Synthesis of [4-13C]-isoflavonoid phytoestrogens

Article abstract of DOI:10.1016/S0040-4020(99)01014-5

Efficient syntheses are described for ¹³C-labelled derivatives of the isoflavonoid phytoestrogens, genistein, biochanin A, daidzein and formononetin, for use in metabolic studies. The synthetic procedure employs ¹³C-labelled cyanide as the source of the label to produce the isoflavones with a single ¹³C atom at the C-4 position. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(99)01014-5

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 455–460
Synthesis of [4-13C]-Isoflavonoid Phytoestrogens1
*
Jacqueline L. Whalley, Mark F. Oldfield and Nigel P. Botting
School of Chemistry, University of St. Andrews, St. Andrews, Fife, KY16 9ST, UK
Received 20 September 1999; revised 28 October 1999; accepted 11 November 1999
Abstract—Efficient syntheses are described for 13C-labelled derivatives of the isoflavonoid phytoestrogens, genistein, biochanin A, daidzein
and formononetin, for use in metabolic studies. The synthetic procedure employs 13C-labelled cyanide as the source of the label to produce
the isoflavones with a single 13C atom at the C-4 position. ᭧ 2000 Elsevier Science Ltd. All rights reserved.
There is currently considerable interest in phytoestrogens
with respect to their impact on human health. The iso-
flavonoid phytoestrogens, daidzein (1; R1ˆH, R2ˆH) and
genistein (1; R1ˆOH, R2ˆH), 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.2 These compounds have also been
implicated in the prevention of cardiovascular disease,3
lessening the symptoms of the menopause4 and protection
against osteoporosis.5 Furthermore, recent evidence
suggests a role in the central nervous system, stimulating
nerve growth, and action as an antioxidant against endogen-
ous toxins that produce free radicals in the CNS which are
associated with the development of Alzheimer’s disease.6 In
order to better understand, quantify and deduce the impor-
tance of these biological effects there is a need for the devel-
opment of an efficient synthesis of isotopically labelled
genistein and daidzein. Such materials are of great value
as both internal standards for analysis and in metabolic
studies. Indeed there are still many unanswered questions
regarding the bioavailability and mammalian metabolism of
daidzein and genistein.
13C-labelled daidzein (1; R1ˆH, R2ˆH), formononetin (1;
R1ˆH, R2ˆCH3), genistein (1; R1ˆOH, R2ˆH), and
biochanin A (1; R1ˆOH, R2ˆCH3) would be prepared by
adapting existing methodologies. The final step of most
isoflavone syntheses involves the formylation and cyclisa-
tion of a suitable deoxybenzoin precursor 2. Various
reagents have been used to provide the necessary one carbon
fragment including dimethyl formamide dimethylacetal in
THF,7 dimethyl formamide followed by mesyl chloride8 and
1,3,5-triazine with boron trifluoride etherate.9 (Scheme 1)
Preliminary studies were therefore carried out to investigate
the first two methods to examine the possibility of using
13C-labelled dimethyl formamide to introduce the label at
the C-2 position in the final step. Unfortunately preliminary
studies with unlabelled daidzein demonstrated that a large
excess of dimethylformamide, or its dimethyl acetal were
required in order to obtain reasonable yields. This would be
feasible if recovery of unreacted labelled starting material
was also efficient but this was not the case.
An alternative strategy was therefore sought. The deoxy-
benzoins are normally prepared via condensation of a
phenol and either a substituted phenylacetic acid, using
boron trifluoride as catalyst, or benzyl nitrile via a Hoesch
reaction.10 An alternative route thus involved the use of
13C-labelled cyanide to prepare the nitrile, which would
A number of synthetic routes towards the isoflavones
have been previously developed and it was proposed that
Scheme 1. (a) DMF dimethylacetal, THF, reflux or DMF then mesyl chloride or BF3·Et2O, 1,3,5-triazine.
Keywords: isoflavones; labelling; antitumour compounds; plants.
* Corresponding author. Tel.: ϩ44-334-463856; fax: ϩ44-334-463808; e-mail: npb@st-andrews.ac.uk
0040–4020/00/$ - see front matter ᭧ 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(99)01014-5

456
J. L. Whalley et al. / Tetrahedron 56 (2000) 455–460
Scheme 2. (a) TMSBr, Et2O (98%); (b) K13CN, 18-Crown-6, MeCN (86%); (c) 2M NaOH (aq), reflux (79%); (d) Resorcinol, BF3·Et2O (66%); (e) DMF(OMe)2
(62%); (f) BBr3, CH2Cl2 (89%).
lead to labelling at the C-4 position of the isoflavone
(Scheme 2). The proposed route was first optimised with
unlabelled material and then used to prepare both 13C
labelled formononetin and daidzein.
and so it was deduced that the contamination was due to
inorganic material, probably boron salts. Unfortunately,
attempts to remove the boron salts by extraction, recrystal-
lisation, normal phase column chromatography and reverse
phase column chromatography failed. In order to obtain
pure 8 it was therefore clear that the best strategy
would be to modify the synthetic route to avoid the final
demethylation step.
