A new short synthesis of coumestrol and its application for the synthesis of [6,6a,11a-13C3]coumestrol

Article abstract of DOI:10.1016/j.tet.2003.11.089

A convenient and simple two-step method for the synthesis of coumestrol has been established, which involves a base catalysed condensation of phenyl acetate with benzoyl chloride, followed by demethylation and subsequent tandem intramolecular cyclisation. This method was then employed for the efficient synthesis of multiply ¹³C-labelled coumestrol.

Full text of DOI:10.1016/j.tet.2003.11.089

Tetrahedron 60 (2004) 1637–1642
A new short synthesis of coumestrol and its application for the
synthesis of [6,6a,11a-13C3]coumestrol
*
Nawaf Al-Maharik and Nigel P. Botting
School of Chemistry, University of St Andrews, St Andrews, Fife, KY16 9ST, Scotland, UK
Received 29 September 2003; revised 3 November 2003; accepted 27 November 2003
Abstract—A convenient and simple two-step method for the synthesis of coumestrol has been established, which involves a base catalysed
condensation of phenyl acetate with benzoyl chloride, followed by demethylation and subsequent tandem intramolecular cyclisation. This
method was then employed for the efficient synthesis of multiply 13C-labelled coumestrol.
q 2003 Elsevier Ltd. All rights reserved.
1. Introduction
plants and biological fluids resulting in considerable
improvements in both sensitivity and reproducibility.
Thus, a similarly 13C-labelled version of coumestrol is a
key synthetic target and may also have application in the
deduction of the metabolic pathway for coumestrol 1 in
mammals.
Coumestans represent a fully oxidised version of the
flavonoid pterocarpans and share the same systematic
numbering.1 A number of coumestans were isolated from
Pueraria and Glycyrrhia.2 The most common and the most
representative, coumestrol 1 (3,9-dihydroxy-6-benzo-
furo[3,2-c][1]benzopyran-6-one), which is related to the
coumarins, was isolated from the roots of Pueraria lubata
and mirifica,3 roots of soybeans,4 the whole plants of
Tephrosia purpurea,5 Phaseolus coccineus6 and lunatus.7 It
is found mainly in red clover sprouts, mature alfalfa
(lucerne) and alfalfa sprouts, soybean sprouts, and kudzu
leaf. However, only small amounts are present in the human
diet.8 Low-coumestrol varieties of alfalfa and red clover are
bred for forage and for commercial extraction of other
phytoestrogens. Coumestrol 1 is an estrogen agonist that has
been shown to be effective in reducing bone loss in model
systems.9 It has a higher binding affinity for the estrogen
receptor than genistein,10 consistent with the receptor
binding model that appears to depend upon the0 phenolic
group in the 40-position of genistein and in the 12 -position
of coumestrol. Its useful biological activities have made it
an attractive synthetic target. In order to better understand,
quantify and deduce the importance of these biological
effects there is a need for the development of stable
isotopically labelled coumestrol derivatives. Recently
multiply 13C-labelled derivatives of isoflavones, a related
class of phytoestrogens which includes daidzein and
genistein, have been employed as internal standards for
LC–MS11,12 and GC–MS13 analysis of these compounds in
Three methods for the synthesis of coumestrol 1 have been
previously described. The first method involved a conden-
sation of methyl-(2-hydroxy-4-methoxyphenyl) glyoxylate
and 2,4-dimethoxybenzyl alcohol, followed by photo-
cyclisation, cyclisation and demethylation.14 The second
synthesis was based on a Pd-catalysed coupling reaction of
aryl iodides and phenylacetylene, followed by deprotection
and PdCl2 catalysed intramolecular carbonylative cyclisa-
tion under an atmosphere of CO.15 In the third procedure,
coumestrol was constructed by an annulation onto the
corresponding coumarin, forming the furan ring in the last
step.16–18 However, these methods all proved to be poor
yielding, and are not suitable for the synthesis of coumestrol
labelled with three 13C atoms as is required for an internal
standard for LC–MS and GC–MS analysis.11–13
In search of a route for preparation of coumestrol containing
three 13C-labelled atoms, a new, facile, short and easy
procedure has been established. This has then been
employed for the synthesis of coumestrol labelled with
three 13C atoms located in positions C-6, C-6a and C-11a.
