M. Bian et al.
Bioorganic Chemistry 107 (2021) 104598
Yield 91%, m. p. = 227.3–228.2 ℃; [
α
]25 D20.3 (c 0.07, CH3OH); 1H
Hz, 1H), 7.42 (dd, J = 8.3, 4.3 Hz, 1H), 7.31 (d, J = 2.3 Hz, 1H), 5.75 (s,
1H). 13C NMR (75 MHz, CDCl3) δ 216.89, 199.11, 174.84, 168.97,
151.55, 150.77, 148.45, 136.09, 128.96, 128.66, 126.30, 122.12,
120.86, 120.13. ESI-HRMS calcd for C39H50NO+4 ([M + H]+): 596.3734;
found:596.3725.
NMR (300 MHz, cdcl3) δ 8.22 (s, 1H), 7.66 (s, 1H), 7.63 (d, J = 8.3 Hz,
2H), 7.31 (d, J = 8.3 Hz, 2H), 5.84 (s, 1H), 4.25 (d, J = 16.9 Hz, 1H). 13C
NMR (75 MHz, CDCl3) δ 206.48, 199.53, 182.14, 170.22, 144.32,
139.02, 134.79, 134.44, 130.24, 128.64, 125.82, 121.54, 120.58. ESI-
HRMS calcd for C40H52N3O+4 ([M + H]+): 638.3952; found:638.39387.
(2S,4aS,6aS,6bR,11E,12aS)-
(2S,4aS,6aS,6bR,12aS)-quinolin-8-yl
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b, 13,14b- icosahydro-
2,4a,6a,6b,9,9,12a-heptamethyl-10,13-dioxopicene-2-carboxylate(6e).
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydro-11-
((1-(4-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methylene)-
White powder; Yield 75%, m. p. = 229.8–231 ℃; [α]25 D15.46 (c 0.06,
2,4a,6a,6b,9,9,12a-heptamethyl-10,13-dioxopicene-2-carboxylic acid
CH3OH); 1H NMR (300 MHz, CDCl3) δ 8.90 (dd, J = 4.1, 1.6 Hz, 1H),
8.18 (dd, J = 8.3, 1.6 Hz, 1H), 7.74 (d, J = 8.2 Hz, 1H), 7.55 (t, J = 7.9
Hz, 1H), 7.49 – 7.37 (m, 2H), 5.88 (s, 1H). 13C NMR (75 MHz, CDCl3) δ
217.22, 199.60, 175.31, 170.30, 150.10, 147.48, 135.92, 129.48,
128.38, 126.08, 125.76, 121.73, 121.25. ESI-HRMS calcd for
(5 g). White powder; Yield 90%, m. p. = 204.8.2–205.7 ℃; [α]25 D
10.29 (c 0.06, CH3OH); 1H NMR (300 MHz, CDCl3) δ 8.21 (s, 1H), 7.69
(d, J = 9.0 Hz, 2H), 7.67 (s, 1H), 7.05 (d, J = 9 Hz, 2H), 5.84 (s, 1H), 4.26
(d, J = 17.2 Hz, 1H), 3.87 (s, 3H). 13C NMR (75 MHz, CDCl3) δ 206.48,
199.57, 182.16, 170.29, 159.90, 144.25, 134.72, 130.13, 128.62,
125.85, 122.26, 121.69, 114.79. ESI-HRMS calcd for C40H52N3O+5 ([M
+ H]+):654.3902; found:654.3887.
C
39H50NO+4 ([M + H]+): 596.3734; found: 596.3725
(2S,4aS,6aS,6bR,12aS)-2-oxo-2H-chromen-4-yl
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11, 12,12a,12b, 13,14b-icosahydro-
2,4a,6a,6b,9,9,12a-heptamethyl-10,13-dioxopicene-2-carboxylate(6f).
