Synthesis of four natural prenylflavonoids and their estrogen-like activities

Article abstract of DOI:10.1002/ardp.200700057

Four prenylflavonoids, bavachin 1, isobavachin 2, 7,4′-dihydroxy-8- prenylflavone 3, and 8-prenylapigenin 4 were synthesized and recognized for possessing estrogen-like activity in MCF-7/BOS cells, as evaluated by an estrogen-screening assay. All compound

Full text of DOI:10.1002/ardp.200700057

372
Arch. Pharm. Chem. Life Sci. 2007, 340, 372–376
Full Paper
Synthesis of Four Natural Prenylflavonoids and Their
Estrogen-like Activities
Xiaowu Dong, Yongjian Fan, Lingjun Yu, and Yongzhou Hu
ZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang University,
Hangzhou, China
Four prenylflavonoids, bavachin 1, isobavachin 2, 7,49-dihydroxy-8-prenylflavone 3, and 8-prenyl-
apigenin 4 were synthesized and recognized for possessing estrogen-like activity in MCF-7/BOS
cells, as evaluated by an estrogen-screening assay. All compounds significantly stimulated the
proliferation of MCF-7/BOS cells in a dose-dependent manner. Isobavachin 2 showed the most
potent activity, while bavachin 1 was the weakest. The estrogenic potency of these compounds is
ranked as follows: 2 A 4 A 3 A 1.
Keywords: Bavachin / 7,49-Dihydroxyl-8-prenylflavone / Estrogenic activity / 8-C-Prenylapigenin / Isobavachin /
Received: March 17, 2007; accepted: May 7, 2007
DOI 10.1002/ardp.200700057
Introduction
Prenylflavonoids are interesting natural products, iso-
lated from some traditional medicinal plants [1–3]. It
was reported that introduction of a prenyl substituent
into flavonoids could remarkably improve the various
biological activities [4–6]. Among them, bavachin 1, iso-
lated from Posralea corylifolia [7] showed various bioactiv-
ities including inhibition of platelet aggregation, antios-
teoporosis, antibacterial activity [8–11]. Jain et al. have
first synthesized bavachin 1 [12], but the poor yield of pre-
nylation and long time of condensation without protec-
tion of the hydroxyl are problems, and they are not
resolved yet. Recently, we revisited the synthesis of bava-
Figure 1. Structure of presented compounds 1–4.
chin 1, and made some improvements, which could
increase the total yield from 0.5% [12] to 4.2% and with a
more reasonable condensation time [13]. Despite of inten-
sive studies for bavachin 1, no data have been reported
about the estrogen-like effect. In this study, our goal is to
evaluate its effect of stimulating MCF-7/BOS cell prolifera-
tion. Considering the effect of substituents (C-6 prenyl or
C-8 prenyl) and the structural skeleton (flavone or flavo-
none) in prenylflavonoids, compounds 2, 3, and 4, which
are also natural products, were synthesized (Fig. 1) as
well. The antioxidant properties, cytotoxicity, and other
bioactivities of these compounds had already been
reported before [5, 14–18]. However, prenylflavonoids 1,
2, 3, and 4 were also recognized for possessing estrogen-
like activities in MCF-7/BOS cells evaluated by an estro-
gen-screening assay. These effects allow a large variety of
therapeutic uses such as for treating osteoporosis and
the menopause syndrome, according to the potential
value of phytoestrogens [19–21].
Correspondence: Yongzhou Hu, ZJU-ENS Joint Laboratory of Medicinal
Chemistry, College of Pharmaceutical Sciences, Zhejiang University,
Hangzhou, Zhejiang, 310031, China.
E-mail: huyz@zju.edu.cn
Fax: +86 571 8820-8460
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Arch. Pharm. Chem. Life Sci. 2007, 340, 372–376
Prenylflavonoids with Estrogenic Activity
373
(a) 1.3N NaOH, acetone; 3,3-dimethylallyl bromide, benzene, reflux; (b) 3,3-dimethylallyl bromide, 10% KOH; (c) MOMCl, K2CO3, acetone, 08C to
r.t.; (d) p-methoxymethoxylbenzaldehyde, 10% KOH (H2O/EtOH); (e) NaOAc, EtOH, reflux; (f) 3N HCl, MeOH, reflux; (g) I2, pyridine, 908C.
