The synthesis of [2,3,4-13C3]glycitein

Article abstract of DOI:10.1016/j.tet.2004.10.028

Glycitein is one of the soy isoflavones which have attracted considerable interest in recent years due to their possible beneficial effects on human health. However, glycitein has been much less studied than other members of this family due to the lack of

Full text of DOI:10.1016/j.tet.2004.10.028

Tetrahedron 60 (2004) 12211–12216
The synthesis of [2,3,4-13C3]glycitein
*
Qingzhi Zhang and Nigel P. Botting
School of Chemistry, University of St Andrews, St Andrews, Fife KY16 9ST, Scotland, UK
Received 9 August 2004; revised 13 September 2004; accepted 7 October 2004
Available online 28 October 2004
Abstract—Glycitein is one of the soy isoflavones which have attracted considerable interest in recent years due to their possible beneficial
effects on human health. However, glycitein has been much less studied than other members of this family due to the lack of good methods
for its synthesis. Herein we report a short high yielding synthesis of a multiply 13C-labelled glycitein derivative, [2,3,4-13C3]glycitein, which
has been employed as an internal standard in LC–MS analysis. A key feature is a rapid and efficient synthesis of 2,4-dihydroxy-5-methoxy-
[10,20-13C2]acetophenone via acetylation of isovanillin with [13C2]acetyl chloride followed by a Baeyer–Villiger reaction, selective
hydrolysis and finally a BF3 catalysed Fries rearrangement. An aldol reaction using 4-benzyloxy-[carbonyl-13C]benzaldehyde gave a
chalcone and then thallium(III) mediated oxidative rearrangement, deprotection and cyclisation provided the [2,3,4-13C3]glycitein. The
overall yield for the 8 step reaction sequence, based on [13C2]acetyl chloride, was 57%.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
efficient synthesis of an isotopically labelled derivative,
which would be of great value as an internal standard for
analysis and for metabolic studies. We have previously
synthesised a number of [2-13C]isoflavones,4,5 which have
been employed in metabolic studies on menopausal women6
and a number of multiply 13C-labelled phytoestrogens such
as [2,3,8-13C3]daidzein,7 which have been fully validated
as internal standards for analysis by both LC–MS8,9 and
GC–MS10 methods. For isoflavone type structures a
minimum of three extra mass units is required to produce
an optimum internal standard which has a large enough
mass difference to nullify the effect of natural abundance
heavy isotopes in the analyte.7 Herein we describe the
synthesis of multiply 13C-labelled glycitein derivative,
which allows glycitein to be added to the range of analytes
with its own 13C-labelled internal standard. The synthesis
involves a Baeyer–Villiger reaction followed by a key
Fries rearrangement to generate one of the 13C-labelled
precursors and a thallium(III) mediated oxidative rearrange-
ment of a chalcone to construct the isoflavone.
The soy isoflavones, including daidzein 1 (R]H), genistein
1 (R]OH) and glycitein 2, and their glucosides are
associated with important health protective properties
including anti-cancer activity, plasma cholesterol reduction
and reductions in postmenopausal bone loss.1 There have
been many studies on the biological activity of daidzein and
genistein, whereas the first studies on glycitein have
appeared only recently.2 Glycitein was shown to have
weak estrogenic activity comparable to that of daidzein and
genistein, although on an equal mole basis glycitein actually
produced a stronger estrogenic response than genistein and
daidzein in the mice uterine enlargement assay. It has also
been found that glycitein is actually more bioavailable than
daidzein in humans.3 Therefore, although glycitein accounts
for only 5–10% of the total isoflavones in soybeans, its
biological activities and potential health effects cannot be
neglected.
2. Results and discussion
The general synthetic route towards the 13C-labelled
isoflavones in our previous work (Scheme 1) involved the
condensation of an appropriate phenol 3 with either a
substituted phenylacetic acid 4 or a benzyl nitrile 5 to give a
deoxybenzoin 6 which can then undergo formylation and
finally cyclisation to give the isoflavonoid 7.4,5,7
In order to better understand, quantify and evaluate the
biological activities of glycitein, there is a need for an
Keywords: Isoflavones; Glycitein; Phytoestrogens; 13C-labelling.
* Corresponding author. Tel.: C44 1334 463856; fax: C44 1334 463808;
e-mail: npb@st-andrews.ac.uk
This methodology worked well for daidzein and genistein,
and their methylated derivatives formononetin and
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.10.028

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Q. Zhang, N. P. Botting / Tetrahedron 60 (2004) 12211–12216
available, our methodology would allow the rapid synthesis
of 2,4-dihydroxy-5-methoxy-[10,20-13C2]acetophenone 11
which could then be incorporated into the glycitein. Using
protected 4-hydroxy-[carbonyl-13C]benzaldehyde 12 as the
other building block would afford [2,3,4-13C3]glycitein 13
(Scheme 3).