4.2.5. General procedure for the synthesis of compounds 6a-6 g
The solution of compound 2 (140.6 mg, 0.3 mmol) in 10 mL dry DCM
and different hydroxy compounds (0.36 mmol) was treated with EDCI
(0.45 mmol) and 4-dimethylaminopyridine (DMAP) (10 mg). The
mixture was stirred at 25 ◦C for 2–8 h and monitored by TLC. After
completion of the reaction, water was added and the mixture was
extracted with dichloromethane and the organic phase was washed with
saturated sodium bicarbonate solution and brine, and dried over
Na2SO4. The evaporation of the solvents gave the crude products, which
were purified by silica gel column to afford compounds 6a-6 g. Yields of
purified compounds were between 69% and 84%.
White powder; Yield 84%, m. p. = 172.4–173.8 ℃; [α]25 D12.07 (c
0.07, CH3OH); 1H NMR (300 MHz, CDCl3) δ 7.67–7.56 (m, 2H),
7.42–7.31 (m, 2H), 6.48 (s, 1H), 5.71 (s, 1H). 13C NMR (75 MHz, CDCl3)
δ 216.77, 199.00, 172.32, 168.28, 161.18, 158.49, 153.66, 132.81,
128.82, 124.40, 122.32, 117.20, 115.67, 104.94. ESI-HRMS calcd for
C
39H47O+6 ([M + H]+): 613.3524; found: 613.3516.
(2S,4aS,6aS,6bR,12aS)-2-oxo-2H-chromen-7-yl
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11, 12,12a,12b, 13,14b-icosahydro-
2,4a,6a,6b,9,9,12a-heptamethyl-10,13-dioxopicene-2-carboxylate(6 g).
White powder; Yield 82%, m. p. = 180.4–181.7 ℃; [α]25 D30.54 (c
0.08, CH3OH); 1H NMR (300 MHz, CDCl3) δ 7.73 (d, J = 9.6 Hz, 1H),
7.53 (d, J = 8.4 Hz, 1H), 7.09 (d, J = 2.1 Hz, 1H), 7.02 (dd, J = 8.4, 2.2
Hz, 1H), 6.43 (d, J = 9.6 Hz, 1H), 5.72 (s, 1H). 13C NMR (75 MHz,
CDCl3) δ 217.20, 199.36, 174.50, 169.09, 160.24, 154.66, 153.31,
142.85, 128.66, 128.60, 118.28, 116.67, 116.09, 110.33. ESI-HRMS
calcd for C39H49O+6 ([M + H]+): 613.3524; found: 613.3514.
(2S,4aS,6aS,6bR,12aS)-quinolin-3-yl
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b, 13,14b- icosahydro-
2,4a,6a,6b,9,9,12a-heptamethyl-10,13-dioxopicene-2-carboxylate(6a).
White powder; Yield 70%, m. p. = 239.7–241.9 ℃; [α]25 D12.31 (c
0.07, CH3OH); 1H NMR (300 MHz, CDCl3) δ 8.69 (d, J = 2.6 Hz, 1H),
8.16 (d, J = 8.4 Hz, 1H), 7.91 (d, J = 2.6 Hz, 1H), 7.85 (d, J = 8.1 Hz,
1H), 7.79–7.70 (m, 1H), 7.61 (t, J = 7.5 Hz, 1H), 5.76 (s, 1H). 13C NMR
(75 MHz, CDCl3) δ 217.11, 199.31, 174.90, 168.99, 146.10, 145.46,
144.27, 129.38, 129.12, 128.73, 128.13, 127.59, 127.42, 126.04. ESI-
HRMS calcd for C39H50NO+4 ([M + H]+): 596.3734; found: 596.3723.
(2S,4aS,6aS,6bR,12aS)-quinolin-5-yl-
4.3. Cell culture
Mouse RAW264.7 cells were cultured in Dulbecco’s Modified Eagle
Medium (Hyclone, USA) with 10% fetal bovine serum, 100 U/mL
penicillin and 100 μ
g/mL streptomycin (Beyotime, China) at 37 ◦C in a
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydro-
2,4a,6a,6b,9,9,12a-heptamethyl-10,13-dioxopicene-2-carboxylate(6b).
humid environment containing 5% CO2. All the cells used in the
experiment are in the logarithmic growth stage, and the number of cells
in the culture bottle is about 70–80%.