Scheme 1. Synthesis route of compounds 1–10.
Result and discussion
The synthetic pathway is outlined in Scheme 1. Bavachin
1 and isobavachin 2 were synthesized using 2,4-dihydrox-
yacetophenone 5a as the starting material, which was
prenylated with 3,3-dimethylallyl bromide, to furnish 6a
and 6b, according to the reported method [13]. Com-
pound 6c was achieved by treating 2,4,6-trihydroxyaceto-
phenone 5c with 3,3-dimethylallyl bromide, according to
the approach in the literature [22]. Selective methoxyme-
thylation of 6a, 6b, and 6c with chloromethoxylmethyl
Figure 2. Relative Proliferative Effect (RPE) of compounds 1–4
ether and anhydrous K2CO3 in dry acetone gave com-
pounds 7a, 7b, and 7c, respectively. Condensation of 7a,
(7b or 7c) with p-methoxymethoxylbenzaldehyde, pro-
ceeded in aqueous alcoholic alkali, to obtain chalcone 8a,
8b, or 8c. Compound 8a, (8b or 8c) was cyclized by reflux-
ing in a solution of NaOAc in EtOH, to afford the flavo-
none 9a, 9b, or 9c. Demethoxymethylation of 9a, 9b, and
9c was carried out in 3N HCl/MeOH to obtain the prod-
ucts 1, 2, and 10c, respectively. Finally, 3 and 4 were
obtained by dehydrogenation of the corresponding pre-
nylflavanones 2 and 10c with iodine and pyridine in
good yields (76% and 80%, respectively).
The estrogenic effects of the selected compounds were
examined by an E-SCREEN assays in MCF-7/BOS cells. The
maximum cell proliferative effect was observed with
1 nM 17b-estradiol. The proliferative effect of these com-
pounds is expressed as relative proliferative effect (RPE)
(Fig. 2), relative to 17b-estradiol's one (1 nM, 100%).
relative to that of 17ß-estradiol (1 nM, 100%).
estrogen-like activities were ascribed to their structural
similarity to mammalian estrogens [23]. Insight into the
observed effects revealed that the 8-prenyl substituent
could markedly improve the estrogenic activity because
the maximal proliferative effect of compounds 2–4 (8-
prenyl) was more potent than compound 1 (6-prenyl). Le
Bail et al. reported that structural spatial conformation
could play an important role in estrogenic activity. For
example, naringenin (flavonone) and apigenin (flavone)
have a 49,5,7-trihydroxyl substituent, however, the for-
mer conformation is much more estrogen-like [24]. The
result could be well validated by the maximal prolifera-
tive effect of compound 2 (flavonone), which was more
efficient than compounds 3 (flavone). Furthermore, a 5-
hydroxyl substituent in compound 4 appeared to slightly
increase the estrogenic activity compared to that of 3.
In conclusion, the synthesized compounds 1–4 exhib-
ited moderate or great estrogenic activity, which could
Previous studies of the structure-activity relationship
of flavone and isoflavone indicated that their potent
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

374
X. Dong et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 372–376
column chromatography using petroleum ether : ethyl acetate
(18 : 1). Yellow oil (680 mg, 70%); IR (KBr) m (cm– 1): 3190, 2932,
1632, 1478, 1355 cm– 1. 1H-NMR (CDCl3, 400 MHz, d): 1.67 (s, 3H),
1.77 (s, 3H), 2.65 (s, 3H), 3.30 (d, 1H, J = 7 Hz), 3.47 (s, 3H), 3.50 (s,
3H), 5.20 (m, 1H), 5.23(s, 2H), 5.24 (s, 2H), 6.39 (s, 1H), 13.81 (s,
1H). ESI-MS: m/z 325 [M+H]+.
be further studied as a potent non-steroidal phytoestro-
gen used for the treatment of certain hormone-depend-
ent diseases, such as osteoporosis and the menopause
syndrome.
