H. Zhou, et al.
Fitoterapia 146 (2020) 104670
activities and preclinical profiles of the hybrid.
4.10 (s, 2H), 3.82 (s, 6H), 1.41 (s, 6H); 13C NMR (75 MHz, CDCl
2
3
) δ
02.4, 160.2, 159.8, 149.1, 147.9, 131.4, 128.2, 126.9, 121.6, 115.7,
113.1, 112.4, 111.4, 108.5, 55.9, 44.3, 28.4; ESI-MS m/z 355.2
M + H]+
4. Materials and methods
[
.
4
4
.1. Chemistry
4
.1.2.4. 1-(5-(4-Bromobutoxy)-2,2-dimethyl-2H-chromen-6-yl)-2-(3,4-
dimethoxyphen yl)Ethan-1-one (10). To a solution of 9 (320 mg,
0.90 mmol, 1.0 equiv.) and K CO (150 mg, 1.08 mmol, 1.2 equiv.)
.1.1. General information
All commercially available reagents were used without further
2
3
purification. Flash column chromatography was carried out on 200–300
mesh silica gel (Qingdao Haiyang Chemical, China). Reactions were
monitored by thin-layer chromatography (TLC) on 0.25 mm silicagel
plates (GF254) and visualized under UV light. H and C NMR spectra
were recorded with a Bruker AV-300 spectrometer (Bruker Company,
in 30 mL MeCN was added 1,4-dibromobutane (389 mg, 1.81 mmol, 2.0
equiv.), the mixture was refluxed for 8 h under nitrogen atmosphere.
The reaction mixture was quenched with 5 mL water and extracted with
ethyl acetate (20 mL × 3). The combined organic extracts were washed
1
13
2 4
with water and brine successively, dried over anhydrous Na SO , and
Germany) in the indicated solvents (CDCl
standard): the values of the chemical shifts are expressed in δ values
ppm) and the coupling constants (J) in Hz. Low-and high-resolution
mass spectrums (LRMS and HRMS) were measured on Finnigan MAT 95
spectrometer (Finnigan, Germany).
3
or DMSO‑d
6
, TMS as internal
concentrated. The crude residue was purified by column
chromatography with petroleum/ethyl acetate (10:1) to give 10 as a
(
white solid (0.26 g, 84%). 1H NMR (300 MHz, CDCl
3
) δ 7.91 (d,
J = 8.7 Hz, 1H), 7.59 (s, 1H), 7.44 (d, J = 8.8 Hz, 1H), 6.89 (d,
J = 10.1 Hz, 1H), 6.76 (d, J = 8.8 Hz, 1H), 6.41 (d, J = 8.8 Hz, 1H),
5.74 (d, J = 10.1 Hz, 1H), 4.01 (d, J = 4.2 Hz, 6H), 3.92 (d,
4
4
.1.2. Synthesis of compound 4
.1.2.1. 1-(2,4-Dihydroxyphenyl)-2-(3,4-dimethoxyphenyl)Ethan-1-one
J = 3.6 Hz, 2H), 3.83 (m, 2H), 3.34 (m, 2H), 1.82 (m, 4H), 1.49 (s, 6H);
13
C NMR (75 MHz, CDCl
125.7, 118.9, 116.3, 113.8, 111.5, 56.2, 50.1, 33.2, 29.7, 29.1, 28.2,
28.0; ESI-MS m/z 527.2 [M + K]+
3
) δ 203.8, 168.7, 149.4, 131.6, 130.4, 126.3,
(
7). 3,4-Dimethoxyphenylacetic acid (5, 1.0 g, 5.1 mmol, 1.0 equiv.)
and resorcinol (6, 561 mg, 5.1 mmol, 1.0 euqiv) were added into 10 mL
boron trifluoride ethyl etherate. The mixture was stirred at 90 °C for
.
1
3
.5 h under the protection of nitrogen. 30 mL cold water containing
.0 g sodium acetate was added into the reaction solution to precipitate
4.1.2.5. 4-((6-(2-(3,4-dimethoxyphenyl)acetyl)-2,2-dimethyl-2H-
chromen-5-yl)oxy) butyl nitrate (4). AgNO (104 mg, 0.61 mmol, 2.0
3
the product. The mixture was filtered and the solid was purified by
equiv.) and 10 (150 mg, 0.30 mmol, 1.0 equiv.) were added into a
solution of 20 mL MeCN, and the mixture was refluxed for 12 h. The
reaction mixture was quenched with 10 mL water and extracted with
ethyl acetate (15 mL × 3). The combined organic extracts were washed
with water and brine successively, dried over anhydrous Na SO , and
2 4
concentrated. The obtained residue was purified by flash column
chromatography using petroleum ether/ethyl acetate (3/1, V/V) as an
ethyl acetate to obtained 7 as a yellow solid (1.17 g, 80%). 1H NMR
(
300 MHz, DMSO‑d
J = 8.9 Hz, 1H), 6.91 (d, J = 2.0 Hz, 1H), 6.87 (d, J = 8.2 Hz, 1H),
.80 (dd, J = 8.2, 2.0 Hz, 1H), 6.40 (dd, J = 8.9, 2.4 Hz, 1H), 6.26 (dd,
6
) δ 12.59 (s, 1H), 10.67 (s, 1H), 7.96 (d,
6
1
3
J = 2.4 Hz, 1H), 4.20 (s, 2H), 3.72 (s, 6H); C NMR (75 MHz,
DMSO‑d ) δ 202.9, 165.4, 165.1, 149.1, 148.1, 134.0, 127.9, 122.0,
13.8, 112.6, 112.3, 108.7, 102.9, 55.9, 55.9, 44.1; ESI-MS m/z 311.1
6
1
eluent to afford the target compound (4) as a white solid (72 mg, 50%),
+
1
[
M + Na]
.
