Synthesis of [3,4,8-13C3]daidzein

Article abstract of DOI:10.1016/j.tet.2003.12.033

The biological effects of the soy isoflavones have attracted considerable interest in recent years leading to numerous studies on dietary intake and epidemiology. Such studies require accurate and reproducible analytical methods. Herein we report the firs

Full text of DOI:10.1016/j.tet.2003.12.033

Tetrahedron 60 (2004) 1887–1893
Synthesis of [3,4,8-13C3]daidzein
*
Mark F. Oldfield, Lirong Chen and Nigel P. Botting
School of Chemistry, University of St Andrews, St Andrews, Fife KY16 9ST, Scotland, UK
Received 22 September 2003; revised 24 November 2003; accepted 11 December 2003
Abstract—The biological effects of the soy isoflavones have attracted considerable interest in recent years leading to numerous studies on
dietary intake and epidemiology. Such studies require accurate and reproducible analytical methods. Herein we report the first synthesis of a
multiply 13C-labelled daidzein derivative, [3,4,8-13C3]daidzein, which has been employed as an internal standard in LC-MS and GC-MS
analysis. The synthesis includes an improved three-step method for the synthesis of [2-13C]resorcinol as one building block.
q 2003 Elsevier Ltd. All rights reserved.
1. Introduction
genistein.10 These are undoubtedly rapid methods with
high sensitivity but can suffer from a lack of specificity.11
However, by far the most widely used technique has been
mass spectrometry due to its inherent sensitivity and
selectivity. GC-MS has been used extensively for the
quantification of isoflavones in urine, even though it
requires complex purification procedures and derivatisation
of the analytes.12–14 More recently LC-MS procedures have
become more popular as these can be carried out without
derivatisation and often with little or no purification of the
sample prior to analysis, allowing the use of smaller
samples.15–18
The impact of phytoestrogens on human health is currently a
subject of major interest.1,2 In particular the soy isoflavones,
including daidzein 1 and genistein 2, have attracted
considerable attention.3,4 The soy isoflavones are present
at significant levels in soya beans and soy products. They
thus feature heavily in the diet particularly in Japan and
Asia, where they have been associated with a low incidence
of hormone dependent cancers.5 These compounds have
also been implicated in the prevention of cardiovascular
disease,6 lessening the symptoms of the menopause7 and
protection against osteoporosis.8
An important aspect of any quantitative analytical pro-
cedure is the nature of the internal standard. The optimum
internal standard for LC-MS and GC-MS is a pure, stable,
isotopically labelled analogue of the analyte, which must
have a large enough mass difference to nullify the effect of
natural abundance heavy isotopes in the analyte. This mass
difference will depend on the molecular weight of the
analyte. For isoflavone type structures a minimum of three
extra mass units is required. Initially a number of deuterated
standards were used for the analysis of isoflavones.18 These
were prepared using acid, or base, catalysed exchange
methods to incorporate the deuterium atoms into the phenol
rings.19–21 The problem with these deuterated isoflavones is
that they are always a mixture of species with varying
numbers of deuteriums incorporated into the isoflavone,
which is an inevitable consequence of the exchange
procedures employed. This produces a range of molecular
ions and complicates the analysis procedure unnecessarily.
More significantly it has been shown that under the analysis
conditions back exchange occurs and the deuteriums are
slowly replaced by hydrogen.22,23 This results in a reduction
of the amount of internal standard present and, also, an
apparent increase in the amount of the phytoestrogen being
analysed.
In order to understand the significance of the biological
effects of phytoestrogens, analytical chemistry has had a key
role to play. Accurate analysis has been important in
attempts to establish the exposure of the population to the
soy isoflavones through their diet and also in epidemio-
logical studies to investigate the associations between
isoflavone exposure and disease.
A number of analytical methods have been used to quantify
the low levels of phytoestrogens found in food and
biological fluids. HPLC has been employed with UV
detection, but this suffers from poor selectivity and low
sensitivity.9 Time-resolved fluoroimmunoassay (TR-FIA)
methods have been developed for both daidzein and
Keywords: Daidzein; Isoflavones; Phytoestrogens; 13C-labelling.
*
Corresponding author. Tel.: þ44-1334-463856; fax: þ44-1334-463808;
e-mail address: npb@st-andrews.ac.uk
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2003.12.033

1888
M. F. Oldfield et al. / Tetrahedron 60 (2004) 1887–1893
tion to give the desired product. Firstly, methyl 4-(chloro-
formyl)butyrate 9 was converted into a mixed anhydride by
reaction with 2-methoxybenzoic acid33 and then reacted
with [13C]methyl magnesium iodide to give 8 in a low 30%
yield. This step was thus modified and the methyl
4-(chloroformyl)butyrate 9 was reacted directly with a
cuprate derived from [13C]methyl iodide, giving the product
in a much better 57% yield (Scheme 2).
