A simple synthesis of 7,4′-dihydroxy-6-methoxyisoflavone, glycitein, the third soybean isoflavone

Article abstract of DOI:10.1021/np020320r

4-Methoxyresorcinol (3) was synthesized as the precursor for glycitein (6) synthesis by the oxidation of 3-hydroxy-4-methoxybenzaldehyde (1) to the aryl formate with H₂O₂ and a catalytic amount of SeO₂. Glycitein (6) was synthesized by cyclization of 2,4,4′-trihydroxy-5-methoxydeoxybenzoin (5) with N,N-dimethylformamide, boron trifluoride diethyl ether, and methanesulfonyl chloride in a microwave oven.

Full text of DOI:10.1021/np020320r

J . Nat. Prod. 2003, 66, 149-151
149
A Sim p le Syn th esis of 7,4′-Dih yd r oxy-6-m eth oxyisofla von e, Glycitein , th e Th ir d
Soybea n Isofla von e
Caroline Lang’at-Thoruwa,† Tong T. Song,‡ J iang Hu,‡ Andrean L. Simons,‡ and Patricia A. Murphy*,‡
Department of Chemistry, Kenyatta University, Nairobi, Kenya, and Department of Food Science and Human Nutrition,
2312 Food Sciences Bldg., Iowa State University, Ames, Iowa 50011
Received J uly 12, 2002
4-Methoxyresorcinol (3) was synthesized as the precursor for glycitein (6) synthesis by the oxidation of
3-hydroxy-4-methoxybenzaldehyde (1) to the aryl formate with H2O2 and a catalytic amount of SeO2.
Glycitein (6) was synthesized by cyclization of 2,4,4′-trihydroxy-5-methoxydeoxybenzoin (5) with N,N-
dimethylformamide, boron trifluoride diethyl ether, and methanesulfonyl chloride in a microwave oven.
The soy isoflavones, daidzein, genistein, and glycitein (6)
(Figure 1), and their glucosides are associated with im-
portant health protective properties including cancer pre-
vention, plasma cholesterol lowering when associated with
soy protein, and reduction of postmenopausal bone loss.1
Daidzein and genistein were isolated and identified from
soybean flour by Walter,2 and glycitein (6) was first isolated
from a concentrated ethanol extract of defatted soybeans
by Naim et al.3 Although numerous studies have evaluated
the biological activities of daidzein and genistein, the
biological activity of glycitein (6) only recently was reported
by Song et al.4 They showed that glycitein has weak
estrogenic activity comparable to that of daidzein and
genistein. On an equal mole basis, glycitein (6) actually
F igu r e 1. Structures of soybean isoflavone aglucons genistein,
daidzein, and glycitein.
has a stronger estrogenic response than genistein and
daidzein in the mice uterine enlargement assay. Zhang et
al.5 reported that glycitein (6) was more bioavailable than
genistein, and glycitein (6), we proposed that glycitein could
be synthesized by using a pathway similar to the microwave-
daidzein in humans. Therefore, although glycitein accounts
for only 5-10% of the total isoflavones in soybeans, its
mediated synthesis of daidzein and genistein,7 if the
biological activities and potential health effects cannot be
appropriate reagents were available. Since one of the
neglected. Soy germ contains approximately 10 times the
required starting reagents is not commercially available
concentration of isoflavones compared to the rest of the
for glycitein (6) synthesis, in contrast to those required for
genistein and daidzein, synthesis of the missing glycitein
soybean seed. Soy germ or hypocotyls contain significant
concentrations of the glycitein forms that have lead to
precursor was necessary and is reported here along with
development of nutraceutical supplements derived from soy
the synthesis of glycitein (6). Although others have reported
synthesis of 4-methoxyresorcinol (3),9 no one has reported
glycitein (6) synthesis using the cyclization method of
Chang et al.7
4-Methoxyresorcinol (3) is the required intermediate to
synthesize glycitein (6) using the microwave-mediated