Scheme 1. Deoxybenzoin route to isoflavones.
biochanin A and the 13C-atoms were incorporated into
either or both fragments. It was thus decided to adopt a
similar approach for the glycitein synthesis, especially as
recent work reported the preparation of glycitein in 26%
yield using a microwave-mediated cyclisation of the
corresponding deoxybenzoin.11 The required phenol, 4-
methoxyresorcinol, however, is not commercially available.
An initial synthesis involving the hydrolysis of the
diazonium salt of 5-amino-2-methoxyphenol was found to
be very poor yielding and gave a difficult to purify
product.12 An alternative procedure is the Baeyer–Villiger
oxidation of isovanillin.13–15 Using a literature procedure15
the hydroxy group of isovanillin 8 was first protected by
acetylation with acetic anhydride and oxidative rearrange-
ment of the aldehyde to formate group carried out using
mCPBA (Scheme 2). Partial hydrolysis of the oxidised
product in EtOH–5% aq NaHCO3 gave 3-acetoxy-4-
methoxyphenol 9 in good yield. Further hydrolysis of
3-acetoxy-4-methoxyphenol in aq ammonia led to
4-methoxyrecinol as expected.
Scheme 3. Retrosynthesis for [2,3,4-13C3]glycitein.
Firstly the required 13C-labelled benzaldehyde, was
prepared by a simple two-step procedure. Firstly cyanation
of 4-benzyloxyiodobenzene 14 using potassium
[13C]cyanide and a palladium(II) acetate catalyst in DMF
under basic conditions7 (calcium hydroxide) introduced the
13C-atom. Reduction of the aromatic nitrile 15 with
DIBAL-H. then gave 4-benzyloxy-[carbonyl-13C]benzalde-
hyde 16 (Scheme 4).
Scheme 4. (i) K13CN, Ca(OH)2, Pd(OAc)2, DMF; (ii) DIBAL-H, THF.
The presence of the 13C atoms in the labelled compounds
was demonstrated using a combination of 13C NMR
spectroscopy, IR spectroscopy and mass spectrometry. In
the case of 4-benzyloxybenzo[13C]nitrile an enhanced
signal was clearly observed in the 13C NMR spectrum at
119.3 ppm and the mass spectrum showed the expected
increase of one mass unit compared to the unlabelled
compound giving a molecular ion at 210 mass units. In the
IR spectrum the effect of 13C-labelling was also visible. The
characteristic nitrile vibration is shifted due to the effect of
Scheme 2. (i) Ac2O, (90%); (ii) mCPBA, CH2Cl2; (iii) EtOH–5% aq
NaHCO3 (86% over two steps); (iv) BF3$Et2O, 70 8C.
In an attempt to shorten the synthesis the 3-acetoxy-4-
methoxyphenol was employed directly in the coupling
reaction to form the deoxybenzoin, by reaction with
4-hydroxyphenylacetic acid in the presence of BF3$OEt2
at 70 8C. However, no trace of the expected deoxybenzoin
was found and the only isolated product was 2,4-dihydroxy-
5-methoxyacetophone. It was then discovered that treatment
of 3-acetoxy-4-methoxyphenol with BF3$OEt2 gave 2,4-
dihydroxy-5-methoxyacetophone 10 in quantitative yield
implying that a Fries rearrangement had taken place.
the increase in mass, such that n
is observed at
13C^N
2169 s cmK1 in 15 compared with nC^N 2221 s cmK1 in the
unlabelled molecule. In the 4-benzyloxybenzaldehyde a
similar effect was seen on the carbonyl stretch, giving
n
1723 cmK1, compared to nC]O 1772 cmK1 in the
13CZO
unlabelled aldehyde. In all the 13C-labelled compounds
where nC]O was present in the IR spectrum a shift of around
40 cmK1 was observed due to the isotopic labeling.
Synthesis of the 13C-labelled acetophenone fragment began
with acetylation of isovanillin with [13C2]acetyl chloride to
give the 3-[1,2-13C2]acetoxy-4-methoxybenzaldehye in
94% yield. The Bayer–Villager reaction with mCPBA
followed by selective hydrolysis of the formate gave
3-[1,2-13C2]acetoxy-4-methoxyphenol in 83% yield over
the two steps. The Fries rearrangement catalysed by
BF3$Et2O was a very clean reaction and after work up
This observation led to a change in the synthetic strategy.
An alternative synthesis of isoflavones involves an aldol
condensation of an acetophenone and a benzaldehyde to
give a chalcone which can then undergo a thallium mediated
oxidative rearrangement to give the isoflavone. This method
has been previously used for the synthesis of glycitein in
good yield.16 Since [13C2]acetyl chloride is commercially

Q. Zhang, N. P. Botting / Tetrahedron 60 (2004) 12211–12216
12213
gave a 96% yield of 2,4-dihydroxy-5-methoxy-[10,20-13C2]-
acetophenone 17. The two 13C-atoms in 17 were visible in
the 13C NMR spectrum at 202.5 ppm for the carbonyl and
26.60 ppm for the methyl group and appeared as the
expected doublets with a coupling constant of 43 Hz. With
this compound, as with a number of other 13C-labelled
compounds, it was difficult to see all the unlabelled carbon
atoms in the NMR spectrum due to a combination of poor
solubility and suppression of these signals as a result of the
materials had completely dissolved at reflux, anhydrous
KOH was added in one portion with stirring and this
dissolved completely after about 10 min. The clear yellow
solid of the product precipitated out soon after the
disappearance of the solid KOH. Simple work up by
filtration followed by washing with cold MeOH, copious
water to remove KOH, cold MeOH, cold Et2O and drying
gave the product as a pure yellow solid in excellent yield.