Firstly, commercially available 4-methoxybenzyl alcohol 3
was brominated, so that nucleophilic displacement of the
bromide could be carried out using cyanide ion. A range
of conditions was examined for the nucleophilic displace-
ment, using a number of solvents including DMSO and
DMF. However the optimum conditions that were finally
established employed 13C-labelled potassium cyanide in
acetonitrile in the presence of 18-crown-6 at room tempera-
ture. This gave the desired nitrile 4 in good yield. The
carboxylic acid 5 was then obtained by subsequent basic
hydrolysis. Formation of the deoxybenzoin 6 was effected
by treatment of resorcinol and the 13C-labelled acid with
boron trifluoride etherate in THF.11 Dimethylformamide
dimethylacetal7 was then employed for the formylation
and cyclisation reaction to give [4-13C]-formononetin 7.
This gave identical spectral data to authentic material.
The presence of the 13C-label was confirmed by mass
spectrometry, showing less than 1% unlabelled material,
and by the enhanced signal due to the carbon at the
4-position in the 13C NMR spectrum. Conversion to daid-
zein then required de-methylation of the 40-methoxy group.
The most efficient reagent for this transformation was found
to be boron tribromide in dichloromethane,12 which afforded
the [4-13C]-daidzein 8 in 89% yield. However analysis of the
[4-13C]-daidzein by GC-MS in comparison with a reference
standard of unlabelled material revealed that it was only
54% pure. NMR analysis showed no organic impurities
It was decided to protect the 4-hydroxy group as its benzyl
ether, which would then be removed under the acidic con-
ditions required for the Hoesch reaction10 to form the
deoxybenzoin. This alternative route is shown in Scheme
3. Thus the nitrile 11 was prepared, as described for the
methoxy analogue, from commercially available 4-benzyl-
oxybenzyl alcohol. The Hoesch reaction was achieved by
treatment of a solution of resorcinol, nitrile 11 and catalytic
zinc chloride in diethyl ether with hydrogen chloride to give
the desired deoxybenzoin 12 in good yield. The modifica-
tion of the original Hoesch reaction, employing only
catalytic zinc chloride, was recently reported by Pelter
et al.13 Formylation and cyclisation of 12 afforded the
[4-13C]-daidzein 13 which was then successfully purified
by flash chromatography.
Synthesis of biochanin A and genistein proved to be more
problematic due to differences in their properties which are
a result of the additional hydroxyl group at the 5-position.
This hydroxyl group hydrogen bonds with the C-4 carbonyl
reducing its reactivity, such that some of the formylation/
cyclisation procedures were unsuitable, e.g. the use of
dimethylformamide dimethylacetal in THF. Also the
Scheme 3. (a) TMSBr, Et2O (89%); (b) K13CN, 18-Crown-6, MeCN (92%); (c) Resorcinol, HCl, cat. ZnCl2, Et2O (96%); (d) DMF(OMe)2 (60%).

J. L. Whalley et al. / Tetrahedron 56 (2000) 455–460
457
Scheme 4. (a) Phloroglucinol, HCl, cat. ZnCl2 (75%); (b) BF3·OEt2, DMF, microwave, then MeSO2Cl, microwave (54%).
Scheme 5. (a) Phloroglucinol, HCl, cat. ZnCl2 (75%); (b) BF3·OEt2, DMF, microwave, then MeSO2Cl, microwave (54%).
compounds exhibit reduced solubility in the organic
solvents used in the daidzein synthesis. Various attempts
were made to tackle these problems, including investigation
into protection of all the free hydroxyl groups followed by
deprotection after cyclisation. However these strategies add
extra steps to the synthesis lowering the overall yield of
labelled material and deprotection again resulted in boron
salt contamination as observed in the synthesis of daidzein
from formononetin described above.
prepared on a gram scale and samples of these materials
are now being employed in metabolic studies. Further
work involves the modification of the synthetic procedures
so that two or three 13C atoms can be incorporated into either
daidzein or genistein.