General method for synthesis of compounds 8a, 8b, and
8c
Experiment
To a cold solution of the acetophenone 7a, (7b or 7c) and p-
methoxymethoxylbenzaldehyde in 3 mL H2O-EtOH (1/4), 20%
KOH in 3 mL H2O-EtOH (1/4) was added with stirring. The result-
ing mixture was stirred under argon at room temperature for
36 h. The whole mixture was poured into ice water, acidified to
pH~2 with 1 N HCl, and extracted with ethyl acetate. The
organic phase was washed with saturated NaCl solution, dried
(Na2SO4), and concentrated in vacuo. The residue was purified to
give chalcone 8a, 8b, or 8c.
Chemistry
Melting points were determined on a Bꢀchi B-540 apparatus
(Bꢀchi Labortechnik, Flawil, Switzerland) and are uncorrected.
All 1H-NMR spectra were recorded on Bruker400M spectrometer
(Bruker Bioscience, Billerica, MA, USA) with CDCl3 or DMSO-d6 as
solvent. Chemical shifts were reported in d values (ppm), relative
to internal TMS, and J values were reported in Hertz. Mass spec-
tral data were obtained by Finnigan LCQ DECA spectrometer
(Thermo Electron Corporation, Bremen, Germany). IR spectra
were measured with Bruker vector 200 (Bruker). Reagents and
solvents were purchased from common commercial suppliers
and were used without further purification.
7,49-Dimethoxymethyl-5-(3,3-dimethylallyl)-chalcone 8a
Reagent: compound 7a (470 mg, 1.78 mmol), p-methoxymethox-
ylbenzaldehyde (296 mg, 1.78 mmol); Purification: silica gel col-
umn chromatography using petroleum ether : ethyl acetate
(15 : 1). Yellow oil (550 mg, 75%); IR (KBr) m (cm– 1): 3447, 2923,
1635, 1570, 1364 cm– 1. 1H-NMR (CDCl3, 400 MHz, d): 1.78 (s, 6H),
3.31 (d, 2H, J = 6.8 Hz), 3.50 (s, 6H), 5.25 (s, 4H,), 5.31 (m, 1H), 6.67
(s, 1H), 7.11 (d, 2H, J = 8.8 Hz), 7.62 (d, 2H, J = 8.8 Hz), 7.65 (s, 1H),
7.47 (d, 1H, J = 15.6 Hz), 7.86 (d, 1H, J = 15.6 Hz), 13.32(s, 1H, OH).
ESI-MS: m/z 413 [M+H]+.
General method for synthesis of compounds 7a–c
To a cool solution of 6a, (6b or 6c), dry potassium carbonate in
anhydrous acetone, chloromethoxylmethyl ether was added.
After stirring for 2 h at room temperature, the potassium carbo-
nate was filtered off. The filtrate was concentrated to give a resi-
due, which was purified by silica gel column chromatography to
afford 7a, 7b, or 7c.
7,49-Dimethoxymethyl-3-(3,3-dimethylallyl)-chalcone 8b
Reagent: compound 7b (470 mg, 1.78 mmol), p-methoxymethox-
ylbenzaldehyde (296 mg, 1.78 mmol); Purification: silica gel col-
umn chromatography using petroleum ether : ethyl acetate
(15 : 1). Yellow oil (572 mg, 72%); IR (KBr) m (cm– 1): 3433, 2955,
1640, 1574, 1360 cm– 1. 1H-NMR (CDCl3, 400 MHz, d): 1.72 (s, 3H),
1.82 (s, 3H), 3.44 (d, 2H, J = 6.8 Hz), 3.51 (s, 6H), 5.25 (s, 2H), 5.27
(m, 1H), 5.31 (s, 2H), 6.70 (d, 1H, J = 8.4 Hz), 7.08 (d, 2H, J = 8.8 Hz),
7.51 (d, 1H, J = 15.6 Hz), 7.64 (d, 2H, J = 8.8 Hz), 7.80 (d, 1H, J =
8.4 Hz), 7.88 (d, 1H, J = 15.6 Hz), 12.8 (s, 1H, OH). ESI-MS: m/z 413
[M+H]+.