m.p. 89–91 °C: H NMR (300 MHz, CDCl
7
3
) δ 7.89 (d, J = 8.7 Hz, 1H),
.57 (s, 1H), 7.47 (d, J = 8.8 Hz, 1H), 6.94 (d, J = 10.1 Hz, 1H), 6.85
4
.1.2.2. 2-(3,4-Dimethoxyphenyl)-1-(2-hydroxy-4-((2-methylbut-3-yn-2-
(d, J = 8.8 Hz, 1H), 6.41 (d, J = 8.8 Hz, 1H), 5.72 (d, J = 10.1 Hz,
1H), 4.35 (m, 2H), 3.99 (d, J = 4.2 Hz, 6H), 3.91 (d, J = 3.6 Hz, 2H),
yl)oxy)phenyl) Ethan-1-one (8). Compound 7 (0.59 g, 2.03 mmol, 1.0
equiv.), 3-chloro-3-methyl-1-butyne (268 μL, 2.44 mmol, 1.2 equiv.),
copper iodide (38 mg, 0.2 mmol, 0.1 equiv.), potassium iodide (34 mg,
0
1
1
3
3.83 (m, 2H), 1.84 (m, 2H), 1.60 (m, 2H), 1.51 (s, 6H); C NMR
(75 MHz, CDCl
3
) δ 193.3, 160.2, 157.8, 149.7, 149.0, 131.7, 130.6,
.2 mmol, 0.1 equiv.) and potassium carbonate (336 mg, 2.44 mmol,
.2 equiv.) were added into 15 mL acetonitrile. The mixture was stirred
125.6, 121.4, 116.2, 114.2, 113.9, 112.3, 111.2, 110.4, 75.3, 63.9,
56.2, 55.9, 40.9, 28.0, 25.7, 24.9; HR-MS ESI, [M + H]+ m/z: calcd for
at room temperature for 10 h. The solvent was removed at reduced
pressure, and the residue was treated with 10 mL water and extracted
with ethyl acetate (3 × 30 mL). The combined organic phases were
25 8
C H30NO : 472.1966, found 472.1989.
4.2. Cell lines and cell culture
2 4
dried (Na SO ), and the solvent was removed at reduced pressure. The
crude residue was purified by column chromatography with petroleum/
Human lung cancer cell lines A549, H596, and H1299 were pur-
chased from the cell bank of Shanghai Biological Institute, Chinese
Academy of Sciences (Shanghai, China). Cells were grown in RPMI
1640 medium (Life Technologies, USA). The media for all cell lines
were supplemented with 10% fetal bovine serum (FBS), 1% strepto-
mycin and 1% penicillin (Life Technologies, USA), and all cells were
maintained at 37 °C in a humidified atmosphere with 5% CO2.
ethyl acetate (10:1) to give 8 as a white solid (0.36 g, 50%). 1H NMR
(
300 MHz, DMSO‑d
J = 2.4 Hz, 1H), 6.91–6.86 (m, 2H), 6.85 (s, 1H), 6.71 (dd, J = 8.7,
.4 Hz, 1H), 4.22 (s, 2H), 4.02 (s, 1H), 3.92 (s, 6H), 1.80 (s, 6H); 13
NMR (75 MHz, CDCl ) δ 202.4, 165.0, 162.7, 149.1, 148.2, 131.6,
26.8, 121.6, 113.7, 112.5, 111.4, 111.1, 106.5, 84.7, 75.2, 72.4, 55.9,
6
) δ 12.66 (s, 1H), 7.81 (d, J = 9.0 Hz, 1H), 6.95 (d,
2
C
3
1
4
+
4.5, 29.6; ESI-MS m/z 377.1 [M + Na]
.
4.3. Cell viability assay
4
.1.2.3. 2-(3,4-Dimethoxyphenyl)-1-(5-hydroxy-2,2-dimethyl-2H-
chromen-6-yl)Ethan-1-one (9). Compound 8 (0.36 g) was dissolved in
Cells were seeded at a density of 4 × 103 cells/well into 96-well
3
2
mL N,N-dimethylaniline, and the mixture was refluxed at 130 °C for
4 h. After the reaction was completed, 10 mL concentrated HCl was
plates. After attachment, medium was removed and 100 μL of fresh
medium containing the compounds at different concentrations was
added to each well and incubated at 37 °C for indicated time. After
treatment, MTT solution was added and incubated at 37 °C for 4 h.
Then, 100 μL DMSO was added and the number of living cells was di-
rectly proportional to the intensity of the blue, which was quantita-
tively measured at 570 nm by a Microplate reader (Bio-Tek, Vermont,
USA). Cell morphology was observed and captured using a phase con-
trast microscopy (Olympus, Japan).
added and extracted with ethyl acetate (3 × 30 mL). The combined
organic phases were dried (Na SO ), and the solvent was removed at
reduced pressure. The crude residue was purified by column
chromatography with petroleum/ethyl acetate (10:1) to give 9 as a
white solid (0.34 g, 95%). H NMR (300 MHz, CDCl
2
4
1
3
) δ 12.92 (s, 1H),
7.61 (d, J = 8.8 Hz, 1H), 6.76 (d, J = 13.0 Hz, 3H), 6.66 (d,
J = 10.1 Hz, 1H), 6.30 (d, J = 8.8 Hz, 1H), 5.53 (d, J = 10.1 Hz, 1H),
6