Experimental
General
Recently a procedure was described,14 involving reaction of
the appropriate deoxybenzoin with BF3·Et2O and DMF
using a short burst of microwave irradiation as the heat
source. Mesyl chloride was then added and the microwave
treatment repeated. Addition of water then resulted in pre-
cipitation of the product. Initially, this method appeared to
work for the synthesis of unlabelled genistein on a small
scale, but the results were very variable. However, it was
found that longer periods of microwave heating produced
larger yields and a more reliable procedure, especially when
working on a larger scale. With this reaction optimised, the
overall synthesis of [4-13C]-biochanin A was achieved using
a similar procedure to that for formononetin (Scheme 4). In
this case the nitrile 4 was reacted with phloroglucinol under
Hoesch conditions to give the deoxybenzoin 14. Formyl-
ation and cyclisation were then achieved under microwave
conditions using DMF and mesyl chloride to afford [4-13C]-
biochanin A 15. Spectral data confirmed the presence of the
13C atom at the C-4 position.
NMR spectra were recorded on a Varian Gemini FT spec-
trometer (1H, 200 MHz; 13C, 50.31 MHz) or a Varian
Gemini 2000 spectrometer (1H 300 MHz; 13C 75.45 MHz).
1H NMR spectra were referenced to chloroform, TMS,
methanol or d6-DMSO, 13C NMR spectra were referenced
to chloroform, methanol or d6-DMSO. Elemental analyses
were carried out in the departmental microanalytical labora-
tory. IR spectra were recorded on a Perkin–Elmer series
1500 FT IR spectrophotometer. The samples were prepared
as Nujol mulls or thin films between sodium chloride discs
and recorded in cmϪ1. Mass spectra were recorded on a
Kratos MS50. The microwave reactions were carried out
using a Panasonic 650 W machine set to “simmer”. Analysis
by TLC was carried out on Merck 5785 Kieselgel 60F254
fluorescent plates. Flash chromatography was performed
according to the procedure of Still15 using silica gel (Fisons
matrix 35–70 mm). Diethyl ether and dichloromethane were
dried by passing down an alumina column and distilling
from calcium hydride. Dimethylformamide was distilled
from magnesium sulfate. Tetrahydrofuran was passed
down a dry alumina column and distilled from sodium
metal and benzophenone.
Modification of the synthesis then allowed the preparation
of [4-13C]-genistein 17 (Scheme 5). As for daidzein 8, the
O-benzylated bromide was used as starting material and the
protecting group cleaved during the Hoesch reaction to
provide 16. Microwave conditions were then employed for
the final formylation/cyclisation step, giving the product in
54% yield. Spectral data for the [4-13C]-genistein 17
confirmed the presence of the desired 13C atom at the
4-position and the material was shown to be pure by
microanalysis.
4-Methoxybenzyl [13C]cyanide (4). To 4-methoxybenzyl
alcohol 3 (2.0 g, 14.5 mmol) in dry diethyl ether (60 ml)
under nitrogen at 0ЊC was added trimethylsilyl bromide
(4.5 g, 29.6 mmol) and the reaction mixture allowed to
warm slowly to room temperature. After 16 h, water
(120 ml) was added and the aqueous phase extracted with
diethyl ether ꢀ4×60 ml†. The combined organic phases
were washed with brine (150 ml), dried over anhydrous
magnesium sulfate, and concentrated to provide pure
Using the synthetic methodology described above, both
[4-13C]-daidzein and [4-13C]-genistein have now been

458
J. L. Whalley et al. / Tetrahedron 56 (2000) 455–460
bromide (3.0 g, 98%) as a colourless liquid. To the bromide
(3.0 g, 15.0 mmol) was immediately added acetonitrile
(200 ml), 18-crown-6 (5.2 g, 15.0 mmol) and 13C-potassium
cyanide (1.0 g, 15.0 mmol). The resultant mixture was
heated at reflux with the protection of a calcium chloride
tube for 4 h. Concentration of the reaction mixture followed
by dilution with diethyl ether, filtration through a pad of
silica gel (Fisons matrix 35–70 m) and subsequent concen-
tration of the filtrate provided pure nitrile 4 (1.9 g, 86%) as a
colourless oil; (Found: C, 73.08; H, 6.25; N, 9.27.
C813C1H9ON requires C, 72.96; H, 6.12; N, 9.45); nmax
(nujol)/cmϪ1 2191 (CN); dH (200 MHz, CDCl3) 3.60 (2H,
d, JC,Hˆ5.3 Hz, CH2), 3.72 (3H, s, OCH3), 6.84 (2H, d,
J2,3ˆJ5,6ˆ7.7 Hz, 3, 5-H), 7.16 (2H, d, J2,3ˆJ5,6ˆ7.7 Hz,
2, 6-H); dC (75.45 MHz, CDCl3) 22.9 (2H, d, Jˆ28 Hz,
CH2), 55.6 (OCH3), 114.9 (3-C and 5-C), 119.1 (enhanced,
13CN), 122.6 (1-C), 129.6 (2-C and 6-C), 159.8 (4-C); m/z
(EI) 148 (Mϩ, 100%).