(93% for unlabelled chalcone, 94.5% for 13C-labelled
chalcone 19). The 13C NMR spectrum confirmed the
presence of the labelled carbon atoms. The carbonyl carbon
gave a doublet at 189.4 ppm (J1,2Z56 Hz. For the alkene
carbons, there was a doublet at 142 ppm (J2,3Z70 Hz) and a
double doublet at 125.4 (J1,2Z56 Hz, J2,3Z70 Hz). The
electrospray mass spectrum showed the expected increase in
mass of 3 units compared to the unlabelled compound
giving MHCZ560 (Scheme 5).
1
enhanced signals for the 13C-enriched positions. In the H
NMR spectrum the 2-OH group was observed at 12.6 ppm
due to strong intramolecular hydrogen bonding between the
2-OH group and the carbonyl group. This resonance was
also split into a doublet, with JZ0.9 Hz, presumably due to
coupling with the 13C-labelled carbonyl group.
Formation of the chalcone intermediate required an aldol
condensation between the acetophenone and the benzal-
dehyde. Preliminary work with unlabelled material showed
that the reaction did not occur without protection of the
hydroxyl groups of the acetophenone, despite some
literature precedent that claimed otherwise.17 Therefore,
2,4-hydroxy-5-methoxy-[10,20-13C2]acetophenone 17 was
fully benzylated using benzyl bromide in acetone with
anhydrous potassium carbonate to give 2,4-dibenzyloxy-5-
methoxy-[10,20-13C2]-acetophenone 18 in 94% yield. The
aldol condensation between 16 and 18 was first attempted in
EtOH–50% aq NaOH at reflux as described in literature,16
however even after extended reaction times there was
always some starting material left and the by-products made
purification problematic. As a result of the poor yields the
conditions were modified. Reaction in refluxing MeOH–
50% aq KOH aq gave 58% pure chalcone after recrystalli-
sation and refluxing EtOH–NaOEt did not improve the yield
much, giving 69%. It was observed that while the aldehyde
dissolves in EtOH or MeOH well upon heating, the
solubility of acetophenone is limited and the addition of
aq NaOH, or aq KOH, made the solubility even poorer.
Bearing this in mind, a mixed solvent of MeOH/THF (8:3).
was used to improve the solubility. Once the starting
Reaction of the chalcone in MeOH in the presence of
trimethyl orthoformate with 1.13 equiv of thallium nitrate
gave the 13C-labelled acetal 20 in an excellent 97% yield.
The work up involved a simple suction filtration followed
by washing with cold MeOH, copious water to remove
thallium salts, 5% NaHCO3 to neutralize the trace of HNO3
produced in the reaction, cold MeOH and cold Et2O
successively to get a pure white product.
The final step involved deprotection of benzyl groups and
cyclisation in methanol and concd HCl to give
[2,3,4-13C]glycitein in 95% yield. The 13C-labelled carbons
were observed as enhanced signals in the 13C NMR
spectrum at 174.5, 150.9 and 123.0 ppm with the expected
13C–13C coupling(dd, enhanced, JC,HZ72.0, 54.5 Hz, C-3),
while electrospray mass spectrometry (in negative ion
mode) gave MKZ286 as expected. To confirm its purity the
[2,3,4-13C3]glycitein 13 was also subject to microanalysis,
UV absorption and HPLC analysis. All these data compared
satisfactorily with the literature.2,11 The compound has been
used as internal standard for the analysis of glycitein in
plasma samples and it appears to be an excellent standard.
Scheme 5. (i) [13C3]Acetyl chloride, Et3N, THF/Et2O, (94%); (ii) mCPBA, CH2Cl2, reflux, then NaHCO3, EtOH (83%); (iii) BF3$Et2O, 70 8C (96%); (iv)
BnBr, K2CO3, acetone, reflux; (v) 16, KOH, MeOH/THF, reflux; (vi) Tl(NO3)2$3H2O, HC(OMe)3, MeOH; (vii) H2, 5% Pd/C, MeOH/Acetone (93%); (viii)
MeOH, Concd HCl, reflux (95%).

