1
Yield: 48 mg (75%), white solid (mp 189-191 °C). H NMR (400
MHz, DMSO-d ) δ 10.60 (s, 1H), 10.10 (s, 1H), 7.71 (d, J = 8.9
Hz, 1H), 7.18 (t, J = 8.5 Hz, 1H), 6.96 (d, J = 2.4 Hz, 1H), 6.85
according to method C. The product was purified by column
chromatography (0-60% ethyl acetate in petroleum ether). Yield:
6
1
41 mg (72%), green solid (mp 203-206 °C). H NMR (400 MHz,
(
m, 2H), 6.79 (d, J = 2.4 Hz, 1H), 2.60 (m, 1H), 2.43 (m, 1H),
acetone-d
6
) δ 9.37 (s, 1H), 8.54 (s, 1H), 8.24 (s, 1H), 6.97 (d, J =
1
3
1
1
1
1
6
.04 (t, J = 7.5 Hz, 3H). C NMR (100 MHz, DMSO-d ) δ
8.7 Hz, 1H), 6.86 (m, 4H), 6.70-6.66 (m, 3H), 6.64 (m, 1H), 6.54-
13
60.94, 159.73, 158.10, 154.98, 154.57, 133.67, 132.44, 127.05,
6
6.49 (m, 2H). C NMR (100 MHz, acetone-d ) δ 161.99, 161.49,
23.96, 119.69, 115.78, 114.53, 113.21, 110.57, 102.29, 22.48,
158.15, 157.24, 155.72, 152.22, 133.09, 131.77, 129.90, 127.22,
+
3.31. HRMS (ESI) calcd for C17
H14ClO
4
[M + H] , 317.0581;
127.03, 123.83, 115.91, 115.15, 114.57, 113.47, 103.11. HRMS
+
found 317.0518.
.1.2.7. 4-Ethyl-3-(3-fluoro-4-hydroxyphenyl)-7-hydroxy-2H-
(ESI) calcd for C21
15
H O
5
[M + H] , 347.0919; found 347.0908.
4
4.2. Estrogen receptor binding affinity
chromen-2-one (3g)
Relative binding affinities were determined by a competitive
3
9, 42
Compound 3g was prepared by 4-ethyl-3-(3-fluoro-4-
methoxyphenyl)-7-methoxy-2H-chromen-2-one (10e) and boron
tribromide according to method C. The product was purified by
column chromatography (0-40% ethyl acetate in petroleum ether).
radiometric binding assay as previously described,
using 2
3
3
nM [ H]-estradiol as tracer ([2,4,6,7- H]-estra-1,3,5(10)-triene-
3,17β-diol, 70-115 Ci/mmol, Perkin-Elmer, Waltham, MA), and
purified full-length human ERα and ERβ were purchased from
PanVera/Invitrogen (Carlsbad, CA). Incubations were for 18-24 h
at 0 °C. Hydroxyapatite (BioRad, Hercules, CA) was used to
absorb the receptor−ligand complexes, and free ligand was
washed away. The binding affinities are expressed as relative
binding affinity (RBA) values with the RBA of estradiol set to
100%. The values given are the average ± range or SD of two to
three independent determinations. Estradiol binds to ERα with a
1
Yield: 36 mg (65%), white solid (mp 272-275 °C). H NMR (400
MHz, DMSO-d
6
) δ 10.33 (s, 2H), 7.68-7.65 (m, 1H), 7.08 (m,
1
2
H), 7.04–6.97 (m, 1H), 6.89 (m, 1H), 6.84 (m, 1H), 6.75 (d, J =
13
.3 Hz, 1H), 2.58 (m, 3H), 1.07 (t, J = 7.5 Hz, 3H). C NMR
) δ 161.20, 161.05, 154.83, 154.27, 151.06,
44.88, 127.51, 126.45, 126.41, 121.51, 118.16, 117.93, 113.60,
(
100 MHz, DMSO-d
6
1
1
1
19
6
11.28, 102.69, 22.85, 14.52. F NMR (375 MHz, DMSO-d ) δ -
+
36.71. HRMS (ESI) calcd for C17
H
14FO
4
[M + H] , 301.0876;
K
d
of 0.2 nM and to ERβ with a K
d
of 0.5 nM; these values were
found 301.0866.
.1.2.8. 3,4-Bis(3-fluoro-4-hydroxyphenyl)-7-hydroxy-2H-
determined by scatchard analysis using the binding assay
39
4
protocol described previously.
chromen-2-one (3h)
4.3. Gene transcriptional activity
Compound 3h was prepared by 3,4-bis(3-fluoro-4-
The human embryonic kidney cell lines, HEK 293T, was
maintained in Dulbecco’s minimum essential medium (DMEM)
(Gibco by Invitrogen Corp., CA) with 10% fetal bovine serum
(FBS) (Hylcone by Thermo Scientific, UT). Cells were plated in
phenol red-free DMEM with 10% FBS. HEK 293T cells were
transfected with 25 µL of mixture per well, containing 300 ng of
3 × ERE-luciferase reporter, 100 ng of ERα or ERβ expression
vector, 125 mM calcium chloride (GuoYao, China), and 12.5 µL
of 2 × HBS. The next day, the cells were treated with increasing
doses of ER ligands diluted in phenol red-free DMEM with 10%
FBS. After 24 h, luciferase activity was measured using Dual-
Luciferase Reporter Assay System (Promega, MI) according to
the manufacturer’s protocol.
methoxyphenyl)-7-methoxy-2H-chromen-2-one (14a) and boron
tribromide according to method C. The product was purified by
column chromatography (0-60% ethyl acetate in petroleum ether).
1
Yield: 34 mg (70%), green solid (mp 189-192 °C). H NMR (400
MHz, acetone-d
6
) δ 9.55 (s, 1H), 9.01 (s, 1H), 8.70 (s, 1H), 6.99
(
(
d, J = 8.7 Hz, 1H), 6.91–6.82 (m, 3H), 6.77–6.73 (m, 1H), 6.68
13
m, 4H). C NMR (101 MHz, acetone-d
55.80, 153.15, 152.27, 151.40, 149.77, 145.88, 144.89,129.86,
28.25, 127.64, 126.92, 123.09, 119.46, 118.59, 118.22, 117.61,
6
) δ 161.84, 161.54,
1
1
1
1
3
4
19
6
14.08, 113,72, 103.19. F NMR (375 MHz, acetone-d ) δ -
+
13 2 5
37.54, -139.08. HRMS (ESI) calcd for C21H F O [M + H] ,
83.0731; found 383.0717.
.1.2.9. 7-Hydroxy-3,4-bis(4-hydroxyphenyl)-2H-chromen-2-
4.4. Cell culture and cell viability assay
one (3i)
Compound 3i was prepared by 7-methoxy-3,4-bis(4-
methoxyphenyl)-2H-chromen-2-one (14b) and boron tribromide
The human breast cancer cell line MCF-7, MDA-MB-231 and
MDA-MB-453 were obtained from ATCC. Cells were
maintained in RPMI, DMEM and L15 added 10% FBS,
11