The cyclisation to [2-13C]cyclohexane-1,3-dione 10 was
previously32 carried out in DMF with sodium methoxide in
61% yield, but we obtained a better 86% yield by using
t
carefully dried potassium butoxide in THF. Dehydrogena-
Scheme 1.
tion was achieved using a palladium on charcoal catalyst in
refluxing xylene at 137–140 8C giving [2-13C]resorcinol 11
in 49% yield. A lower temperature was employed for this
reaction compared to the 190 8C in refluxing triglyme
previously reported.32 The yields were comparable for the
two procedures, but the lower temperature produced a
cleaner more easily purified product. The spectral data for
the [2-13C]resorcinol were identical to the literature data.32
The 13C atom was clearly observed as an enhanced signal at
103.8 ppm in the 13C NMR spectrum. This represents a
much improved synthesis of [2-13C]resorcinol in 24% yield
over the three steps.
Due to the inherent problems with deuterated internal
standards the aim of this work was to synthesise daidzein
with three 13C atoms incorporated into the carbon frame-
work of the molecule for use as an internal standard. Using
commercially available starting materials with 99% incor-
poration of 13C, it should be possible to obtain the final
product with 99% 13C at the corresponding position in the
phytoestrogen molecule. Thus with three 13C atoms one
major signal three mass units bigger than the unlabelled
compound will be observed. This is in contrast to the
variable levels of deuterium incorporation obtained through
exchange procedures, which give an envelope of signals.
It was then necessary to incorporate two 13C-atoms into the
phenylacetic acid fragment and it was envisaged that these
could both be derived from 13C-labelled potassium cyanide,
a very cheap source of 13C atoms. In our previous work
potassium [13C]cyanide was reacted with a suitably
protected benzyl bromide to give a single labelled
precursor.25 Therefore incorporation of two 13C atoms
required a 13C-labelled benzyl bromide, which could be
synthesised via an aromatic cyanation using another mole of
potassium [13C]cyanide and a suitably substituted aryl
halide, followed by functional group transformations.
Also the 13C standards will be chemically stable and the 13
C
atoms will not exchange back out from the molecule during
the analysis procedures. We had previously prepared both
daidzein and genistein labelled with a single 13C-atom at the
4-position24,25 and these compounds have been employed in
metabolic studies on menopausal women.26 This previous
synthetic strategy was used as a suitable starting point.
2. Results and discussion
4-Iodophenol 12 was used as the starting material and the
hydroxyl group was first protected as the benzyl ether 13
(Scheme 3). A large number of procedures are available for
aromatic cyanation34 but our choice was restricted by the
requirement for 13C-labelled cyanide, of which only the
sodium and potassium salts are commercially available.
Thus all the copper(I) cyanide methods were ruled out and
instead a procedure employing potassium cyanide and a
palladium(II) acetate catalyst in DMF under basic con-
ditions (calcium hydroxide) was identified.35 Only a limited
range of aryl iodides had been examined under these
conditions but we found that 4-benzyloxy-1-iodobenzene 13
reacted smoothly with unlabelled potassium cyanide to give
the desired product. The reaction conditions were then
optimised and using K13CN the purified 13C-labelled nitrile
was obtained in 70% yield. The 13C atom was clearly
identified by the enhanced signal in the 13C NMR spectrum
at 119.6 ppm. The nitrile 14 was then hydrolysed under
The general synthetic route towards the isoflavonoid
phytoestrogens (Scheme 1) involves the condensation of
an appropriate phenol 3 with either a substituted phenyl-
acetic acid24,25,27 4 or a benzyl nitrile 5.24,25,28–30 This gives
a deoxybenzoin 6 which then undergoes formylation and
finally cyclisation to give the isoflavonoid 7.24,25,30,31 The
synthesis presents various opportunities for the incorpor-
ation of 13C atoms. However, the obvious strategy of putting
put one 13C atom into the 2-position in the formylation step
at the end of the synthesis had already been shown to be
unsuitable.24 It was thus decided to incorporate one 13C
atom into the resorcinol and two into the side chain of the
phenylacetic acid.
A previous literature procedure was discovered for the
synthesis of [2-13C]resorcinol32 involving the synthesis of
methyl 5-oxo-[6-13C]hexanoate 8, cyclisation via an
intramolecular Claisen condensation and finally aromatisa-
Scheme 2. (a) H133CI, Li, CuI, Et2O, (57%); (b) KOtBu, THF then aq. HCl (86%); (c) 10% Pd/C, xylene, reflux (49%).