4-Methoxymethyl-5-(3, 3-dimethylallyl)-2-
hydroxylacetonphenone 7a
Reagent: compound 6a (660 mg, 3.0 mmol), chloromethoxyl-
methyl ether (300 mg, 3.73 mmol), dry potassium carbonate
(550 mg, 4.0 mmol), anhydrous acetone (50 mL); Purification:
silica gel column chromatography using petroleum ether : ethyl
acetate (20 : 1). Yellow oil (580 mg, 73%); IR (KBr) m (cm– 1): 3242,
2920, 1636, 1492, 1368 cm– 1. 1H-NMR (CDCl3, 400 MHz, d): 1.74
(s, 6H), 2.54 (s, 3H), 3.28 (d, 2H, J = 6.8 Hz ), 3.47 (s, 3H), 5.24 (s,
2H), 5.27 (m, 1H), 6.60 (s, 1H), 7.44 (s, 1H), 12.51 (s, 1H). ESI-MS: m/
z 265 [M+H]+.
5,7,49-Trimethoxymethyl-3-(3,3-dimethylallyl)-chalcone
8c
4-Methoxymethyl-3-(3, 3-dimethylallyl)-2-
hydroxylacetonphenone 7b
Reagent: compound 7c (500 mg, 1.54 mmol), p-methoxymethox-
ylbenzaldehyde (256 mg, 1.54 mmol); Purification: silica gel col-
umn chromatography using petroleum ether : ethyl acetate
(12 : 1). Yellow oil (552 mg, 71%); IR (KBr) m (cm– 1): 3420, 2940,
1644, 1568, 1359 cm– 1. 1H-NMR (CDCl3, 400 MHz, d): 1.67(s, 3H,),
1.79(s, 3H), 3.34 (d, 2H, J = 6.8 Hz), 3.49 (s, 3H), 3.50 (s, 3H), 3.52 (s,
3H), 5.21 (m, 1H), 5.22 (s, 2H), 5.25 (s, 2H), 5.27 (s, 2H), 6.40 (s,
1H,), 7.06 (d, 2H, J = 8.8 Hz), 7.55 (d, 2H, J = 8.8 Hz), 7.76 (d, 1H, J =
15.6 Hz), 7.83 (d, 1H, J = 15.6 Hz), 13.83 (s, 1H). ESI-MS: m/z 473
[M+H]+.
Reagent: compound 6b (660 mg, 3.0 mmol), chloromethoxyl-
methyl ether (300 mg, 3.73 mmol), dry potassium carbonate
(550 mg, 4.0 mmol), anhydrous acetone (50 mL); Purification:
silica gel column chromatography using petroleum ether : ethyl
acetate (20 : 1). Yellow oil (580 mg, 70%); IR (KBr) m (cm– 1): 3230,
2915, 1628, 1488, 1360 cm-1. 1H-NMR (CDCl3, 400 MHz, d): 1.69 (s,
3H), 1.81 (s, 3H), 2.58 (s, 3H), 3.37 (d, 2H, J = 7.2 Hz), 5.21 (m, 1H),
6.66 (d, 1H, J = 8.8 Hz), 7.60 (d, 1H, J = 8.8 Hz), 12.80 (s, 1H). ESI-
MS: m/z 265 [M+H]+.
4,6-Dimethoxymethyl-3-(3, 3-dimethylallyl)-2-
General method for synthesis of compounds 9a, 9b, and
hydroxylacetonphenone 7c
9c
Reagent: compound 6c (708 mg, 3.0 mmol), methoxymethyl-
chloride (600 mg, 7.45 mmol), dry potassium carbonate (1.1 g,
8.0 mmol), anhydrous acetone (50 mL); Purification: silica gel
A stirred solution of 8a, (8b or 8c) and sodium acetate in 5 mL
ethanol containing three drops of water was refluxed for 24 h.