combined ethereal layers were washed with water (10 ml),
brine ꢀ2×50 ml† and dried over magnesium sulfate to give a
dark red oil. Purification by flash chromatography (employ-
ing gradient elution 6:4 toluene/ethyl acetate to 100% ethyl
acetate) gave 6 as a pale orange solid (3.1 g, 92%); mp
152ЊC; (Found: C, 69.98; H, 5.42. C1413C1H14O4 requires
C, 69.87; H, 5.44); nmax (nujol)/cmϪ1 3350 (OH), 2880
(OCH3), 1617 (CyO); dH (200 MHz, CD3OD), 3.72 (3H,
s, OCH3), 4.10 (2H, d, JC,Hˆ6.2 Hz, –CH2–), 6.26 (1H, d,
0
0
0
0
0
0
0
J3 ,5 ˆ2.2 Hz, 3 -H), 6.36 (1H, dd, J3 ,5 ˆ2.2 Hz, J5 ,6
ˆ
8.9 Hz, 50-H), 6.83 (2H, d, J2,3ˆJ5,6ˆ8.6 Hz, 3, 5-H), 7.15
0
0
(2H, d, J2,3ˆJ5,6ˆ8.6 Hz, 2, 6-H), 7.81 (1H, dd, J5 ,6
ˆ
8.9 Hz, JC,Hˆ4.9 Hz, 60-H); dC (50.3 MHz, CD3OD), 44.9
(d, Jˆ41.2 Hz, –CH2–), 55.9 (OCH3), 104.0 (30-C), 109.4
(50-C), 113.9 (d, Jˆ57 Hz, 10-C), 115.3 (3-C and 5-C),
128.6 (1-C), 131.7 (2-C and 6-C), 134.7 (60-C), 160.3
(4-C), 166.7 (40-C), 167.1 (20-C), 204.4 (enhanced,
13CvO); m/z (EI) 259 (Mϩ, 14%), 138 (100), 121 (17).
4-Benzyloxybenzyl [13C]cyanide (11). Nitrile 11 was pre-
pared in the same manner as nitrile 4 by treating 4-benzyl-
oxybenzyl alcohol 9 with trimethylsilyl bromide to give the
bromide 10 (1.0 g, 89%) which was treated with 18-crown-6
and 13C-potassium cyanide to afford 11 (0.75 g, 92%) as a
white solid; mp 66ЊC; (Found: C, 81.12; H, 7.14; N, 6.25.
C1413C1H13ON requires C, 80.90; H, 7.13; N, 6.24); nmax
(nujol)/cmϪ1 2193 (CN); dH (200 MHz, CDCl3) 3.51 (2H,
d, JC,Hˆ5.4 Hz, CH2), 4.92 (2H, s, OCH2Ph), 6.84 (2H, d,
J2,3ˆJ5,6ˆ7.7 Hz, 3, 5-H), 7.16 (2H, d, J2,3ˆJ5,6ˆ7.7 Hz, 2,
6-H), 7.35 (5H, m, OCH2Ph); dC (50.3 MHz, CDCl3) 21.3
(d, Jˆ28 Hz, CH213CN), 70.0 (OCH2Ph), 115.5 (3-C and
40-Methoxybenzyl-7-hydroxy-[4-13C]isoflavone ([4-13C]-
formononetin) (7). N,N-Dimethylformamide dimethyl ace-
tal (162 mg, 180 ml, 1.36 mmol) was added dropwise to
deoxybenzoin 6 (200 mg, 0.78 mmol) in THF (2 ml) under
nitrogen at reflux. After 4 h, the dark red solution was
cooled to room temperature and methanol (5 ml) was
added. Repeated concentration after addition of further
5 ml portions of methanol, gave an orange solid which
was recrystallised from 75% aqueous methanol to afford
pure 7 (129 mg, 62%) as a pale beige solid; mp 257ЊC
(lit.7 256–257ЊC); (Found: C, 71.09; H, 4.41.
C1513C1H12O4 requires C, 71.37; H, 4.49); nmax (nujol)/
cmϪ1 3350 (OH), 1616 (CyO); dH (200 MHz, d6-DMSO)
3.81 (3H, s, OCH3), 6.9 (2H, m, 6, 8-H), 7.0 (2H, d,
13
5-C), 118.2 (enhanced, CN), 122.0 (1-C), 127.5 (20-C
and 60-C), 128.1 (30-C and 50-C) 128.7 (40-C), 129.1 (2-C
and 6-C), 136.4 (10-C), 159.8 (4-C); m/z (EI) 224 (Mϩ,
14%).