M. F. Oldfield et al. / Tetrahedron 60 (2004) 1887–1893
1889
Scheme 3. (a) BnBr, K2CO3, acetone (90%); (b) K13CN, Pd(OAc)2, Ca(OH)2, DMF (70%); (c) 2 N NaOH, MeOH (83%); (d) LiAlH4, THF (93%); (e) PBr3,
Et2O (93%); (f) K13CN, 18-crown-6, MeCN (80%); (g) 2 M aq. NaOH, reflux (98%); (h) H2, 10% Pd/C, EtOAc (96%).
alkaline conditions, sodium hydroxide in methanol, to give
the acid 15 in 83% yield and reduced to the alcohol 16 in
93% yield using lithium aluminium hydride in THF.
was required to clean up the products from the higher
temperature reactions. This represents the best reported
yield for this procedure in the synthesis of daidzein and we
have found it to be more reproducible than many of the
published methods.25,30,31
A number of procedures were examined for the conversion
of the alcohol to the bromide 17 including trimethylsilyl
bromide and carbon tetrabromide with triphenylphosphine.
However, the best yield was obtained using phosphorus
tribromide. The bromide was found to be reasonably
unstable due to the 4-substituent and so was used without
purification in the next step.
The [3,4,8-13C3]daidzein 22 was found to be identical to
daidzein in all respects except for the expected increase in
mass and NMR spectroscopic data. The three 13C atoms
were observed at 178.8 ppm, for the carbonyl at C-4,
126.0 ppm for C-3 and 103.5 ppm for C-8, with the
expected coupling between C-3 and C-4 (J¼54 Hz). The
1
Reaction of the benzyl bromide 17 with a second mole of
K13CN in acetonitrile, using 18-crown-6 to aid solubility,
effected nucleophilic displacement to give the benzyl nitrile
with 18 two 13C-atoms, observed at 118.6 and 23.2 ppm in
the 13C NMR spectrum. Alkaline hydrolysis to the
carboxylic acid, 19 was followed by hydrogenation to
remove the benzyl ether and provide the precursor 20 for the
synthesis of [3,4,8-13C3]daidzein (Scheme 3).
effect of the 13C incorporation was also evident on the H
NMR spectrum, for example 8-H exhibited a 162 Hz
coupling with the attached 13C atom. The purity of the
compound was confirmed by reverse phase HPLC
(Kingsorb 3m C18 Column (150£4.6 mm)) giving a
retention time of 8 min 40 s with a mobile phase of
acetonitrile:water (1:1) and a 0.3 mL min21 flow rate. This
gave a single peak at the identical retention time to
unlabelled daidzein. Furthermore, the UV spectrum was
measured giving a lmax (EtOH) of 262 nm and 1¼
23,894 dm3 mol21 cm21, compared with literature value36
of 1¼24,739 dm3 mol21 cm21, implying 96% purity within
experimental error.
With the two 13C-labelled building blocks prepared, the
[3,4,8-13C3]daidzein was synthesised using adaptations of
our previous methods (Scheme 4).25 Condensation of the
[2-13C]resorcinol 11 and the phenylacetic acid 20 was
carried out in neat boron trifluoride etherate, giving the
deoxybenzoin intermediate 21 in 55% yield. The formyl-
ation/cyclisation step was achieved using dimethyl-
formamide dimethylacetal in THF. In our previous work
this had been carried out at reflux in 60% yield.24,25
However, the step was carefully optimised by varying the
temperature, time of reaction and solvent (THF, diethyl
ether and DMF) and it was found that the yield increased as
the reaction temperature was decreased, giving an optimum
yield of 80% after stirring at room temperature in THF for
3 h. The crude product from this reaction was found to be
much cleaner than from reactions at higher temperature and
readily purified by simple crystallisation, whereas HPLC
The compound has since been used as an internal standard in
both GC-MS37 and LC-MS22,38 using isotope dilution
methods. To be used over an extended dynamic range the
isotopically labelled internal standard must have no
significant mass overlap with the unlabelled analyte. This
is obviously complicated by the natural abundance distri-
bution of carbon, hydrogen and oxygen. When isotope
distributions are taken into consideration the predicted mass
intensity pattern for daidzein examined by negative
electrospray ionisation is 253 (100%):254 (18%):255(3%).
For quantitative analysis selective ion monitoring (SIM) is
used at 253 for optimal instrumental sensitivity, so the
Scheme 4. (a) BF3·Et2O (55%); (b) DMF·(OMe)2, DMF (80%).

1890
M. F. Oldfield et al. / Tetrahedron 60 (2004) 1887–1893
daidzein internal standard must not give any significant
signal at this mass. When the isotopic purity of the
[3,4,8-13C3]daidzein was examined by LC-MS, the major
signal was observed, as expected, at 256 along with a signal
of 4% relative intensity at 255 for daidzein molecules
having only two 13C atoms.22 However, there were no
discernible ions due to molecules with zero or one 13C per
molecule, thus comfortably meeting the criteria for use as an
internal standard for isotope dilution mass spectrometry.