The mixture was poured into cold water and extracted with
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2007, 340, 372–376
Prenylflavonoids with Estrogenic Activity
375
ethyl acetate. The organic phase was washed with brine, dried,
and filtered. After removing the solvent, the residue was puri-
fied to afford flavanone 9a, 9b, or 9c.
7.45 (s, 1H), 9.55 (s, 1H, OH), 10.60 (s, 1H, OH). ESI-MS: m/z 325
[M+H]+.
(l)-Isobavachin 2
7,49-Dimethoxymethyl-6-(3,3-dimethylallyl)-flavanone 9a
Reagent: compound 8a (500 mg, 1.21 mmol), sodium acetate
(500 mg); Purification: silica gel column chromatography using
petroleum ether : ethyl acetate (10 : 1). Pale yellow syrup
(360 mg, 72%); Purification: silica gel column chromatography
using petroleum ether: ethyl acetate (10 : 1). IR (KBr) m (cm– 1):
2960, 1682, 1574, 1238 cm– 1. 1H-NMR (CDCl3, 400 MHz, d): 1.73,
1.76 (3H, each s), 2.81 (dd, 1H, J = 16.8 Hz, 0.8 Hz), 3.03 (d, 1H, J =
12.0 Hz, 16.8 Hz), 3.29 (d, 2H, J = 7.2 Hz), 3.49 (s, 6H), 5.20-5.29 (m,
5H ), 5.41 (dd, 1H, J = 12.0 Hz, 0.8 Hz), 6.70 (s, 1H), 7.10 (d, 2H, J =
8.4 Hz), 7.41(d,2H, J = 8.4 Hz), 7.72 (s, 1H). ESI-MS: m/z 413 [M+H]+.
Reagent: compound 9b (300 mg, 0.73 mmol); Purification: silica
gel column chromatography using petroleum ether : ethyl ace-
tate (4 : 1). White amorphous powder (165 mg, 70%), mp.: 188-
1890C (lit. [26] mp. 187-1880C); IR (KBr) m (cm– 1): 3266, 2966,
1646, 1520, 1441 cm– 1. H-NMR (DMSO-d6, 400 MHz, d): 1.64 (s,
1
3H), 1.67 (s, 3H), 2.75 (dd, 1H, J = 16.8 Hz, 0.8 Hz), 3.12 (d, 1H,
J =12.0 Hz, 16.8 Hz), 3.26 (d, 2H, J = 7.0 Hz), 5.20 (t, 1H, J = 7.0 Hz),
5.50 (dd, 1H, J = 12.0 Hz, 0.8 Hz), 6.64 (d, 1H, J = 8.8 Hz), 6.86 (d,
2H, J = 8.4 Hz), 7.39 (d, 2H, J = 8.4 Hz), 7.58 (d, 1H, J = 8.8 Hz), 9.63
(s, 1H), 10.53 (s,1H). ESI-MS: m/z 325 [M+H]+.
(l)-8-Prenylnaringenin 10c
7,49-Dimethoxymethyl-8-(3,3-dimethylallyl)-flavanone 9b
Reagent: compound 8b (500 mg, 1.21 mmol), sodium acetate
(500 mg); Purification: silica gel column chromatography using
petroleum ether : ethyl acetate (10 : 1). Pale yellow syrup
(373 mg, 70%); IR (KBr) m (cm– 1): 2965, 1675, 1568, 1250 cm– 1. 1H-
NMR (CDCl3, 400 MHz, d): 1.67 (s, 3H), 1.78 (s, 3H), 2.70 (d, 1H, J =
16.8 Hz), 3.07 (d, 1H, J = 12.0 Hz, 16.8 Hz), 3.21 (d, 2H, J = 7.2 Hz),
3.40(s, 3H), 3.40 (s, 3H), 5.15 (t, 1H, J = 7.2 Hz), 5.28 (s, 2H), 5.35 (s,
2H), 5.47 (d, 1H, J = 12.0 Hz), 6.60 (d, 1H, J = 8.8 Hz), 6.83 (m, 2H, J
= 8.4 Hz), 7.37 (m, 2H, J = 8.4 Hz), 7.55 (d, 1H, J = 8.8 Hz). ESI-MS:
m/z 413 [M+H]+.