0
0
0
0
0
0
0
0
J2 ,3 ˆJ5 ,6 ˆ8.6 Hz, 3 ,5 -H), 7.5 (2H, d, J2,3ˆJ5 ,6
ˆ
8.6 Hz, 20,60-H), 8.0 (1H, dd, J5,6ˆ9 Hz, J4,5ˆ4 Hz
(13C-1H coupling), 5-H), 8.4 (1H, d, J2,4ˆ6 Hz (13C-1H
coupling), 2-H); dC (50.31 MHz, d6-DMSO) 55.2 (OCH3),
102.2 (8-C), 113.7 (30-C and 50-C), 115.2 (6-C), 116.3 (d,
Jˆ57 Hz, 4a-C), 122.6 (10-C), 123.4 (1-C), 124.2 (d,
Jˆ56 Hz, 3-C), 127.3 (5-C), 130.1 (20-C and 60-C), 153.0
(2-C), 157.1 (7-C), 159.0 (8a-C), 162.6 (40-C), 174.7
(enhanced, 4-13C); m/z (EI) 269 (Mϩ, 62%), 254 (14,
MϩϪCH3). m/z (EI) 269 (Mϩ, 100%), 254 (14%,
MϩϪCH3).
40-Methoxyphenyl [1-13C]acetic acid (5). 4-Methoxy-
benzyl [13C]cyanide 4 (945 mg, 6.43 mmol), was heated to
reflux with aqueous 2 M sodium hydroxide (45 ml) for 1 h.
Upon cooling the solution was acidified to pH 2 using
concentrated hydrochloric acid and the solution boiled to
dissolve the precipitate. After cooling overnight at 4ЊC
(refrigerator), the solid was collected and dried over phos-
phorus pentoxide to afford 5 as a white solid (742 mg, 79%);
mp 79ЊC; (Found: C, 66.03; H, 4.82. C813C1H8O3 requires C,
66.05; H, 4.88); nmax (nujol)/cmϪ1 2810 (OCH3), 1654
(13CO2H); dH (200 MHz, CD3OD) 3.55 (2H, d,
JC,Hˆ7.0 Hz, –CH2–), 3.78 (3H, s, OCH3), 6.86 (2H, d,
4-Hydroxybenzyl-20,40-dihydroxyphenyl [13C]ketone (12).
To a vigorously stirred solution of the [13C]-nitrile 11
(500 mg, 2.23 mmol) and resorcinol (297 mg, 2.70 mmol)
in dry diethyl ether (15 ml) under nitrogen was added
freshly fused zinc chloride (37 mg, 0.03 mmol) via a dry
addition funnel. The nitrogen inlet was then replaced with
a calcium chloride drying tube and the reaction mixture
saturated with dry hydrogen chloride at 0ЊC for 4 h, during
which time a solid crust had formed on the edge of the
reaction vessel. After stirring for 16 h at room temperature
the ether layer was decanted and the residue washed twice
with ether (10 ml). 1N aqueous hydrochloric acid was added
to the residue and the solution heated at reflux for 2 h under
nitrogen, then cooled and the resultant orange solid filtered
off and purified by flash chromatography eluting with
diethyl ether to afford [13C]-deoxybenzoin 12 (543 mg,
96%): mp 189ЊC (lit. 188–190ЊC); (Found: C, 67.13; H,
12.08. C1313C1H12O4 requires C, 67.15; H, 12.09); nmax
0
0
0
0
0
0
0
0
J2,3ˆJ5 ,6 ˆ8.7 Hz, 3 ,5 -H), 7.19 (2H, d, J2 ,3 ˆJ5 ,6
ˆ
8.7 Hz, 20,60-H); dC (50.3 MHz, CD3OD) 40.8 (d,
Jˆ41.6 Hz, –CH2–), 55.9 (OCH3), 115.2 (30-C and 50-C),
128.1 (10-C), 131.1 (20-C and 60-C), 164.0 (40-C), 178.3
(enhanced, 13CO2H); m/z (EI) 165 (Mϩ, 36%),
147 ((MϪH2O)ϩ, 25), 120 ((MϪCO2H)ϩ, 17).
4-Methoxybenzyl-20,40-dihydroxyphenyl [13C]ketone (6).