2. Results and discussion
A retrosynthetic analysis illustrates that coumestrol 1 can be
constructed by condensation of a phenyl acetate 2 and a
benzoyl chloride 3 to give methyl 2,3-bis(2,4-dimethoxy-
phenyl)-3-oxopropanoate 4. Demethylation and subsequent
Keywords: Coumestrol; Coumestans; Pterocarpans; 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.2003.11.089

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N. Al-Maharik, N. P. Botting / Tetrahedron 60 (2004) 1637–1642
C18 Column (150£4.6 mm)) giving a retention time of
8.25 min with a mobile phase of acetonitrile/water (1:1) and
a 0.5 mL min21 flow rate.
Treatment of 4b under the same conditions gave a mixture
of compounds, which was shown by 1H NMR spectroscopy
to be a result of only partial removal of the benzyl group.
Two steps were required to obtain coumestrol 1 from the
silyl protected precursor 4c. The TBDMS group was first
removed with TBAF to give 4d in 90% yield, which on
standard treatment with BBr3, as for 4a, gave 1 in almost
quantitative yield (Scheme 2).
It was thus clear that use of the two fully methoxylated
precursors 2 and 4a gave the most high-yielding synthesis of
coumestrol, superior to any of the previous literature
methods. Therefore this procedure was then adapted for
the synthesis of [6,6a,11a-13C3]coumestrol 5 starting from
Scheme 1. Retro-synthesis of coumestrol.
Scheme 2. (i) n-BuLi, i-Pr2NH, THF, 278 8C for 2 h then 0 8C for 3 h; (ii) TBAF, THF, 15 min; (iii) 10 equiv. of 1 M BBr3 in CH2Cl2, rt, 72 h (100% from 4a).
intramolecular cyclisation would then afford coumestrol 1
(Scheme 1). Both the starting materials are amenable to
C-labelling as the side chains could be built up using readily
available C-labelled precursors, such as [1,2-13C2]acetyl
chloride, [1-13C1]acetyl chloride or K13CN.
20,40-dimethoxy[1-13C1]acetophenone
6
and 20,40-
dimethoxy[1,2-13C2]acetophenone 7. The two 13C-labelled
acetophenones can be readily synthesised from 1,3-
dimethoxybenzene 8 through acetylation with commercially
available [1-13C1]acetyl chloride or [1,2-13C2]acetyl
chloride (Schemes 3 and 4). Firstly, 8 was acetylated with
[1-13C1]acetyl chloride using aluminium trichloride in
nitroethane to give 6 in 88% yield. Oxidative cleavage of
the acetophenone 6 with O2 using a catalytic amount of
Co(NO3)3 and Mn(NO3)3 in glacial acetic acid at 110 8C,
afforded the 2,4-dimethoxybenzoic[carboxy-13C]acid 9 in
75% yield, which on treatment with an excess of oxalyl
chloride in CH2Cl2 at room temperature resulted in the
formation of 2,4-dimethoxy[carboxy-13C]benzoyl chloride
The synthesis of 1 is depicted in Scheme 2, starting from
commercially available 2,4-dimethoxybenzoyl chloride 3
and a variety of protected methyl 2-hydroxy-4-methoxy-
phenyl acetates 2a–c. The conditions for the C-acylation
reaction were optimised using three different methyl 2-O-
substituted 4-methoxyphenylacetates, namely the 2-methoxy
2a, 2-benzyloxy 2b, and 2-tert-butyldimethylsilyloxy 2c
derivatives, under a range of conditions using dry diethyl
etheranddryTHFassolvent(Scheme 2). Thebestresultswere
obtained by reaction of the methyl acetates 2a–c with a slight
excess of 3 in the presence of LDA in dry THF at 278 8C and
then warming to 0 8C to afford the methyl 2-methoxy-,
benzyloxy- and methyl tert-butyldimethylsilyloxy 3-oxo-
propanoates 4a–c in 71–80% yield.