Reagent: compound 9c (300 mg, 0.64 mmol); Purification: silica
gel column chromatography using petroleum ether : ethyl ace-
tate (5 : 1). White amorphous powder (98 mg, 72%), mp.: 183–
1848C (lit. [27], mp. 183–1848C); IR (KBr) m (cm– 1): 3334, 2964,
1
1654, 1524, 1441 cm-1. H-NMR (DMSO-d6, 400 MHz, d): 1.70 (s,
3H), 1.77 (s, 3H), 2.68 (dd, 1H, J = 16.4 Hz, 0.8 Hz ), 2.92 (d, 1H, J =
12.0 Hz, 16.4 Hz), 3.30 (d, 2H, J = 7.0 Hz), 5.08 (m, 1H, J = 7.0 Hz),
5.41 (dd, 1H, J = 12.0 Hz, 0.8 Hz), 5.93 (s, 1H), 7.05 (d, 2H, J =
8.0 Hz), 7.35 (d, 1H, J = 8.0 Hz), 10.38 (s, 1H), 10.65 (s, 1H), 12.31 (s,
1H). ESI-MS: m/z 341 [M+H]+.
General method for synthesis of compounds 3 and 4
A stirred solution of corresponding prenylflavonone and iodine
in dry pyridine (4 mL) was heated to 908C for 6 h. The mixture
was cooled and poured into cold water. The precipitate was sepa-
rated and the mixture was extracted with ethyl acetate. The com-
bined organic phases were washed with saturated sodium thio-
sulfate and water, successively. Then, the organic layer was dried
with sodium sulfate and concentrated. The residue was purified
by silica gel column chromatography using eluent mixtures of
solvents in the proportions indicated for each case.
5,7,49-Trimethoxymethyl-8-(3,3-dimethylallyl)-flavanone
9c
Reagent: compound 8c (500 mg, 1.06 mmol), sodium acetate
(500 mg); Purification: silica gel column chromatography using
petroleum ether: ethyl acetate (8 : 1). Pale yellow syrup (350 mg,
1
70%); IR (KBr) m (cm– 1): 2980, 1655, 1565, 1245 cm– 1. H-NMR
(CDCl3, 400 MHz, d): 1.64 (s, 3H), 1.65 (s, 3H), 2.80 (dd, 1H, J =
16.4 Hz, 2.8 Hz), 2.98 (dd, 1H, J = 13.4 Hz, 16.4 Hz), 3.31 (d, 2H, J =
6.8 Hz), 3.48 (s, 3H), 3.49 (s, 3H), 3.54 (s, 3H), 5.17 (m, 1H), 5.20 (s,
2H), 5.24 (s, 2H), 5.26 (s, 2H), 5.36 (dd, 1H, J = 2.8 Hz, 13.4 Hz), 6.57
(s, 1H), 7.08 (d, 2H, J = 8.4 Hz), 7.38 (d, 1H, J = 8.4 Hz). ESI-MS: m/z
473 [M+H]+.
7,49-Dihydroxy-8-prenylflavone 3
Reagents: isobavachin 2 (100 mg, 0.31 mmol), iodine (78 mg,
0.31 mmol). Purification: silica gel column chromatography
using petroleum ether : ethyl acetate (1 : 1). Light yellow solid
(76 mg, 76%), mp.: 240–2418C (lit. [27], mp. 240–2418C); IR (KBr)
General method for synthesis of compounds 1, 2, and 10c
To a solution of 9a, (9b or 9c) in methanol (10 mL), 3 N HCl (2 mL)
was added. The resulting mixture was refluxed for 45 min, then
poured into cold water and extracted with ethyl acetate. The
organic phase was washed with saturated NaCl solution and
then dried (Na2SO4). After removal of the solvent, the residue was
chromatographed over silicon gel. Elution with petroleum
ether-ethyl acetate gave 1, 2, or 10c.