To acid 5 (2.2 g, 13.0 mmol) in BF3·OEt2 (0.5 ml) under
nitrogen was added resorcinol (2.9 g, 26.0 mmol) portion-
wise over 10 min. The solution was heated to reflux for
10 min, cooled and then aqueous saturated sodium acetate
(30 ml) and saturated aqueous sodium hydrogen carbonate
(15 ml) were added sequentially to give an orange oily layer
which was extracted with diethyl ether ꢀ3×50 ml†. The

J. L. Whalley et al. / Tetrahedron 56 (2000) 455–460
459
(nujol)/cmϪ1 3450 (OH), 1636 (CyO); dH (200 MHz, d6-
40-Methoxybenzyl-5,7-dihydroxy-[4-13C]isoflavone
([4-13C]biochanin A) (15). Boron trifluoride etherate
(10 ml, 11.5 g, 81.3 mmol) was added to a solution of
deoxybenzoin 14 (500 mg, 1.82 mmol) in dimethylforma-
mide (20 ml) in a beaker covered with a watch glass and
heated employing a microwave (800 W) for 2 × 15 s on
‘simmer’ setting. The solution was swirled, methane sul-
fonyl chloride (10 ml, 14.8 g, 129.2 mmol) was added and
the reaction mixture heated in short bursts of 2 × 30 s then
2×15 s on simmer. After cooling, water (100 ml) was added
and the reaction mixture stirred for 4 h. The solid was
filtered off and dried over phosphorus pentoxide. Purifica-
tion by flash chromatography (gradient elution 100%
diethyl ether to 10:90 methanol/diethyl ether) provided
pure [4-13C]-biochanin A 15 (279 mg, 54%) as a white
solid: mp 194–196ЊC; (Found: C, 67.71; H, 4.22.
C1513C1H12O5 requires C, 67.37; H, 4.24%); nmax (nujol)/
cmϪ1 3395 (OH), 1610 (CyO); dH (200 MHz, d6-DMSO)
3.80 (3H, s, OCH3), 6.26 (1H, d, J6,8ˆ2.5 Hz, 8-H), 6.41
DMSO) 4.11 (2H, d, JC,Hˆ6.2 Hz, –CH2–), 6.26 (1H, d,
0
0
0
0
0
J3 ,5 ˆ2.2, 3 -H), 6.38 (1H, dd, Jˆ8.8 Hz, J5 ,6 ˆ2.3 Hz,
50-H), 6.83 (2H, d, J2,3ˆJ5,6ˆ8.6 Hz, 3, 5-H), 7.12 (2H, d,
0
0
J2,3ˆJ5,6ˆ8.6 Hz, 2, 6-H) 7.84 (1H, dd, J5 ,6 ˆ8.7 Hz,
JC,Hˆ4.7 Hz, 60-H); dC (50.3 MHz, d6-DMSO) 44.0 (d,
Jˆ41 Hz, CH2), 96.1 (30-C and 50-C), 112.9 (10-C), 116.3
(3-C and 5-C), 128.5 (1-C), 132.1 (2-C and 6-C), 157.2
(4-C), 166.2 (20-C and 60-C), 166.6 (40-C), 205.4 (enhanced,
13CyO); m/z (EI) 245 (Mϩ, 12%), 121 (32), 107 (12).
40-Hydroxybenzyl-7-hydroxy-[4-13C]isoflavone ([4-13C]-
daidzein) (13). N,N-Dimethylformamide dimethyl acetal
(291 mg, 324 ml, 2.44 mmol) was added dropwise to deoxy-
benzoin 12 (200 mg, 0.82 mmol) in THF (2 ml) under nitro-
gen, at reflux. After 16 h, the solution was cooled to ambient
temperature, and methanol (5 ml) was added. Repeated
concentration under reduced pressure after addition of
further 5 ml portions of methanol, gave an orange solid
which was purified by column chromatography (employing
gradient elution 5:1 diethyl ether/petroleum ether 40–60ЊC
to 100% diethyl ether) to provide [4-13C]-daidzein 13 as a
very pale yellow solid (125 mg, 60%): mp 219ЊC (lit.7 212–
214ЊC); (Found: C, 70.34; H, 4.30. C1413C1H10O4 requires C,
70.59; H, 4.00%); nmax (nujol)/cmϪ1 3360 (br, OH), 1735
(CyO); dH (200 MHz, d6-DMSO) 6.89 (3H, m, 30-H, 50-H
and 8-H), 6.95 (1H, dd, J6,8ˆ2 Hz, J5,6ˆ9 Hz, 6-H), 7.4 (2H,
0
0
0
00
(1H, d, J6,8ˆ2.5 Hz, 6-H), 7.03 (2H, d, J2 ,3 ˆJ5 ,6 ˆ7.4 Hz,
0
20,60-H), 7.51 (2H, d, J2 ,3 ˆJ5 ,6 ˆ7.4 Hz, 3 ,5 -H), 8.37
(1H, d, J2,4ˆ6 Hz (13C-1H coupling), 2-H), 9.6 (1H, br s,
7-OH); dC (50.3 MHz, d6-DMSO) 55.4 (OCH3), 102.4
(8-C), 114.0 (30-C and 50-C), 115.4 (6-C), 116.9 (4a-C),
122.8 (10-C), 123.75 (3-C), 127.6 (5-C), 130.4 (20-C
and 60-C), 154.5 (2-C), 157.85 (7-C), 159.4 (8a-C), 164.6
(40-C), 180.4 (enhanced, 4-13C); m/z (CI) 286 (MHϩ,
76%).