1
10 in 92% yield as indicated by H NMR spectroscopy
When O-demethylation of 4a was carried out using an
excess of BBr3 in CH2Cl2 at room temperature for 72 h a
spontaneous tandem intramolecular cyclisation took place
as envisaged to afford coumestrol 1 in almost quantitative
yield. The product gave identical spectral data to that in the
literature. The melting point (360–365 8C (dec)) was a poor
indicator of purity due to decomposition of the sample and
the wide variation in literature data, encompassing values
from 290–293 8C5 to 385 8C.16 However, the compound
was shown to be pure by reverse phase HPLC (Kingsorb 3m
Scheme 3. (i) AlCl3, [1-13C1]AcCl, nitroethane, 45 8C, 30 min (88%); (ii)
O2, 0.04 equiv. of Mn(NO3)3, 0.04 equiv. of Co(NO3)3, AcOH, 110 8C,
24 h (75%); (iii) 2 equiv. of CO2Cl2, a drop of DMF, CH2Cl2, rt, 24 h.

N. Al-Maharik, N. P. Botting / Tetrahedron 60 (2004) 1637–1642
1639
while for 13C NMR (75 MHz) spectra the central peak of
CDCl3 (77.0 ppm) and that of CD3SOCD3 (39.95 ppm)
were used as reference. Chemical shifts are given in d and J
values in Hz. Peak assignments were performed for the new
compounds with the aid of the 2D COSY, GHSQCTOCSY
and GHMBC spectra. Mass spectra were recorded at 70 eV.
HRMS were recorded on a Finnigan VG AutoSpec
instrument. The O-substituted methyl 2-hydroxy-4-
methoxyphenyl acetates 2a,19 2b20 and 2c20 were
synthesized according to literature procedures.
Scheme 4. (i) AlCl3, [1,2-13C]AcCl, nitroethane, 45 8C, 30 min (89%);
(ii) TTN, 70% HClO4, MeOH, rt, 2 h (80%).
(Scheme 3). The crude 10 was used in the condensation
reaction without further purification.
3.1.1. Methyl 2,3-bis(2,4-dimethoxyphenyl)-3-oxo-
propanoate (4a). Under an Ar atmosphere, a solution of
the acetate 2a (1.00 g, 4.76 mmol) in dry THF (5 mL) was
slowly added at 278 8C to a solution of LDA in THF
(20 mL), prepared from diisopropylamine (0.53 g,
0.734 mL, 5.24 mmol) and n-BuLi (2.5 M in hexane,
0.21 mL, 5.24 mmol) at 0 8C. The light yellow solution
was stirred for 30 min at 278 8C, and then transferred via
cannula to a solution of 2,4-dimethoxybenzoyl chloride 3
(1.146 g, 5.71 mmol) in THF (5 mL) at 278 8C. The
solution was stirred for 2 h at 278 8C, and then was allowed
to warm to 0 8C. After 2 h stirring, the solution was poured
into 2% aqueous HCl (20 mL), and extracted with ethyl
acetate (3£50 mL). The combined extracts were washed
with water (50 mL), dried over MgSO4, and solvent was
removed at reduced pressure. The resulting orange viscous
oil was subjected to flash chromatography on silica
(CH2Cl2/EtOAc 97:3) to give title compound 4a (1.43 g,
80%) as a light yellow solid: mp 107–108 8C; nmax (nujol)/
cm21 1743 (CO2Me), 1654 (CvO), 1597; 1H NMR
(CDCl3, 300 MHz): d 3.73 (s, 3H, OCH3), 3.78 (s, 9H,
OCH3), 3.83 (s, 3H, OCH3), 5.95 (s, 1H, H-2), 6.37 (d,
J¼2.4 Hz, 1H, H-300), 6.43 0(dd, J¼8.7, 2.4 Hz, 1H, H-50),
6.43 (d, J¼2.4 Hz, 1H, H-3 ), 6.52 (dd, J¼8.7, 2.4 Hz, 1H,
H-500), 7.04 (d, J¼8.7 Hz, 1H, H-60), 7.91 (d, J¼8.7 Hz, 1H,
H-600); 13C NMR (CDCl3, 75 MHz): 52.2 (CO2CH3), 55.2
(OCH3), 55.3 (OCH3), 55.5 (2£OCH3), 57.2 (C-2), 98.1
(C-300), 98.6 (C-30), 104.2 (C-50), 105.5 (C-050 00), 115.7 (C-100),
119.7 (C-100), 130.3 (C-60), 133.59 (C-6 ), 157.8 (C-2 ),
160.3 (C-40), 160.6 (C-400), 164.8 (C-200), 170.8 (C-1), 193.0
(C-3); m/z (EI) 374.1361 (Mþ, requires 374.1365), 374 (5)
and 165 (100); Anal. calcd for C20H22O7: C, 64.16; H, 5.92.