1
m (cm– 1): 3197, 2923, 1656, 1507, 1422 cm– 1. H-NMR (DMSO-d6,
400 MHz, d): 1.65 (s, 3H), 1.79(s, 3H), 3.58 (d, 2H, J = 5.6 Hz), 5.24
(m, 1H), 6.72 (s, 1H), 6.93 (d, 2H, J = 8.4 Hz), 6.97(d, 1H, J = 8.4 Hz),
7.74 (d, 1H, J = 8.4 Hz), 7.89 (d, 2H, J = 8.4 Hz), 10.28(s, 1H),
10.65(s, 1H). ESI-MS: m/z 323 [M+H]+.
8-C-Prenylapigenin 4
(l)-Bavachin 1
Reagents: 8-C-prenylnaringenin 10c (100 mg, 0.29 mmol), iodine
(75 mg, 0.29 mmol). Purification: silica gel column chromatog-
raphy using petroleum ether : ethyl acetate (1 : 1). Light yellow
solid (78 mg, 80%), mp. 239–2408C (lit. [28], mp. 173–1748C,
decomposed); IR (KBr) m (cm– 1): 3385, 3197, 2925, 1622, 1508,
Reagent: compound 9a (300 mg, 0.73 mmol); Purification: silica
gel column chromatography using petroleum ether: ethyl ace-
tate (4 : 1). White amorphous powder (180 mg, 76%), mp.: 184-
1850C (lit. [25] mp. 1890C); IR (KBr) m (cm– 1): 3126, 2961, 1654,
1518, 1461 cm– 1.1H-NMR (DMSO-d6, 400 MHz, d): 1.66(s, 3H), 1.71
(s, 3H), 2.63 (dd, 1H, J = 16.8 Hz, 0.8 Hz), 3.03 (d, 2H, J = 7.2 Hz),
3.17 (d, 1H, J =12.0, 16.8 Hz), 5.26 (m,1H), 5.40 (dd, 1H, J = 12.0,
0.8 Hz), 6.38 (s, 1H), 6.78 (d, 2H, J = 8.4 Hz), 7.31 (d, 2H, J = 8.4 Hz),
1
1435 cm– 1. H-NMR (DMSO-d6, 400 MHz, d): 1.62 (s, 3H), 1.75 (s,
3H), 3.43 (d, 2H, J = 6.4 Hz), 5.19 (m,1H), 6.28 (s, 1H), 6.77 (s, 1H),
6.93 (d, 2H, J = 8.0 Hz), 7.89 (d, 2H, J = 8.0 Hz), 10.38 (s, 1H), 10.79
(s, 1H), 12.90 (s, 1H). ESI-MS: m/z 339 [M+H]+.
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

376
X. Dong et al.
Arch. Pharm. Chem. Life Sci. 2007, 340, 372–376
[8] W. Tsai, W. Hsin, C. Chen, J. Nat. Prod. 1996, 59, 671–672.
Biological assay
Cell culture
[9] S. I. Ayabbe, M. Kobayashi, Phytochemistry 1980, 19, 2179–
Estrogen receptor-positive human breast adenocarcinoma MCF-
7/BOS cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) containing antibiotics (100 IU/mL– 1 penicillin
and 100 Ag/mL– 1 streptomycin), supplemented with 10% fetal
bovine serum (FBS). Cells were grown at 378C in a humidified
atmosphere of 5% CO2 in air. Medium was renewed 2–3 times
per week.
2183.
[10] J. Guo, X. Weng, H. Wu, Q. Li, K. Bi, Food Chem. 2005, 91,
287–292.
[11] S. Yin, C. Fan, Y. Wang, L. Dong, J. Yue, Bioorg. Med. Chem.
2004, 12, 4387–4392.
[12] A. C. Jain, P. Lal, T. R. Seshadri, Indian J. Chem. 1969, 7,
1072.