0
0
0
0
0
0
0
0
d, J2 ,3 ˆJ5,6ˆ8.5 Hz, 2 ,6 -H), 7.98 (1H, dd, J4,5ˆ4 Hz,
J5,6ˆ9 Hz, 5-H), 8.3 (1H, d, J2,4ˆ6 Hz, 2-H); dC
(50.3 MHz, d6-DMSO) 102.1 (8-C), 114.9 (30-C and 50-
C), 115.1 (6-C), 116.7 (d, Jˆ46 Hz, 4a-C), 122.5 (10-C),
123.5 (d, Jˆ56 Hz, 3-C), 127.2 (5-C), 130.0 (20-C and 60-
C), 153.1 (2-C), 157.1 (40-C), 156.4 (8a-C), 162.4 (7-C),
175.0 (enhanced, 4-13C); m/z (EI) 255 (Mϩ, 54%), 138
(100, C7H4Oϩ3 ).
4-Hydroxybenzyl-20,40,60-trihydroxyphenyl [13C]ketone
(16). 13C-Deoxybenzoin 16 (872 mg, 75%) was prepared
as for 14 by treatment of nitrile 11 (1.0 g, 4.47 mmol) and
phloroglucinol (845 mg, 6.71 mmol) in diethyl ether with
freshly fused zinc chloride (61 mg, 0.05 mmol) followed by
saturation of the reaction mixture with hydrogen chloride.
Purification was carried out by column chromatography
eluting with diethyl ether: mp 258ЊC (lit.13 258–262ЊC);
nmax (nujol)/cmϪ1 3395 (OH), 1610 (CyO); dH (200 MHz,
d6-DMSO) 4.28 (2H, d, JC,Hˆ6.2 Hz, –CH2–), 5.82 (2H, s,
30, 50-H), 6.70 (2H, d, J2,3ˆJ5,6ˆ8.4 Hz, 3, 5-H), 7.06 (2H,
d, J2,3ˆJ5,6ˆ8.4 Hz, 2, 6-H); dC (50.3 MHz, d6-DMSO)
44.04 (d, Jˆ45.1 Hz, CH2), 96.7 (30-C and 50-C), 116.2
(3-C and 5-C), 128.5 (1-C), 132.0 (2-C and 6-C), 157.2
(4-C), 164.6 (20-C and 60-C), 167.4 (40-C), 205 (enhanced
13CyO); m/z (EI) 261 (Mϩ, 7%), 107 (12).
4-Methoxybenzyl-20,40,60-trihydroxyphenyl [13C]ketone
(14). To a vigorously stirred solution of the [13C]-nitrile 4
(1.15 g, 7.76 mmol) and phloroglucinol (1.47 g, 11.64 mmol)
in dry diethyl ether (35 ml) under nitrogen was added
freshly fused zinc chloride (106 mg, 0.78 mmol) via a dry
addition funnel. The nitrogen inlet was then replaced with a
calcium chloride drying tube and the reaction mixture satu-
rated with dry hydrogen chloride at 0ЊC for 4 h, during
which time a solid crust had formed on the surface of the
reaction vessel. After stirring for 16 h at room temperature
the ether layer was decanted and the residue washed twice
with ether (10 ml). 1N aqueous hydrochloric acid was added
to the residue and the solution heated at reflux for 2 h under
nitrogen, then cooled and the resultant orange solid filtered
off and purified by flash chromatography eluting with
diethyl ether to afford [13C]-deoxybenzoin 14 (1.56 g,
75%): mp 158–160ЊC; (Found C, 65.38; H, 5.12.
C1413C1H14O5 requires C, 65.81; H, 5.12.); nmax (nujol)/
cmϪ1 3350 (OH) dH (200 MHz, d6-DMSO) 3.73 (3H, s,
OCH3), 4.30 (2H, d, JC,Hˆ6.3 Hz, –CH2–), 5.82 (2H, s,
30,50-H), 6.80 (2H, d, J2,3ˆJ5,6ˆ8.7 Hz, 3, 5-H), 7.1 (2H,
d, J2,3ˆJ5,6ˆ8.7 Hz, 2, 6-H); dC (50.3 MHz, d6-DMSO) 48.2
(d, Jˆ43.2 Hz, –CH2–), 55.6(OCH3), 96.08 (30-C and 50-
C), 113.5 (10-C), 114.9 (3-C and 5-C), 128.6 (1-C), 132.0 (2-
C and 6-C), 160.4 (4-C), 166.2 (40-C), 166.6 (20-C and 60-
C), 205.2 (enhanced, 13CyO); m/z (EI) 275 (Mϩ, 13%), 184
(100), 126 (32), 121 (27), 86 (17).