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Q. Zhang, N. P. Botting / Tetrahedron 60 (2004) 12211–12216
In conclusion, we have developed an efficient synthetic
method for [2,3,4-13C3]glycitein by employing the Baeyer–
Villiger oxidation, Fries rearrangement, aldol condensation
and thallium oxidative rearrangement. Each step gave an
excellent yield of the corresponding product. The total yield
of [2,3,4-13C3]-glycitein over an 8 step reaction sequence
was 57% based on the starting reagent [13C2]-acetyl
chloride.
dd, JC,HZ130.3, 7.0 Hz, 13CH313CO); dC (75 MHz, CDCl3)
190.1 (CHO), 168.8 (d, enhanced, JC,CZ60.0 Hz,
13COCH3), 156.5 (C-1), 140.4 (C-3), 130.3 (C-6), 130.1
(C-4), 123.6 (C-2), 112.2 (C-5), 56.4 (CH3O), 20.7
(d, enhanced, JC,CZ60.0 Hz, 13CH313CO); m/z (EI)
196.0640 (MC, C813C2H10O4 requires 196.0646).
3.1.2. 3-[1,2-13C2]Acetoxy-4-methoxyphenol. A clear
solution of 3-[1,2-13C2]acetoxy-4-methoxybenzaldehyde
(4.52 g, 23.0 mmol) and m-chloroperbenzoic acid
(mCPBA) (7.95 g, 46.1 mmol) in dry CH2Cl2 (60 mL) was
heated under reflux with stirring for 5 h. The reaction
mixture was then cooled to room temperature. Filtration and
removal of the solvent at reduced pressure afforded an oily
residue which was diluted with EtOAc and washed with 5%
NaHCO3 and brine. The residue was then dissolved in EtOH
(75 mL), after addition of 5% NaHCO3 aq (100 mL) and the
solution was stirred for 17 h at room temp. The reaction
mixture was acidified to pH 2 with 2 M HCl, salted out and
extracted with EtOAc. The extracts were washed with brine,
5% aq NaHCO3 and brine successively, dried over
anhydrous MgSO4 and filtered. After removal of the solvent
at reduced pressure, the residue was purified by flash
column chromatography on silica eluting with CH2Cl2/
AcOEt (40:1 to 20:1) and then recrystallised from 4:1
CH2Cl2/petroleum ether (bp 40–60) to give colourless
needles (3.54 g, 83%); nmax (KBr disc)/cmK1 1700
(13C]O); dH (300 MHz, CDCl3) 6.80 (1H, d, J5,6Z9 Hz,
H-5), 6.60 (1H, dd, J5,6Z9 Hz, J2,6Z3 Hz, H-6), 6.52 (1H,
d, J5,6Z3 Hz, H-2), 5.81 (1H, br s, OH), 3.76 (3H, s, CH3O),
2.31 (3H, dd, JC,HZ130.3, 6.8 Hz, 13CH313C); dC (75 MHz,
CDCl3) 170.0 (d, enhanced, JC,CZ59.9 Hz, 13CH313CO),
150.0 (C-1), 145.1 (C-4), 140.1 (C-3), 113.8 (C-5), 113.4
3. Experimental
3.1. General
Chemical and reagents were obtained from Aldrich and
Lancaster and used without further purification. Chemical
reactions were monitored by thin layer chromatography
using MN precoated silica gel G/UV254 (0.2 mm thickness)
plates. Silica Gel 60A (35–70 units) was utilized for column
chromatography. THF was freshly distilled from sodium/
benzophenone under nitrogen. DCM was refluxed over
powdered calcium hydride and distilled under nitrogen.
Methanol was heated to reflux over magnesium and distilled
under nitrogen. Infrared spectra were recorded as KBr
pellets in the range of 400–220 cmK1 on a Perkin–Elmer
system 2000 spectrometer, UV spectra on a Kontron
1
UVIKON spectrophotometer. H NMR spectra (300 MHz)
and 13C NMR (75 MHz) on a Varian Gemini 2000
spectrometer with the residue peak of CHCl3 (7.27 ppm)
and the central peak of CDCl3 (77.2 ppm) as reference,
respectively. Chemical shifts are given in d and J values in
Hz. Due to the poor solubility of the target glycitein, the
smallest amount of d6-DMSO was used in addition to
CDCl3 for NMR spectra recording. HREIMS were
measured on a Finnigan VG AutoSpec instrument,
HRESMS on a Waters LCT-ES spectrometer. Microana-
lysis was performed by the St Andrews University Service
within this Department. HPLC spectra were obtained from a
Waters 600 Multisolvent Delivery System using Kingsorb
3m C-18 silica-gel on a 150!4.60 mm2 3 micron sized
column eluted with a mixture of acetonitrile/water (75:25)
at a run rate of 0.6 mL/min.
(C-6), 111.0 (C-2), 56.7 (CH3O), 20.9 (d, enhanced, JC,C
59.9 Hz, 13CH313CO); m/z (EI) 184.0644 (MC,
Z
C713C2H10O4 requires 184.0646).
3.1.3. 2,4-Dihydroxy-5-methoxy-[1,2-13C2]acetophenone
(17). To 3-[1,2-13C2]acetoxy-4-methoxyphenol (3.30 g,
17.9 mmol) was added neat boron trifluoride diethyl
etherate BF3$Et2O (10 mL, 48.4 mmol). The mixture was
stirred at 70 8C for 2 h and then cooled to room temperature.