synthesis of Chang et al.7 (Figure 2). It was prepared by
the oxidation of 3-hydroxy-4-methoxybenzaldehyde (1) to
the aryl formate (2) with H2O2 and a catalytic amount of
SeO2 in methylene chloride (CH2Cl2). Treatment of 2 with
K2CO3 in MeOH at room temperature yielded 3. Guzman
germ. However, the ratio of genistein/daidzein/glycitein in
germ is 1:5:4, in contrast to the ratio in soybean seeds, 5:4:
1.6
To evaluate the biological activities of these isoflavones,
a stable and inexpensive source of these phytochemicals
is desirable. Isolation of isoflavones from soybean products
is time-consuming and produces low yields. Chemical
synthesis is a practical way to produce these compounds
in large, high-purity quantities. A simple, high-yield syn-
thesis approach using conventional microwave ovens to
produce genistein and daidzein was reported by Chang et
et al.9 reported a 75% yield over a 12 h oxidation reaction
al.7 and modified by Song et al.6 Chemical synthesis of
in tert-butyl alcohol for 3. They provided no other spectral
glycitein (6) was reported in 1995 by No´gra´di and Szo¨llo¨sy8
information to compare with our data. Our reaction scale
using an oxidative rearrangement of the fully protected
was about 10 times larger than that of Guzman et al.9
corresponding chalcone by TI (NO3)3 in methanol (MeOH)
Hauer et al.10 report NMR spectra similar to our data and
report 3 as an oil.
4-Methoxyresorcinol (3) was refluxed with 4-hydroxy-
phenylacetic acid (4) and boron trifluoride etherate
(BF3‚Et2O) to furnish compound 5, 2,4,4′-trihydroxy-5-
methoxydeoxybenzoin (TMD). Cyclization of TMD was
achieved by placing a mixture of TMD, N,N-dimethyl-
followed by deprotection and ring closure. However, their
procedure to synthesize glycitein (6) involved multistep
protection and deprotection, making the synthetic process
complicated and hard to perform for the nonorganic chem-
ist. Because of the structural similarity between daidzein,
* To whom correspondence should be addressed. Tel: 515-294-1970.
Fax: 515-294-8181. E-mail: pmurphy@iastate.edu.
formamide (DMF), and BF3‚Et2O under medium microwave
energy for 21 s before adding methane sulfonyl chloride
† Kenyatta University.
‡ Iowa State University.
10.1021/np020320r CCC: $25.00
© 2003 American Chemical Society and American Society of Pharmacognosy
Published on Web 12/13/2002

150 J ournal of Natural Products, 2003, Vol. 66, No. 1
Notes
0.44 g). The reaction mixture was stirred at room temperature
in a water bath for 16 h. The mixture was filtered through
Whatman No. 42 paper, and the filtrate was washed with 100
mL of water. The organic layer was separated and washed with
100 mL of 10% NaHSO3, followed by 100 mL of 10% Na2CO3,
and dried over anhydrous Na2SO4. The residue obtained after
evaporation of the solvents was dissolved in 30 mL of MeOH.
Then, 25 mL of a 14% solution of K2CO3 in water was added.
The mixture was stirred at room temperature for 1 h, and the
solvent was removed using a rotary evaporator. To the residue,
50 mL of water was added and extracted with ethyl ether (2
× 60 mL). The aqueous layer was saturated with NaCl and
extracted again with ethyl ether (2 × 40 mL). The combined
ether layers were washed with 200 mL of saturated NaCl and
dried over anhydrous Na2SO4. The ether was removed by
evaporation to give 0.034 mol (4.7818 g) (68%) of 4-methoxy-
resorcinol (3) as a clear, yellow oil: M+ 140 [EI 140(96%),
125(100%), 97(57%), 73(20%), 61(25%), 45(21%)]; 1H NMR (d6-
DMSO, 300 MHz) δ 3.67 (s, 3H, OCH3); 6.17 (d, J ) 8.5 Hz,
1H, 6-H); 6.35 (s, 1H, 2-H); 6.67 (d, J ) 8.6 Hz, 1H, 5-H); 8.80
(s, 1H, OH); 8.77 (s, 1H, OH).
2,4,4′-Tr ih yd r oxy-5-m eth oxyd eoxyben zoin (TMD) (5).