40-Hydroxybenzyl-5,6-dihydroxy-[4-13C]isoflavone
([4-13C]genistein) (17). [4-13C]-Genistein (112 mg, 54%)
was prepared using the same method as for biochanin A
15. Microwave heating of a solution of deoxybenzoin 16
(200 mg, 0.77 mmol) in dimethylformamide (8 ml) and
borontrifluoride etherate (5.0 ml, 5.77 g, 40.7 mmol) addi-
tion of methane sulfonyl chloride (5.0 ml, 7.4 g, 64.6 mmol)
and further irradiation: mp 307ЊC (decomp) (lit.13 291–
296ЊC (decomp));16 (Found: C, 66.20; H, 4.02.
C1413C1H10O5 requires C, 66.42; H, 3.72%); nmax (nujol)/
cmϪ1 3395, 1610 (CyO); dH (300 MHz, d6-DMSO) 6.25
(1H, d, Jˆ2 Hz, 8-H), 6.41 (1H, d, Jˆ2 Hz, 6-H), 6.81
0
0
0
0
0
0
(2H, d, J2 ,3 ˆJ5 ,6 ˆ8.2 Hz, 3 -H and 5 -H), 7.4 (2H, d,
0
0
0
0
0
0
J2 ,3 ˆJ5 ,6 ˆ8.2 Hz, 2 -H and 6 -H), 8.4 (1H, d, J2,4ˆ6 Hz
(13C-1H coupling), 2-H); dC (50.3 MHz, d6-DMSO) 94.0
(8-C), 99.1 (6-C), 104.6 (4a-C), 115.8 (30 and 50-C), 121.4
(3-C), 122.4 (10-C), 130.3 (20-C and 60-C), 154.1 (2-C),

460
J. L. Whalley et al. / Tetrahedron 56 (2000) 455–460
157.5 (40-C), 157.7 (5-C), 162.25 (8a-C), 164.6 (7-C), 180.5
(enhanced 4-13C); m/z (CI) 272 (MHϩ, 100%).
5. Anderson, J. J. J. Nutr. 1995, 125, 799.
6. Simpkins, J. W. Pharmacological Treatment of Alzheimer’s
Disease: Molecular and Neurobiological Foundations; Wiley:
New York, 1997.
Acknowledgements
7. Pelter, A.; Foot, S. Synthesis 1976, 326.
8. Bass, R. J. J. Chem. Soc., Chem. Commun. 1976, 78–79.
9. Jha, H. D.; Zilliken, F.; Breitmaier, E. Angew. Chem., Int. Ed.
Engl. 1981, 20, 102–103.
10. Chadderton, J.; Baker, W.; Harborne, J. B.; Ollis, W. D.
J. Chem. Soc. 1953, 1853–1860.
This work was funded by the Ministry of Agriculture
Fisheries and Food (MAFF Contract FS2060). We wish to
thank Dr L. Bluck and Dr J. Liggins at the Dunn Nutritional
Laboratory for carrying out GC-MS analysis.
11. Hase, T.; Wahala, K. J. Chem. Soc., Perkin Trans 1 1991,
3005–3008.
References
12. McOmir, J. F. W.; West, D. E. Org. Synth. 1973, 5, 412.
13. Pelter, A.; Ward, R. S.; Whalley, J. L. Synthesis 1998, 1793–
1802.
14. Cheng, Y.-C.; Nair, M. G.; Santell, R. C.; Helferich, W. G.
J. Agric. Food. Chem. 1994, 42, 1869–1871.
15. Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43,
2923–2925.
1. A preliminary account of some of this work has been published:
Whalley, J. L.; Bond, T. J.; Botting, N. P. Bioorg. Med. Chem. Lett.
1998, 8, 2569–2572.
2. Barnes, S.; Peterson, T. G. Proc. Soc. Exp. Biol. Med. 1995,
208, 103–108.
3. Wu, S. Y.; Brewer, M. S. J. Food Sci. 1994, 59, 702–706.
4. Colditz, G. A.; Stampfer, M. J.; Willett, W. C.; Hunter, D. J.;
Manson, J. E.; Hennekens, C. H.; Rosner, B. A.; Speizer, F. E.
Cancer Causes Control 1992, 3, 433–439.
16. Melting point indistinct due to decomposition but purity
established by both microanalysis and HPLC analysis.