MHz (CD3Cl),
d
8.00 (dd,
4.4.3. Cell apoptosis analysis
J ¼ 15.6,1.6 Hz, 2H), 7.71e7.77 (m, 4H), 7.56 (t, J ¼ 7.6 Hz, 2H), 7.46 (t,
HepG2 cells (5 ꢂ 105/well) were plated in six-well plates and
incubated for 24 h to allow exponential growth, then treated with
test compounds for 24 h. When necessary, the cells were pretreated
with GSH or NAC for 1 h before adding test compounds. The treated
cells were collected and labeled with Annexin V-FITC/PI according
to the manufacturer's instructions. A total of 10,000 cells per
sample were collected and analyzed by FACSDiva software.
J ¼ 7.6 Hz, 2H), 6.59 (d, J ¼ 15.6 Hz, 2H), 5.92 (s, 1H); 13C NMR
100 MHz (CD3Cl),
d 182.9, 136.2, 136.1, 133.9, 132.0, 129.4, 129.1,
128.8, 128.5, 128.1, 127.8, 126.3, 126.2, 125.4, 122.6, 101.6; HRMS
(ESI) m/z 413.0971 [M þ H]þ, calcd for 412.0898, error ¼ 1.0 ppm.
4.3.8. 1,7-Bis(3-trifluoromethyphenyl)-heptane-3,5-dione (3b)
Yield: 47%, yellow powder; Rf ¼ 0.42 (petroleum ether/EtOAc
(5:1)); mp 154e156 ꢁC; 1H NMR 400 MHz (CD3Cl),
d
7.81 (s, 2H),
4.4.4. Measurement for intracellular ROS levels
7.67e7.74 (m, 4H), 7.63 (d, J ¼ 7.6 Hz, 2H), 7.52 (t, J ¼ 8.0 Hz, 2H),
Intracellular ROS levels were determined according to 20,70-
dichlorofluorescein fluorescence assay as described previously [24].
After 3 or 6 of treatment with test compounds, HepG2 cells were
6.68 (d, J ¼ 15.6 Hz, 2H), 5.90 (s, 1H); 13C NMR 100 MHz (CD3Cl),
d
182.9, 139.0, 135.7, 132.0, 131.7, 131.3, 131.0, 129.5, 127.9, 126.5,
125.7, 125.6, 125.2, 124.5, 124.4, 122.5, 102.4, 102.3; HRMS (ESI) m/z
incubated with 3
m
M DCFH-DA for 30 min at 37 ꢁC in the dark. Then
413.0979 [M þ H]þ, calcd for 412.0898, error ¼ 1.9 ppm.
the cells were washed with PBS and analyzed immediately for 20,70-