[13] L. Yu, Y. Hu, zhongguo yaoxue zazhi 2004, 40, 1029–1031.
Proliferation assay of cell
Confluent MCF-7/BOS cells were washed twice with D-Hanks sol-
ution before the addition of 0.25% trypsin–EDTA and seeded
into 24-well plates at a density of 16104 cells/well in normal
growth medium. After 48 h, the cells were washed with D-Hanks
solution and the estrogen-free medium (phenol red-free DMEM
with 5% charcoal-dextran stripped human serum) was added.
Following another 48 h pretreatment, the cells were exposed to
increasing concentrations of test compounds. Cell proliferation
was assessed by MTT method after seven days, during which the
medium was changed every three days. The results are expressed
as proliferation compared, with the treatment by 1 nM 17b-
estradiol as reference.
[14] C. L. Miranda, G. L. M. Aponso, J. F. Stevens, M. L. Deinzer,
D. R. Buhler, Cancer Lett. 2000, 149, 21–29.
[15] B. F. Rasulev, N. D. Abdullaev, V. N. Syrov, J. Leszczynski,
QSAR Comb. Sci. 2005, 24, 1056–1065.
[16] K. Fujimoto, I. Mikoshiba, N. Tanaka, Y. Iwano, Y. Iijima,
M. Taki, JP 09301915. 1997.
[17] J. Daskiewicz, F. Depeint, L. Viornery, C. Bayet, et al., J.
Med. Chem. 2005, 48, 2790–2804.
[18] K. Zou, Y. Zhao, J. Chin. Pharm. Sci. 1996, 5, 182–185.
[19] D. Prakash, S. Suri, Indian J. Agr. Biochem. 2005, 18, 1–8.
[20] A. L. Ososki, E. J. Kennelly, Phytother. Res. 2003, 17, 845–
869.
[21] S. F. Fan, P. E. Jonathan, Phytochemistry 2004, 65, 1317–
References
1330.
[22] R. A. Diller, H. M. Riepl, O. Rose, C. Frias, et al., Chem.
Biodivers. 2005, 2, 1331–1337.
[1] K. Jiyamas, A. Emizuy, U. Iraga, J. Nat. Prod. 1992, 55,
1197–1203.
[23] B. M. Collins-Burow, M. E. Burow, B. N. Duong, J. A. McLa-
[2] W. R. Phillips, N. J. Baj, A. A. Leslie Gunatilaka, David G. I.
chlan, Nutr. Cancer 2000, 38, 229–244.
Kingston, J. Nat. Prod. 1996, 59, 495–497.
[24] J. C. L. Bail, F. Varnat, J. C. Nicolas, G. Habrioux, Cancer
Lett. 1998, 130, 209–216.
[3] M. Chung, C. Lu, P. Huang, C. Lin, Phytochemistry 1995, 40,
1279–1282.
[25] J. W. Critchfied, C. J. Welsh, Biochem. Pharmacol. 1994, 48,
[4] H. Y. Sohn, K. H. Son, C. S. Kwon, G.-S. Kwon, S. S. Kang,
1437–1445.
Phytomedicine 2004, 11, 666–672.
[26] D. E. Lincoln, M. D. Walla, Biochem. Syst. Ecol. 1986, 14,
[5] W. Waetjen, N. Weber, Y.-J. Lou, Z.-Q. Wang, et al., Food
Chem. Toxicol. 2007, 45, 119–124.
195–198.
[27] B. Cheon, H. Young, Planta Med. 2000, 66, 596–600.
[28] D. Wang, R. Li, Z. Jiang, Planta Med. 2001, 67, 748–749.
[6] G. Comte, J.-B. Daskiewicz, C. Bayte, G. Conseil, et al., J.
Med. Chem. 2001, 44, 763–768.
[7] V. K. Bhalla, U. R. Nayak, S. Dev, Tetrahedron Lett. 1968, 20,
[29] N. Al-Maharik, N. P. Botting, Tetrahedron 2003, 59, 4177–
2401–2406.
4181.
i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com