Found: C, 63.99; H, 5.75.
The other fragment for the condensation reaction, methyl
20,40-dimethoxy[1,2-13C2]phenylacetate 11, was synthesised
in 80% yield via oxidative rearrangement of 20,40-
dimethoxy[1,2-13C2]acetophenone 7 using thallium(III)
nitrate (TTN) in MeOH and in presence of 70% HClO4 as
reported previously for the unlabelled analogue (Scheme
4).19
Condensation between 2,4-dimet0hoxy[carboxy-13C]benzoyl
chloride 10 and methyl 2 ,40-dimethoxy[1,2-13C2]-
phenylacetate 11, employing LDA for enolate generation,
afforded
methyl
2,3-bis(2,4-dimethoxyphenyl)-3-
[1,2,3-13C3]oxopropanoate 12 in 83% yield (Scheme 5).
Demethylation and subsequent intramolecular cyclisation of
12 using BBr3 in CH2Cl2 resulted in [6,6a,11a-13C3]-
coumestrol 5 in almost quantitative yield. The presence of
the three 13C-atoms was confirmed by mass spectrometry
and by the enhanced signals for C-6,6a and 11a in the 13C
NMR spectrum, observed at 157.5, 102.0 and 159.4 ppm,
respectively.
Scheme 5. (i) n-BuLi, i-Pr2NH, THF, 278 8C for 2 h then 0 8C for 3 h
(83%); (ii) 10 equiv. of 1 M BBr3 in CH2Cl2, rt, 72 h (82%).
In conclusion, we have established a simple, two-step, high
yielding method for the synthesis of coumestrol 1 from
commercially available starting materials. This synthetic
route is readily adaptable for the synthesis of different
analogues as it is flexible with regard to substitution on both
the phenyl acetate and benzoyl chloride. The method has
been employed for the synthesis of multiply 13C-labelled
coumestrol for use as an internal standard in LC–MS and
GC–MS analysis.
3.1.2. Methyl 2-(2-benzyloxy-4-methoxyphenyl)-3-(2,4-
dimethoxyphenyl)-3-oxopropanoate (4b). Reaction of
methyl 2-benzyloxy-4-methoxyphenyl acetate 2b (0.28 g,
0.97 mmol) sequentially with LDA (1.07 mmol) and 3
(0.214 g, 1.07 mmol), as described for the preparation of 4a,
afforded the title compound 4b (0.31 g, 71%) as a colorless
waxy solid: mp 59–60 8C; nmax (nujol)/cm21 1734
(CO2Me), 1664 (CvO), 1604, 834, 737; 1H NMR
(CDCl3, 300 MHz): d 3.69 (s, 3H, OCH3), 3.72 (s, 3H,
OCH3), 3.76 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 5.03 (s, 2H,
OCH2C6H5), 6.08 (s, 1H, C-2), 6.35 (d, J¼2.4 Hz, 1H,
H-300), 6.47 (dd, 0J0 ¼8.7, 2.4 Hz, 1H, H-50), 6.49 (dd, J¼8.7,
2.4 Hz, 1H, H-5 ), 6.52 (d, J¼2.4 Hz, 1H, H-30), 7.10 (d,
J¼8.7 Hz, 1H, H-60), 7.28–7.34 (m, 5H, C6H5), 7.87 (d,
J¼8.7, 2.4 Hz, 1H, H-600); 13C NMR (CDCl3, 75 MHz): 52.2
(CO2CH3), 55.2 (OCH3), 55.3 (OCH3), 55.5 (OCH3), 57.2
(C-2), 70.1 OCH2C6H5), 98.2 (C-300), 99.8 (C-30), 104.6
(C-50), 105.5 (C-500), 116.2 (C-10), 119.9 (C-100), 126.9,
3. Experimental
3.1. General
Melting points were determined in open capillary tubes with
an electrothermal apparatus and are uncorrected. THF was
1
freshly distilled from sodium/benzophenone. For H NMR
(300 MHz) spectra the residual peak of CHCl3 (7.26 ppm)
and CH3SOCH3 (2.59 ppm) were used as internal reference,

1640
N. Al-Maharik, N. P. Botting / Tetrahedron 60 (2004) 1637–1642
127.7, 128.4, 130.3 (C-6000), 133.6 (C-600), 136.8, 156.9 (C-20),
160.2 (C-40), 160.6 (C-4 ), 164.7 (C-200), 170.8 (C-1), 193.1
(C-3); m/z (CIþ) 451 (Mþ, 100%), 361 (10), 165 (16); Anal.