Recent isoflavone analysis by GC-MS37 demonstrated
improved linearity over a range of concentrations due to
the use of the 13C-labelled internal standards, along with
lower limits of detection. Quality control was also improved
with lower intra- and interassay coefficients of variation
(CV) for isoflavones where a 13C-labelled standard was
available.
J¼7.2 Hz, CH2-3), 2.31 (2H, t, J¼7.2 Hz, CH2-4), 2.11 (3H,
d, J¼127 Hz, CH2-6), 1.86 (2H, quin, J¼7.2 Hz, CH2-2); dC
(75.45 MHz, CDCl3) 202 (d, J5,6¼45 Hz, C-5), 174.4 (C-1),
52.0 (OCH3), 42.9 (d, J4,6¼22 Hz, C-4) 33.4 (C-2), 30.2
(enhanced, C-6), 26.4 (C-6); m/z (ESþ) 168 [(MþNa)þ,
100%]; (EI) 145 (Mþ, 15%), 114 (40, [M2OCH3]þ), 86
(30, [M2COOCH3]þ), 44 (100, CH3COþ).
3.1.2. [2-13C]Cyclohexane-1,3-dione (10). To the solution
of methyl 5-oxo-[6-13C]hexanoate 8 (758 mg, 5.22 mmol)
t
in dry THF (100 mL), was added potassium butoxide
(2.345 g, 20.9 mmol). The mixture was heated under reflux
for 6 h, then the THF was removed at reduced pressure. The
residue was dissolved in water (20 mL), acidified to pH 1
with concentrated hydrochloric acid, extracted with ethyl
acetate (6£20 mL) and dried (MgSO4). After removal of the
solvent at reduced pressure, the desired product was
obtained as a pale yellow solid (510 mg, 86%) mp 102–
103 8C (lit.32 103–104 8C); nmax (nujol)/cm21 2480, 1627,
1600, 1180; dH (300 MHz, CDCl3) 3.36 (2H, d, J¼130 Hz,
CH2-2), 2.55 (4H, t, J¼6.7 Hz, CH2-4 and 6), 2.0–1.8 (2H,
m, CH2-5); dC (75.45 MHz, CDCl3) 58.8 (enhanced, C-1);
m/z (EI) 113 (Mþ, 50%), 85 (50, [M2CO]þ).
3. Experimental
3.1. General
NMR spectra were recorded on a Varian Gemini 2000 (1H
300 MHz, 13C 75.45 MHz) or a Bruker Avance 300 (1H
300 MHz, 13C 75.45 MHz) spectrometer chemical shifts (d)
in ppm are given relative to Me4Si, coupling constants (J) in
Hz. Elemental analyses were carried out in the departmental
microanalytical laboratory. IR spectra were recorded on a
Perkin–Elmer series 1420 FT IR spectrophotometer. The
samples were prepared as Nujol mulls or thin films between
sodium chloride discs and recorded in cm21. EI and CI mass
spectra were recorded on a VG Autospec. Electrospray mass
spectra recorded on Micromass LC-T UV spectra were
recorded on a Kontron Uvikon 930 spectrometer. Melting
points were recorded on an electrothermal melting point
apparatus and are uncorrected. Analytical TLC was carried
out on Merck 5785 Kieselgel 60F254 fluorescent plates.
Flash chromatography was performed according to the
procedure of Still39 using silica gel of 35–70 m particle size.
DMF was distilled from magnesium sulfate. Diethyl ether
and tetrahydrofuran were distilled from sodium metal and
benzophenone.
3.1.3. [2-13C]Resorcinol (11). To a flask containing
[2-13C]cyclohexane-1,3-dione 10 (500 mg, 4.42 mmol)
and xylene (75 mL), palladium on carbon (10%, 2.5 g)
was added. The mixture was heated under reflux for 3 h,
then the palladium catalyst was filtered off through celite.
The reaction mixture was extracted with aqueous sodium
hydroxide (20%, 4£20 mL). The combined aqueous layers
were cooled to 0 8C, acidified to pH 2 with concentrated
hydrochloric acid, and extracted with diethyl ether
(3£25 mL). After removal of the solvent at reduced
pressure, red oil was obtained. Further purification was
carried out by column chromatography on silica, eluting
with diethyl ether/petroleum ether (40–60 8C) (2:1) to give
the desired product as a white solid (238 mg, 49%) mp 105–
108 8C (lit.32 108–109 8C); nmax (nujol)/cm21 3240 (OH),
1615, 966; dH (300 MHz, acetone-d6) 8.04 (2H, s, –OH),
6.84 (1H, dt, J¼8.1, 1.4 Hz, H-5), 6.21 (1H, dt, JC,H¼156,
J2,4¼J2,6¼2.3 Hz, H-2), 6.26–6.1 (2H, m, H-4 and 6); dC
(75.45 MHz, acetone-d6) 103.8 (enhanced, C-2); m/z (CI)
112.0480 (MHþ, 12C513CH6O2 requires 112.0484).