The suspension was taken up in saturated aq NaOAc
(50 mL) and saturated aq NaHCO3 was added until no
further CO2 was evolved. The suspension was then
extracted with EtOAc/Et2O (1:1). The extracts were washed
with brine, dried (MgSO4) and the solvent removed at
reduced pressure to give the product (3.16 g, 96%); nmax
(KBr disc)/cmK1 1634 (13C]O); dH (300 MHz, CDCl3)
12.58 (1H, d, 2JC–O/HZ0.9 Hz, D2O exchangeable, OH-2),
7.06 (1H, d, 3JC,HZ4.3 Hz, H-6), 6.53 (1H, d, JC,HZ1.2 Hz,
H-3), 6.23 (1H, s, D2O exchangeable, OH-4), 3.92 (3H, s,
3.1.1. 3-[1,2-13C2]Acetoxy-4-methoxybenzaldehyde. To a
solution of isovanillin (3.75 g, 24.64 mmol) in dry THF
(40 mL) was added Et3N (5 mL, 36 mmol). The mixture
was cooled to 0 8C. [13C2]Acetyl chloride (2.0 g,
24.8 mmol) in Et2O (8 mL) was added dropwise over
30 min. The reaction mixture was stirred at room tempera-
ture for 14 h and then the solvent was removed at reduced
pressure. The residue was taken up in water (200 mL) to
dissolve the salt. Filtration followed by successive washings
with water, cold MeOH and cold Et2O afforded the first crop
of product. The filtrates were then extracted with EtOAc.
The extracts were combined, washed with brine, dried
(MgSO4) and the solvent removed at reduced pressure. The
residue was subject to flash column chromatography (SiO2
eluted with gradient solvent CH2Cl2/EtOAcZ20:1 to 10:1)
to give the second crop of the product. Total yield: 4.75 g,
94%. nmax (KBr disc)/cmK1 1723 (13C]O); dH (300 MHz,
CDCl3) 9.89 (2H, s, CHO), 7.77 (1H, dd, J5,6Z8.7 Hz,
J2,6Z2.0 Hz, H-6), 7.60 (1H, d, J2,6Z2.0 Hz, H-2), 7.09
(1H, d, J5,6Z8.7 Hz, H-5), 3.93 (3H, s, CH3O), 2.34 (3H,
0
CH3O), 2.57 (3H, dd, JC,H Z127.8 Hz, JC,HZ5.8 Hz,
13CH133C); dC (75 MHz, CDCl3) 202.5 (d, enhanced,
JC,CZ43.2 Hz, CH313CO), 56.9 (CH3O), 26.60 (d,
enhanced, JC,CZ43.2 Hz, 13CH313CO); m/z (EI) 184.0650
(MC, C173C2H10O4 requires 184.0646).
3.1.4. 20,40-Dibenzyloxy-5-methoxy-[1,2-13C2]aceto-
phenone (18). To 17 (3.50 g, 19.0 mmol), 18-crown-6
(0.50 g, 1.90 mmol) and anhydrous K2CO3 (30 g,
217 mmol) in acetone (200 mL) was added benzyl bromide
(5.0 mL, 42.9 mmol). The reaction mixture was heated to

Q. Zhang, N. P. Botting / Tetrahedron 60 (2004) 12211–12216
12215
reflux for 5 h and then cooled to room temperature.
Removal of solvent at reduced pressure and recrystallisation
from acetone/Et2O and petroleum ether afforded the first
(4.93 g) crop of product as colourless needles. The mother
liquors were subject to column chromatography [SiO2,
petroleum ether (bp 40–60)/Et2OZ6:1 to 3:1] to give more
product (1.57 g). Total yield: 6.50 g, 94%. nmax (KBr disc)/
cmK1 1623 (13C]O); dH (300 MHz, CDCl3) 7.45 (1H,d,
JC,HZ3.9 Hz, H-6), 7.45–7.33 (10H, m, PhH), 6.55 (1H, d,
solution of 18 (2.35 g, 6.45 mmol) and 4-benzyloxy-
[carbonyl-13C]benzaldehyde 16 (1.46 g, 213 mmol) in a
mixture of THF (20 mL) and MeOH (60 mL) was added
anhydrous KOH (3.6 g, 64.3 mmol). The solution was
then heated to reflux for 6 h during which time the
product precipitated out as bright yellow solid. After
cooling in an ice bath, the suspension was filtered washing
successively with cold MeOH, water, cold MeOH and
cold Et2O to give the product as tiny yellow crystals
(3.40 g, 94%); nmax (KBr disc)/cmK1 1603, 1589, 1572,
1533, 1521, 1507; dH (300 MHz, CDCl3) 7.89–7.74 (m,
1H, 1/2 CH]CH), 7.41 (1H, d, JC,HZ4.3 Hz, H-60),
7.38–7.15 (18H, m, PhHC1/2 CH]CHCH-200 and 600),
JC,HZ1.4 Hz, H-3), 5.17, 5.02 (4H, 2!s, 2!CH2Ph), 3.89
2
(3H, s, CH3O), 2.57 (3H, dd,, JC,HZ128.3 Hz, JC,H
Z
6.0 Hz, 13CH313C); dC (75 MHz, CDCl3) 197.6 (d,
enhanced, JC,CZ42.6 Hz, 13CH313CO), 154.3, 152.9,
143.9, 136.4, 128.9, 128.4, 128.3, 127.7, 127.3, 120.5 (d,
C-1), 113.2, 100.6, 71.7 (CH2), 71.3 (CH2), 56.6 (CH3O),
32.5 (d, enhanced, JC,CZ42.6 Hz, 13CH313CO); m/z
(ES) 387.1480 (MNaC, C2113C2H22NaO4 requires 387.1484).