4-Methoxyresorcinol (3) (0.0157 mol, 2.1975 g) was added to
a mixture containing 4-hydroxyphenylacetic acid (4) (0.0148
mol, 2.2521 g) and BF3‚Et2O (4.5 mL). The reaction mixture
was refluxed for 10 min and cooled, and 60 mL of water was
added; the aqueous layer was extracted with ethyl ether (3 ×
50 mL). The combined ether layers were washed with satu-
rated aqueous sodium acetate (30 mL) and saturated NaHCO3
(15 mL), respectively. The layers were separated, and the ether
layer was dried with anhydrous Na2SO4. Removal of ether by
evaporation gave 0.01135 mol (3.1106 g) (77%) of a dark yellow
oil of 2,4,4′-trihydroxy-5-methoxydeoxybenzoin (5), C15O5H14
(MW 274): MS spectrum showed M+ 274; 1H NMR (d6-DMSO,
400 MHz) δ 3.75 (s, 3H, OCH3); 4.15 (s, 2H, -CH2); 6.30 (s,
1H, 5-H); 6.68 (d, J ) 11.2 Hz, 2H, 2′,6′-H); 7.07 (d, J ) 11.2
Hz, 2H, 3′,5′-H); 7.39 (s, 1H, 8-H).
Glycitein (6). TMD (5) (1.55 mmol, 0.4247 g) was dissolved
in 8 mL of DMF in a 500 mL glass beaker. BF3‚Et2O (4 mL)
was added, and a vigorous exothermic reaction occurred. The
reaction mixture was heated in microwave oven for 21 s using
40% energy. Then 4 mL of methanesulfonyl chloride was added
to the beaker, and the mixture was heated for 70 s in the
microwave oven using 40% energy. Addition of cold water (400
mL) led to the formation of a dark yellow precipitate. The
supernatant and precipitate were extracted separately with
ethyl ether (3 × 60 mL), the ethyl ether fractions were
combined, and the ether fraction was dried with anhydrous
Na2SO4. Evaporation of ether afforded 7,4′-dihydroxy-6-meth-
oxyisoflavone (glycitein) (6) as pale yellow crystals. Glycitein
(6) was recrystallized from 80% methanol, yielding 0.381 mmol
(0.1082 g) (26%), C16O5H12 (MW 284): MS spectrum showed
M+ 284 [EI 284 (100%), 283(42%), 167 (11%), 166(24%); 1H
NMR (d6-DMSO, 300 MHz) δ 3.86 (s, 3H, OCH3); 6.78 (d, J )
8.7 Hz, 2H, 3′,5′-H); 6.93 (s, 1H, 8-H), 7.36 (d, J ) 8.4 Hz, 2H,
2′,6′-H); 7.42 (s, 1H, 5-H), 8.27 (s, 1H, 2-H); mp 337-339 °C,
uncorrected; λmax (methanol) 257, 319 nm; λmax (sodium meth-
oxide) 259, 344 nm; λmax (AlCl3) 257, 317 nm; λmax (AlCl3-HCl)
257, 317 nm; λmax (sodium acetate) 255, 346 nm; λmax (sodium
acetate-H3BO3) 255, 320 nm.
F igu r e 2. Reaction scheme for synthesis of 4-methoxyresorcinol
followed by cyclization to glycitein.
and heating it for an additional 70 s under medium
microwave energy. Pale yellow crystals of glycitein (6) were
obtained after recrystallization from 80% MeOH. The UV
spectra, HPLC, mass spectrum, and 1H NMR conformed
to those reported for authentic glycitein.3,4,8,11 The melting
point for our synthesized glycitein (337-339 °C) compared
well with the melting point reported for glycitein synthe-
sized by No´gra´di and Szo¨llo¨sy8 (337-339 °C) and for
glycitein isolated from soy germ by Song et al.4 (337-339
°C).