3.1.1. Methyl 5-oxo-[6-13C]hexanoate (8).32 Under the
protection of a nitrogen atmosphere, lithium pieces
(489 mg, 70.4 mmol) were added to dry diethyl ether
(70 mL). 13C-Methyl iodide (1.8 mL, 4.104 g, 28.91 mmol)
was then introduced and the reaction mixture stirred at room
temperature for 1 h, then cooled to 0 8C and copper iodide
(3.352 g, 17.6 mmol) was added. After 0.5 h stirring at 0 8C,
the resulting lithium dimethylcuprate was cooled to 220 8C.
Precooled methyl 4-chloroformyl butyrate (2 mL, 2.386 g,
14.5 mmol) was then added dropwise with vigorous stirring.
The reaction mixture was maintained at 220 8C for 1 h, then
room temperature overnight. The reaction was quenched
with saturated ammonium chloride (45 mL) and filtered to
remove the copper residue. The filtrate was extracted with
diethyl ether (3£20 mL), washed with brine (20 mL) and
dried (MgSO4). Further purification was carried out by
column chromatography, eluting with petroleum ether
(40–60 8C)/ethyl acetate (1:1) to give a colourless oil
(1.19 g, 57%); nmax (nujol)/cm21 1725 (CvO); dH
(300 MHz, CDCl3) 3.64 (3H, s, –OCH3), 2.48 (2H, t,
3.1.4. 4-Benzyloxyiodobenzene (13). To a solution of
4-iodophenol (10 g, 45.5 mmol) in acetone (250 mL) were
added potassium carbonate (30.273 g, 227.3 mmol) and
benzyl bromide (4.5 mL, 6.471 g, 37.9 mmol). The mixture
was heated under reflux for 6 h, then cooled and filtered to
remove the solid. The solvent was evaporated at reduced
pressure and the crude product dissolved in diethyl ether
(50 mL), washed with water (3£20 mL) and dried (MgSO4).
Further purification was carried out by column chromato-
graphy, eluting with ethyl acetate/petroleum ether
(40–60 8C) (1:10) to give the product as white crystals
(11.1 g, 94%) mp 60–62 8C (lit.40 62–63 8C) (found: C,
50.51; H, 3.58, C13H11IO requires C, 50.35; H, 3.58%); nmax
(nujol)/cm21 1600, 1119, 972; dH (300 MHz, CDCl3) 7.57
(2H, d, J2,3¼J5,6¼8.9 Hz, H-2, 6), 7.54–7.32 (5H, m, H-20
to H-60), 6.76 (2H, d, J2,3¼J5,6¼8.9 Hz, H-3, 5), 5.04 (2H, s,
OCH2); dC (75.45 MHz, CDCl3) 159.0 (C-4), 138.6 (C-2
and 6), 136.9 (C-10), 129.0 (C-30 and 50), 128.5 (C-40), 127.8

M. F. Oldfield et al. / Tetrahedron 60 (2004) 1887–1893
1891
(C-20 and 60), 117.7 (C-3 and 5), 83.4 (C-1), 70.4 (C-7); m/z
(CI) 311 (MHþ, 35%), 310 (23, Mþ), 184 (97, [M2I]þ), 91
(58, C7H7þ).
(3£30 mL) and dried (MgSO4). After removal of the solvent
at reduced pressure the desired product was obtained as a
pale white solid (3.0 g, 93%); mp 94–96 8C (lit.41 94–
96 8C); nmax (nujol)/cm21 3424 (OH), 1601, 972; dH
(300 MHz, CDCl3) 7.38–7.18 (7H, m, H-20 to 60, H-2 and
3.1.5. 4-Benzyloxybenzo[13C]nitrile (14). To a solution of
4-benzyloxyiodobenzene 13 (20.04 g, 64.62 mmol) in dry
DMF (250 mL) were added calcium hydroxide (2.33 g,
31.5 mmol), potassium [13C]-cyanide (4.30 g, 65 mmol)
and palladium acetate (2.25 g, 10.0 mmol). Under a
nitrogen atmosphere, the mixture was heated at reflux for
4 h, then the DMF was removed under reduced pressure.
The crude product was extracted with diethyl ether
(3£50 mL). The combined organic extracts were then
washed with water (3£50 mL) and dried (MgSO4). Further
purification was carried out by column chromatography,
eluted with ethyl acetate/petroleum ether (40–60 8C) (3:10).