00
00
00 00
00 00
6.78 (d, 2H, J2 ,3 ZJ5 ,6 Z8.7 Hz, H-3 and 5 ), 6.54 (d,
1H, JC,HZ1.4 Hz, H-30), 5.14, 5.01, 5.00 (3!s, 3!2H,
3!CH2Ph), 3.84 (3H, s, CH3O); dC (75 MHz, CDCl3)
189.4 (d, enhanced, J1,2Z56.4 Hz, 13C]O), 141.98
(d, enhanced, J2,3Z70.2 Hz, 13C-3), 125.4 (dd, enhanced,
J1,2Z56.4 Hz, J2,3Z70.2 Hz, 13C-2); m/z (ES) 560.2436
(MHC, C3413C3H33O5 requires 560.2430).
3.1.5. 4-Benzyloxybenzo-[1-13C]nitrile (15). To a solution
of 4-benzyloxyphenyl iodide (3.71 g, 11.96 mmol),
Ca(OH)2 (0.443 g, 5.98 mmol) and Pd(OAc)2 (0.27 g,
1.12 mmol) in anhydrous DMF (40 mL) was added
potassium [13C]cyanide (0.79 g, 12.0 mmol). The reaction
mixture was heated to reflux for 16 h and then cooled to
room temperature. After filtration washing with CH2Cl2 and
acetone, the filtrates were concentrated, diluted with
copious water (500 mL) and extracted with EtOAc. The
extracts were washed with brine and dried over anhydrous
MgSO4. Removal of the solvent at reduced pressure
afforded a brown solid, which was purified by column
chromatography (SiO2, petroleum ether (bp 40–60)/Et2OZ
10:1) to give the product as a white solid (1.63 g, 65%); nmax
(KBr disc)/cmK1 2169 (13CN); dH (300 MHz, CDCl3) 7.59
(2H, dd, J2,3Z3J5,6Z8.7 Hz, JC,HZ4.8 Hz, H-2 and 6),
7.45–7.35 (5H, m, PhH), 7.03 (2H, d, J2,3ZJ5,6Z8.7 Hz,
H-3 and 5), 5.13 (2H, s, CH2Ph); dC (75 MHz, CDCl3) 162.1
(C-4), 135.9 (C-10), 134.1 (d, JC,CZ2.0 Hz, C-3 and 5),
128.9 (C-20 and 60), 128.5 (C-40), 127.6 (C-30 and 50), 119.3
(enhanced, 13CN), 115.71 (d, JC,CZ5.7 Hz, C-2 and 6),
104.3 (d, JC,CZ74.0 Hz, C-1), 70.4 (CH2Ph); m/z (EI)
210.0871 (MC, C1313CH11NO requires 210.0874).
3.1.8. 2-(4-Benzyloxyphenyl)-1-[2,4-bis(benzyloxy)-5-
methoxyphenyl]-3,3-dimethoxy-[1,2,3-13C3]-1-propa-
none (20). To a suspension of 19 (1.12 g, 2.00 mmol) in dry
MeOH (10 mL) and trimethyl orthoformate (7 mL,
64.0 mmol) was added Tl(NO3)3$3H2O (1.00 g,
2.25 mmol). The mixture was stirred for 24 h at room
temperature and then cooled in an ice bath. Filtration
washing with cold MeOH, copious H2O, 5% NaHCO3 aq,
H2O, cold MeOH and Et2O successively afforded the
product as a white solid. (1.20 g, 97%); nmax (KBr disc)/
cmK1 1627 (13C]O); dH (300 MHz, CDCl3) 7.42–7.28 (m,
16 H, PhHCH-60), 7.11 (dd, 2H, J2 ,3 ZJ5 ,6 Z8.7 Hz,
00 00
00 00
JC,HZJC,HZ3.4 Hz, H-200 and 600), 6.80 (d, 2H, J2 ,3
Z
00 00
00
00
00 00
J5 ,6 Z8.7 Hz, H-3 and 5 ), 6.44 (d, 1H, JC,HZ1.4 Hz, H-
30), 5.16 (dm, 1H, JC,HZ130.7 Hz, H-20), 5.09 (s, 2H,
CH2Ph), 5.04 (dd, JC,HZ166.9 Hz, JH,HZ7.7 Hz, H-3),
5.01 (d, 1H, JH,HZ12 Hz, OCHaPh-20), 4.99 (s, 2H,
CH2Ph), 4.92 (d, 1H, JH,HZ12 Hz, OCHbPh-20), 3.86
(s, 3H, CH3O-50), 3.39, 3.05 (2!d, 2!3H, 13CH(OCH3)2,
JC,HZ4.8 Hz). dC (75 MHz, CDCl3) 197.5 (d, enhanced,
13C]O, J1,2Z41.4 Hz), 107.0 (d, enhanced, J2,3Z47.2 Hz,
13C-3), 58.7 (dd, enhanced, J1,2Z41.4 Hz, J2,3Z47.2 Hz,
13C-2); m/z (ES) 644.2616 (MNaC, C3613C3H38O7Na requires
644.2617).