Yellow powder; Yield 70%, m. p. = 248.2–249 ℃; [α]25 D10.46 (c 0.07,
CH3OH); 1H NMR (300 MHz, CDCl3) δ 8.98 (dd, J = 4.2, 1.6 Hz, 1H),
8.20 (d, J = 8.4 Hz, 1H), 8.05 (d, J = 8.5 Hz, 1H), 7.75 (t, J = 8.1 Hz,
1H), 7.48 (dd, J = 8.5, 4.2 Hz, 1H), 7.31 (d, J = 7.7 Hz, 1H), 5.76 (s, 1H).
13C NMR (75 MHz, CDCl3) δ 217.06, 199.27, 174.82, 168.98, 150.84,
148.90, 146.08, 129.55, 128.78, 128.67, 127.44, 122.34, 121.38,
118.41. ESI-HRMS calcd for C39H50NO+4 ([M + H]+): 596.3734;
found:596.3726.
4.4. Assessment of toxicity
Cell viability studies induced by synthesized compounds were eval-
uated by MTT assay. RAW264.7 macrophages were seeded in 96-well
plates at a density of 1 × 104 cells/mL in complete medium and incu-
bated for 24 h (100 µL/well). Then the cells were treated with synthe-
sized compounds (25 µg/mL) for 24 h. MTT (5 mg/mL in PBS, 10% total
volume) was added to each well and the cells were further incubated for
(2S,4aS,6aS,6bR,12aS)-quinolin-6-yl-
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b,13,14b-icosahydro-
2,4a,6a,6b,9,9,12a-heptamethyl-10,13-dioxopicene-2-carboxylate(6c).
4 h. The supernatant was removed and the cells were lysed with 150 μL/
Brown powder; Yield 74%, m. p. = 249.4–250.6 ℃; [α]25 D24.16 (c
well DMSO. The optical density was measured at 570 (Measurement
wavelength) and 630 (reference wavelength) nm on a microplate reader
(Thermo Scientific, Waltham, MA, USA).
0.08, CH3OH); 1H NMR (300 MHz, CDCl3) δ 9.01–8.87 (m, 1H), 8.15 (d,
J = 8.6 Hz, 2H), 7.53 (d, J = 2.4 Hz, 1H), 7.49–7.38 (m, 2H), 5.75 (s,
1H). 13C NMR (75 MHz, CDCl3) δ 217.12, 199.38, 175.06, 169.23,
150.14, 148.60, 146.14, 135.83, 131.04, 128.61, 124.63, 121.63,
118.24. ESI-HRMS calcd for C39H50NO+4 ([M + H]+): 596.3734;
found:596.3727.
4.5. Measurement of TNF-α and IL-6
The RAW 264.7 macrophages were seeded at 1 × 104 cells in 96-well
(2S,4aS,6aS,6bR,12aS)-quinolin-7-yl
plates. Cells were incubated for 24 h and treated with 2.5, 5, 10 or 25
μ
g/
1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,10,11,12,12a,12b, 13,14b- icosahydro-
2,4a,6a,6b,9,9,12a-heptamethyl-10,13-dioxopicene-2-carboxylate (6d).
mL of LA derivatives 1 h and then stimulated with LPS (1
μg/mL) for 24
h. Cell culture supernatants were centrifuged at 5000g for 10 min at 4℃
White powder; Yield 69%, m. p. = 273.2–274.4 ℃; [α]25 D12.48 (c
to remove insoluble material and the supernatants were collected and
0.06, CH3OH); 1H NMR (300 MHz, CDCl3) δ 8.94 (dd, J = 4.2, 1.6 Hz,
1H), 8.20 (d, J = 8.1 Hz, 1H), 7.87 (d, J = 8.8 Hz, 1H), 7.79 (d, J = 2.0
stored at-20℃ until assayed for cytokines. Secreted TNF-α, IL-6 were
measured in cell culture supernatants using commercially available
13