After removal of the solvent, a white solid was obtained
0
0
0
0
6), 6.89 (2H, d, J2 ,3 ¼J5 ,6 ¼8.5 Hz, H-3 and 5), 5.00 (2H, s,
OCH2), 4.54 (2H, d, JC,H¼143 Hz, 13CH2O); dC
(75.45 MHz, CDCl3) 158.8 (C-4), 137.3 (C-10), 133.7 (d,
J¼48 Hz, C-1), 129.1 (C-2 and 6), 129.0 (C-30 and 50), 128.4
(C-40), 127.8 (C-20 and 60), 115.3 (C-3 and 5), 70.4 (C-7),
65.5 (þenhanced, 13CH2O); m/z (EI) 215 (Mþ, 25%), 91 (100,
C7H7 ).
3.1.8. 4-Benzyloxy[methylene-13C]benzyl bromide (17).
To a solution of 4-benzyloxy[methylene-13C]benzyl alcohol
16 (2.50 g, 11.61 mmol) in dry diethyl ether (50 mL) was
added phosphorus tribromide (3.3 mL, 34.83 mmol). The
reaction mixture was stirred at room temperature overnight.
The pale yellow solution formed was then poured into ice
water (200 mL), extracted with diethyl ether (5£30 mL) and
dried (MgSO4). After removal of the solvent at reduced
pressure a white solid was obtained (3.02 g, 93%) and this
product was employed in next step immediately without
further purification. dH (300 MHz, CDCl3) 7.37–7.23 (7H,
(9.52 g, 70%) mp 96–99 8C; (found: C, 80.31; H, 5.05; N,
12
6.99.
C
13
13CH11NO requires C, 80.45; H, 5.27; N, 6.66%);
nmax (KBr)/cm21 2169 (CN); dH (300 MHz, CDCl3) 7.60
(2H, dd, J2,3¼J5,6¼9.0 Hz, JC,H¼5.0 Hz, H-2 and 6), 7.43–
7.34 (5H, m, H-20 and 60), 7.03 (2H, d, J2,3¼J5,6¼9.0 Hz,
H-3 and 5), 5.12 (2H, s, OCH2); dC (75.45 MHz, CDCl3)
162.3 (C-4), 136.1 (C-10), 134.0 (C-2 and 6), 129.2 (C-30 and
50), 128.8 (C-40), 127.9 (C-20 and 60), 119.6 (enhanced
13CN), 116.0 (C-3 and 5), 104.6 (d, JC,C¼83 Hz, C-1), 70.7
(C-7); m/z (CI) 211 (MHþ, 100%), 91 (10, C7H7þ).
m, H-20 to 60, H-2 and 6), 6.86 (2H, d, J2 ,3 ¼J5 ,6 ¼8.6 Hz,
H-3 and 5), 4.99 (2H, s, OCH2), 4.43 (2H, d, JC,H¼153 Hz,
13CH2Br); m/z (EI) 277/279 (Mþ, 5%), 198 (50, [M2Br]þ),
91 (100, C7H7þ).
0
0
0
0
3.1.6. 4-Benzyloxy[carboxy-13C]benzoic acid (15).
4-Benzyloxybenzo[13C]nitrile 14 (8.53 g, 40.6 mmol) was
mixed with sodium hydroxide (2 N, 500 mL) and methanol
(50 mL). The reaction was followed by TLC (silica, ethyl
acetate/methanol (50:1)) and heated under reflux until the
starting material had disappeared. The resulting mixture was
cooled and adjusted to pH 1 with concentrated hydrochloric
acid. The solid that precipitated was collected, then heated
to reflux with sodium hydroxide (2 N, 500 mL) for another
8 h. The resulting mixture was then cooled and adjusted to
pH 1 with concentrated hydrochloric acid again, extracted
with diethyl ether/methanol (6:1). The organic layers were
combined, washed with brine (2£50 mL), dried (MgSO4)
and the solvent removed at reduced pressure to give the
3.1.9. 40-Benzyloxy[1,2-13C2]phenylacetonitrile (18).
40-Benzyloxy[methylene-13C]benzyl bromide 17 (2.80 g,
10.06 mmol) was dissolved in acetonitrile (120 mL) then
18-crown-6 (2.66 g, 10.06 mmol) and potassium 13C-
cyanide (666 mg, 10.06 mmol) were added. The mixture
was heated under reflux for 4 h, then cooled, and the solvent
was removed to give a pale white residue, which was then
extracted with diethyl ether (3£30 mL). The combined ether
layers were washed with water (2£30 mL), dried (MgSO4)
and the solvent removed at reduced pressure. Purification by
column chromatography on silica, eluting with petroleum
ether (40–60 8C)/ethyl acetate (10:3 to 2:1) gave the desired
product as a white solid (1.82 g, 80%); mp 66–69 8C;
12
product as white crystals (7.74 g, 83%) mp 192–196 8C;
12
(found: C, 80.46; H, 5.82; N, 6.15.