3.1.6. 4-Benzyloxy-[carbonyl-13C]benzaldehyde (16). To
a solution of 4-benzyloxybenzo[13C]nitrile 15 (1.62 g,
7.72 mmol) in THF (50 mL) was added diisobutylaluminum
hydride (DIBAL-H, 1.0 M in THF, 15.4 mL, 15.4 mmol).
The reaction mixture was stirred at room temperature
overnight and then quenched with water and 2 M HCl until
pHZ1. Extraction with CH2Cl2, drying over anhydrous
MgSO4 and removal of solvent gave the off-white product.
(1.46 g, 89%); nmax (KBr disc)/cmK1 1653 (13C]O); dH
(300 MHz,CDCl3) 9.86 (1H, d, JC,HZ172.2 Hz, 13CHO),
7.81 (2H, dd, J2,3ZJ5,6Z8.7 Hz, JC,HZ4.1 Hz, H-2 and 6),
7.42–7.31 (5H, m, PhH), 7.05 (2H, d, J2,3ZJ5,6Z8.7 Hz,
H-3 and 5), 5.13 (2H, s, CH2Ph); dC (75 MHz, CDCl3) 190.9
3.1.9. 1-(2,4-Dihydroxy-5-methoxyphenyl)-2-(4-hydro-
xyphenyl)-3,3-dimethoxy-[1,2,3-13C3]-1-propanone.
A solution of the acetal 20 (1.13 g, 1.81 mmol) in
methanol/acetone (1:1, 120 mL) was hydrogenated over
palladium on charcoal (0.56 g, 5%). The mixture was
stirred overnight at room temperature. Filtration through a
celite pad and removal of solvent afforded the product as
an off-white foam (0.57 g, 93%); nmax (nujol)/cmK1 1636
(13C]O); dH (300 MHz, d60 -acetone) 12.66 (1H, s,
OH-20), 8.59 (2H,0 br s, OH-4 and OH-400), 7.53 (1H, d,
13
(enhanced, CHO), 162.1 (C-4), 136.1 (C-10), 132.1
00 00
00 00
(d, JC,CZ3.4 Hz, C-2 and 6), 130.3 (d, JC,CZ56.4 Hz,
C-1), 128.9 (C-30 and 50), 128.4 (C-40), 127.6 (C-30 and 50),
115.3 (d, JC,CZ4.6 Hz, C-3 and 5), 70.5 (CH2Ph); m/z (EI)
213.0870 (MC, C1313CH12O2 requires 213.0871).
JC,HZ4.0 Hz, H-6 ), 7.37 (2H, dd, J2 3 ZJ5 ,6 Z8.6 Hz,
JC,HZ3.3 Hz, H-200 and 600), 6.80 (2H, d, J2 ,3 ZJ5 ,6
Z
00 00
00 00
8.6 Hz, H-300 and 500), 6.34 (1H, d, JC,HZ1.1 Hz, H-30),
5.09 (1H, dd, JC,HZ166.1 Hz, J2,3Z8.2 Hz, H-3), 4.87
(1H, ddt, JC,HZ130.0 Hz, JZ4.3 Hz, J2,3Z8.2 Hz, H-2),
3.1.7. 3-(4-Benzyloxyphenyl)-1-[2,4-bis(benzyloxy)-5-
3.86 (3H, s, CH3O-50), 3.38, 3.18 (6H, 2!d, JC,H
Z
methoxyphenyl]-[1,2,3-13C3]-2-propen-1-one (19). To a
4.4 Hz, 13CH(OCH3)2); dC (75 MHz, CDCl3) 203.2

12216
Q. Zhang, N. P. Botting / Tetrahedron 60 (2004) 12211–12216
(d, enhanced, JC,CZ42.6 Hz, 13C]O), 107.1
(d, enhanced, J2,3Z47.2 Hz, 13C-3), 58.7 (dd, enhanced,
J1,2Z42.6 Hz, J2,3Z47.2 Hz, 13C-2); m/z (ESK) 350.1233
(MK, C1513C3H19O7 requires 350.1233).