The yield of glycitein (6) by this microwave-mediated
method was 26%, which compared well with the 36% yield
reported by Chang et al.7 for genistein but lower than the
55% yield of Nogradi and Szollosy.7 We routinely produce
glycitein in a one-pot synthesis style similar to Balasubra-
manian and Nair,12 although they did not report an
attempt to produce glycitein. We isolated and evaluated
the intermediates for the purposes of this paper to confirm
we were producing the correct compounds. However, for
routine work, this is not necessary. The one-pot synthesis
method can be routinely scaled up to produce gram
quantities of glycitein for biological studies.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. All chemicals used
in the synthesis were purchased from Sigma Chemical Com-
pany (St. Louis, MO). A Kenmore U88-332 (1350 W) microwave
oven was used in the cyclization step. The identification and
purity of glycitein were confirmed by using HPLC, UV spectral
analysis, melting point, mass spectrum, and NMR. Glycitein
was analyzed by using HPLC on a Beckman System Gold
chromatography system including a Model 507 autosampler,
Model 126 dual pumps, a Model 168 photodiode array detector,
and an IBM 486 computer with Beckman Gold System HPLC
data processing software (version 8, 1993) according to Murphy
et al.11 UV spectral analysis was performed according to Mabry
et al.13 using a Beckman DU 7400 spectrophotometer. The
melting point of glycitein was measured with a Perkin-Elmer
7 series differential scanning calorimeter (DSC) (Perkin-Elmer
Inc., Norwalk, CT). Mass spectral analysis, using chemical
ionization, was performed on a Finnigan Model TSQ-700 mass
spectrometer (Finnigan Inc., Piscataway, NJ ). 1H NMR was
performed in deuterated DMSO on a Bruker DRX 400 MHZ
for 5 and a Varian VXR 300 MHz for 3 and 6.
Ack n ow led gm en t. We thank Cassie Thoen Keppel and
Michelle Yan for independently confirming the synthetic
protocol as part of their Ph.D. rotation. This article was
supported in part by USDA Fund for Rural America Grant
No. 97-362155190 and by the Iowa Agricultural and Home
Economics Experiment Station and published as J -19775,
project 3526.
Refer en ces a n d Notes
(1) Murphy, P. A.; Hendrich, S. Adv. Food Nutri. Res. 2002, 44, 195-
246.
(2) Walter, E. D. J . Am. Chem. Soc. 1941, 63, 3273-3276.
(3) Naim, M.; Gestetner, B.; Kirson, I.; Birk, Y.; Bondi, A. Phytochemistry
1973, 12, 169-170.
4-Meth oxyr esor cin ol (3). 3-Hydroxy-4-methoxybenzalde-
hyde (1) (0.05 mol, 7.603 g) was added to a mixture of CH2Cl2
(100 mL), 0.11 mol of H2O2 (13 mL), and SeO2 (3.96 mmol,

Notes
(4) Song, T. T.; Hendrich, S.; Murphy, P. A. J . Agric. Food Chem. 1999,
J ournal of Natural Products, 2003, Vol. 66, No. 1 151
(10) Hauer, H.; Ritter, T.; Grotemeier, G. Arch. Pharm. (Weinheim) 1995,
328, 737-738.
47, 1607-1610.
(5) Zhang, Y.; Wang, G. J .; Song, T. T.; Murphy, P. A.; Hendrich, S. J .
Nutr. 2001, 131, 147-148.
(11) Murphy, P. A.; Song, T. T.; Buseman, G.; Barua, K. J . Agric. Food
Chem. 1997, 45, 4635-4638.
(6) Song, T.; Barua, K.; Buseman, G.; Murphy, P. Am. J . Clin. Nutr. 1998,
68 (Suppl.), 1474S-1479S.
(12) Balasubramanian, S.; Nair, M. G. Synth. Commun. 2000, 30, 469-
484.
(7) Chang, Y.; Nair, M.; Santell, R.; Helferich, W. J . Agric. Food Chem.
1994, 42, 1869-1871.
(13) Mabry, T. J .; Markham, K. R.; Thomas, M. B. In The Systematic
Idenification of Flavanoids; Springer-Verlag: New York, 1970; pp
165-180.
(8) Nogradi, M.; Szollosy, A. Liebigs Ann. 1996, 1651-1652.
(9) Guzman, J . A.; Mendoza, V.; Garcia, E.; Garibay, C. F.; Olivares, L.
Z.; Maldonado, L. A. Synth. Commun. 1995, 25, 2121-2133.
NP020320R