C
13C2H13NO
13
(found: C, 73.58; H, 5.08.
C
13CH12O3 requires C, 73.78;
requires C, 80.86; H, 5.82; N, 6.22%); nmax (nujol)/cm21
13
H, 5.28%); nmax (nujol)/cm21 1648 (CO2H), 1601, 972; dH
(300 MHz, CDCl) 7.94 (2H, dd, J2,3¼J5,6¼9.0 Hz,
JC,H¼3.9 Hz, H-2 and 6), 7.38–7.24 (5H, m, H-20 and 60),
6.92 (2H, d, J2,3¼J5,6¼9.0 Hz, H-3 and 5), 5.04 (2H, s,
OCH2); dC (75.45 MHz, CDCl3) 168.9 (enhanced,
13COOH), 0162.7 0(C-4), 136.70 (C-10), 132.2 (C-20 and 6),
129.0 (C-3 and 5 ), 128.5 (C-4 ), 127.8 (C-20 and 6 ), 123.7
(d, JC,C¼74 Hz, C-1), 114.7 (C-3 and 5), 70.4 (C-7); m/z
(EI) 229 (Mþ, 10%), 91 (100, C7H7þ).
2192 (CN); dH(300 MHz, CDCl3) 7.46–7.21 (H, m, H-20 to
60, H-2 and 6), 6.98 (2H, d, J2 ,3 ¼J5 ,6 ¼8.7 Hz, H-3 and 5),
5.08 (2H, s, OCH2), 3.69 (2H, dd, JC,H¼136, 10.6 Hz,
13CH123CN); dC (75.45 MHz, CDCl3) 158.9 (C-4), 137.0
(C-10), 129.05 (C-2 and 6), 129.0 (C-30 and 50), 128.5 (C-40),
0
0
0
0
127.8 (C-2 and 60), 122.4 (d, J2,1 ¼42 Hz, C-1), 118.6
0
(enhanced d, J1,2¼58 Hz, 13CN), 115.9 (C-3 and 5), 70.5
(C-7), 23.2 (enhanced d, J1,2¼58 Hz, 13CH2); m/z (EI) 225
(Mþ, 10%), 91 (100, C7H7þ).
3.1.7. 4-Benzyloxy[methylene-13C]benzyl alcohol (16).
Under the protection of a nitrogen atmosphere, 4-benzyl-
oxy[carboxy-13C]benzoic acid 15 (3.50 g, 15.27 mmol) in
dry THF (50 mL) was added dropwise to a suspension of
lithium aluminium hydride (2.277 g, 60 mmol) in dry THF
(60 mL). The mixture was stirred at room temperature
overnight, then sulfuric acid (10%, 150 mL) was added
carefully to quench the reaction. The resulting mixture was
extracted with diethyl ether (4£50 mL), washed with water
3.1.10. 40-Benzyloxy[1,2-13C2]phenylacetic acid (19).
40-Benzyloxy[1,2-13C2]phenylacetonitrile 18 (1.666 g,
7.40 mmol) was dissolved in aqueous sodium hydroxide
(2 N, 90 mL) and heated to reflux for 6 h, then cooled and
adjusted pH to 1 with concentrated hydrochloric acid. The
precipitate formed was collected by filtration, washed with
plenty of water and then diethyl ether. After drying under
vacuum, the desired product was obtained as a white solid
(1.77 g, 98%) mp 120–122 8C; (lit.42 120–122 8C) nmax

1892
M. F. Oldfield et al. / Tetrahedron 60 (2004) 1887–1893
(nujol)/cm21 1654 (CO2H), 1600, 970; dH (300 MHz,
CDCl3) 7.37–7.21 (5H, m, H-20 and 60), 7.12 (2H, m,
hydrochloric acid (1 M, 70 mL) and stirred for a further
2 h. The suspension formed was left at room temperature
overnight. The precipitate was then collected by filtration,
washed with water (3£5 mL), then diethyl ether (3£2 mL)
and dried under vacuum. Further recrystallisation of the
product from methanol gave the pure product as white solid
(72 mg, 80%) mp 221 8C (lit.31 212–214 8C); lmax
(EtOH)/nm 262 (1/23894 dm3 mol21 cm21, lit.36 24,739);
0
0
0
0
H-2, 6), 6.86 (2H, d, J2 ,3 ¼J5 ,6 ¼8.7 Hz, H-3 and 5), 4.97
(2H, s, OCH2), 3.49 (2H, dd, JC,H¼129, 7.7 Hz, 13CH2); dC
(75.45 MHz, CDCl3) 177.2 (enhanced d, J1,2¼56 Hz,
13COOH), 0158.4 0(C-4), 137.30 (C-10), 130.8 (C-20 and 6),
129.0 (C-3 and 5 ), 128.4 (C-4 ), 127.9 (C-20 and 6 ), 126.2
(d, J¼48 Hz, C-1), 115.3 (C3, 5), 70.4 (C-7), 40.5
(enhanced d, J1,2¼56 Hz, 13CH2); m/z (EI) 244 (Mþ,
15%), 91 (100, C7H7þ).