References and notes
1. Murphy, P. A.; Hendrich, S. Adv. Food Nutri. Res. 2002, 44,
195–246.
2. Song, T. T.; Hendrich, S.; Murphy, P. A. J. Agric. Food Chem.
1999, 47, 1607–1610.
3.1.10. [2,3,4-13C3]Glycitein (13). A solution of the
hydroxyacetal (0.59 g, 1.68 mmol) in a mixture of
methanol (20 mL) and concentrated hydrochloric acid
(2.0 mL) was heated to reflux for 4 h and then cooled to
room temperature. Filtration followed by washing with
cold MeOH, water, 5% aq NaHCO3, water, cold MeOH
and diethyl ether successively afforded the crude product
as off-white solid. Recrystallisation from EtOH gave a
3. Zhang, Y.; Wang, G. J.; Song, T. T.; Murphy, P. A.; Hendrich,
S. J. Nutr. 2001, 131, 147–148.
4. Whalley, J. L.; Bond, T. J.; Botting, N. P. Bioorg. Med. Chem.
Lett. 1998, 8, 2569–2572.
5. Whalley, J. L.; Oldfield, M. F.; Botting, N. P. Tetrahedron
2000, 56, 455–460.
6. Setchell, K. D. R.; Faughnan, M. S.; Avades, T.; Zimmer-
Nechemias, L.;Wolfe, B.;Brashear, E.;Desai, P.;Oldfield,M. F.;
Botting, N. P.; Cassidy, A. Am. J. Clin. Nutr. 2003, 77, 411–419.
7. Oldfield, M. F.; Chen, L.; Botting, N. P. Tetrahedron 2004, 60,
1887–1893.
white solid (0.455 g, 95%); (Found: C, 66.81; H, 4.08,
13
C C3H11O5 requires C, 66.90; H 4.21%); lmax (EtOH)/
13
nm 260 (3/30,225 dm3 molK1 cmK1, and 320 (3/13,134
dm3 molK1 cmK1); literature2 unlabelled glycitein in
MeOH 257 (3/31,622 dm3 molK1 cmK1); nmax (KBr
disc)/cmK1 3405, 1614, 1548, 1515; dH (300 MHz,
CDCl3 and d6-DMSO) 9.58 (1H, br s, OH-7), 8.66 (1H,
br s, OH-40), 7.51 (1H, dt, JC,HZ194.0, 6.5 Hz, H-2), 7.09
8. Grace, P. B.; Taylor, J. I.; Botting, N. P.; Fryatt, T.; Oldfield,
M. F.; Bingham, S. A. Anal. Biochem. 2003, 315, 114–121.
9. Clarke, D. B.; Lloyd, A. S.; Oldfield, M. F.; Botting, N. P.;
Needs, P. W.; Wiseman, H. Anal. Chem. 2002, 309, 158–172.
10. Grace, P. B.; Taylor, J. I.; Botting, N. P.; Fryatt, T.; Oldfield,
M. F.; Al-Maharik, N.; Bingham, S. A. Rapid Commun. Mass
Spectrom. 2003, 17, 1350–1357.
0
0
0
0
(1H, d, JC,HZ3.9 Hz, H-5), 6.93 (2H, dd, J2 ,3 ZJ5 ,6
Z
8.7 Hz, JC,HZ3.4 Hz, H-20 and 60), 6.50 (1H, d, JC,H
Z
0
0
0
0
0
1.9 Hz, H-8), 6.42 (2H, d, J2 ,3 ZJ5 ,6 Z8.7 Hz, H-3
and 50), 3.52 (3H, s, OCH3); dC (75 MHz, (CDCl3 and
d6-DMSO) 174.5 (d, enhanced, JC,HZ54.5 Hz, C-4),
150.9 (d, enhanced, JC,HZ72.0 Hz, C-2), 123.0 (dd,
enhanced, JC,HZ72.0, 54.5 Hz, C-3); m/z (ESK)
286.0712 (MK, C1313C3H11O5 requires 286.0707).
11. Lang’at-Thoruwa, C.; Song, T. T.; Hu, J.; Simons, A. L.;
Murphy, P. A. J. Nat. Prod. 2003, 66, 149–151.
12. Baker, W.; Evans, C. J. Chem. Soc. 1938, 372–375.
13. Guzman, J. A.; Mendoza, V.; Garcia, E.; Garibay, C. F.;
Olivares, L. Z.; Maldonado, L. A. Synth. Commun. 1995, 25,
2121–2133.
14. Hauer, H.; Ritter, T.; Grotemeier, G. Arch. Pharm. 1995, 328,
737–738.
15. Aihara, K.; Urano, Y.; Higuchi, T.; Hirobe, M. J. Chem. Soc.,
Perkin Trans. 2 1993, 2165–2170.
Acknowledgements
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17. Screenivasulu, B.; Sarma, P. N. Synth. Commun. 1997, 27,
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This work was funded by the Food Standards Agency (FSA)
under contract T05023.