n
max (nujol)/cm21 3360 (OH), 1735 (CvO0); dH (300 MHz,
d 6-acetone) 9.6 (1H, s, 7-OH), 8.4 (1H, s, 4 -OH), 8.06 (1H,
ddd, J5,6¼8.8 Hz, J4,5¼3.8 Hz (13C–1H coupling),
J5,8¼1.3 Hz, H-5), 8.14 (1H, t, J2,3¼J2,4¼6.4 Hz (13C–1H
3.1.11. 40-Hydroxy[1,2-13C2]phenylacetic acid (20).
40-Benzyloxy[1,2-13C2]phenylacetic acid 19 (300 mg,
1.23 mmol) was dissolved in ethyl acetate (12 mL). After
flushing with nitrogen, palladium on carbon (10%, 100 mg)
was added. The mixture was stirred under a hydrogen
atmosphere at room temperature overnight. The mixture
was then filtered through celite, and the solvent removed at
reduced pressure to give the product as a pale white solid
(181 mg, 96%) mp 149–151 8C (lit.42 149–152 8C); (found:
C, 63.29; H, 5.15. 12C613C2H8O3 requires C, 63.63; H,
5.23%); nmax (nujol)/cm21 3395 (OH), 1654 (CO2H); dH
0
0
0
0 0
0
couplings), H-2), 7.48 (2H, dd, J2 ,3 ¼J5 ,6 ¼8.6 Hz,
13
1
0
0
J3,2 ¼J3,6 ¼3.5 Hz ( C– H coupling), 2 and H-6 ), 7.0
(1H, m, H-6), 6.9 (1H, dt, J¼162 Hz (13C–1H coupling),
0
0
0
0
0
J6,8¼1.9 Hz, H-8), 6.89 (2H, d, J2 ,3 ¼J5 ,6 ¼8.6 Hz, H-3
and 50); dC (75.45 MHz, d 6-acetone) 178.8 (d, enhanced,
J3,4¼54 Hz, C-4), 126.0 (d, enhanced, J3,4¼54 Hz, C-3),
103.5 (enhanced, C-8); m/z (CI) 258.0750 (MHþ,
12
C
12
13C3H11O4 requires 258.0758).
0
0
0
0
(300 MHz, CDCl3, ppm) 7.03 (2H, dd, J2 ,3 ¼J5 ,6 ¼8.4 Hz,
Acknowledgements
JC,H¼4.2 Hz, H-20 and 60), 6.70 (2H, d, J2 ,3 ¼J5 ,6 ¼8.4 Hz,
H-3 and 5), 3.42 (2H, dd, JC,H¼129, 7.6 Hz, 13CH2); dC
(75.45 MHz, CDCl3) 175.0 (enhanced, d, 0 J1,2¼55 Hz,
13COOH), 156.1 (C-40), 130.5 (C-200 and 6 ), 125.5 (d,
0
0
0
0
This work was funded by the FSA under contracts T05001
and T05023. We would also like to thank Dr. Don Clarke at
the Central Science Laboratory, York and Dr. Philip Grace
at the MRC Dunn Human Nutrition Unit for their analytical
support.
0
0
0
J2,1 ¼42 Hz, C-1 ), 115.6 (C-3 and 5 ), 40.5 (enhanced d,
J1,2¼55 Hz, 13CH2); m/z (ESþ) 177 ([MþNa]þ, 100%); m/z
(ES2) 153 [(M2H)2, 65%].
3.1.12. [1,2,30-13C3]-1-(20,40-Dihydroxyphenyl)-2-(400-
hydroxyphenyl)ethanone 0 (21). To [2-13C]resorcinol 11
(100 mg, 0.9 mmol) and 4 -hydroxy[1,2-13C2]phenylacetic
acid 20 (139 mg, 0.9 mmol) was added boron trifluoride
diethyl etherate (5 mL) under a nitrogen atmosphere. The
mixture was heated at 70–80 8C for 3 h, then cooled, and
poured into saturated sodium acetate (50 mL) and basified
with saturated sodium hydrogen carbonate (40 mL). The
mixture was then extracted with diethyl ether (4£20 mL),
the organic layers combined and concentrated at reduced
pressure to yield a pink gum. This crude product was
purified by column chromatography on silica, eluting with
dichloromethane/ethyl acetate (4:1) to give the desired
product as a white solid (114 mg, 55%) mp 186–188 8C
(lit.30 188–190 8C); nmax (nujol)/cm21 3395 (OH), 1610
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