calcd for C26H26O7: C, 69.32; H, 5.82. Found: C, 68.87; H,
6.13.
filtered, and the filtrate was extracted with EtOAc
(3£30 mL), dried over MgSO4. The combined brown solid
was purified by silica gel chromatography (CH2Cl2/EtOAc
8:2) to give title compound 1 (0.29 g, 82%) as a light yellow
solid: mp 360–365 8C (dec); 1H NMR (DMSO-d6,
300 MHz): d 6.99 (d, J¼2.1 Hz, 1H, H-4), 7.02 (dd,
J¼8.7, 2.1 Hz, 1H, H-2), 7.04 (d, J¼8.7, 2.1 Hz, 1H,
H-8), 7.25 (d, J¼2.1 Hz, 1H, H-10), 7.78 (d, J¼8.7 Hz, 1H,
H-7), 7.93 (d, J¼8.7, 2.4 Hz, 1H, H-1), 10.11 (br s, 1H,
9-OH), 10.77 (br s, 1H, 3-OH); 13C NMR (CDCl3, 75 MHz):
98.6 (C-10), 102.0 (C-6a), 103.0 (C-4), 104.1 (C-1a), 113.7
(C-2), 113.9 (C-10), 114.5 (C-7a), 120.6 (C-7), 122.6 (C-1),
154.6 (C-4a), 155.9 (C-9), 156.9 (C-10a), 157.5 (C-6), 159.4
(C-11a), 161.1 (C-3); Anal. calcd for C15H8O5: C, 67.17; H,
3.01. Found: C, 66.83; H, 2.78.
3.1.3. Methyl 2-(2-t-butyldimethylsilyloxy-4-methoxy-
phenyl)-3-(2,4-dimethoxyphenyl)-3-oxopropanoate (4c).
Reaction of methyl 2-t-butyldimethylsilyloxy-4-methoxy-
phenyl acetate 2c (0.30 g, 0.968 mmol) sequentially with
LDA (1.07 mmol) and 3 (0.214 g, 1.07 mmol), as described
for the preparation of 4a, afforded the title compound 4c
(0.37 g, 78%) as a light yellow wax: nmax (nujol)/cm21 1736
(CO2Me), 1670 (CvO), 1600; 1H NMR (CDCl3,
300 MHz): d 0.20 (s, 6H, 2£SiCH3), 0.94 (s, 9H, Si-Bu-t),
3.71 (s, 3H, OCH3), 3.75 (s, 3H, OCH3), 3.78 (s, 3H, OCH3),
3.83 (s, 3H, OCH3), 5.94 (s, 1H, H-2), 6.37 (d, J¼2.4 Hz,
1H, H-300), 6.390 (d, J¼2.4 Hz, 1H, H-30), 6.47 (dd,00J¼8.7,
2.4 Hz, 1H, H-5 ), 6.52 (dd, J¼8.7, 2.4 Hz, 1H, H-5 ), 7.06
3.1.6. 20,40-Dimethoxy[1,2-13C2]acetophenone (7). Finely
powdered anhydrous AlCl3 (1.68 g, 12.6 mmol) was added
to a well-stirred solution of 1,3-dimethoxybenzene 8 (2 g,
14.47 mmol) in freshly distilled nitroethane (10 mL) under a
N2-atmosphere. Then [1,2-13C3]acetyl chloride (1 g,
0.905 mL, 12.58 mmol) was slowly added, and the resulting
red solution was stirred at 45 8C for 30 min. The mixture
was allowed to cool to room temperature, poured into ice
water (100 mL), and extracted with diethyl ether
(3£50 mL). The combined organic layers were washed
with brine (50 mL) and dried over MgSO4. The solvent was
removed at reduced pressure, and the orange oily residue
was purified by column chromatography (silica, CH2Cl2/
hexane, 4:1) to afford the title compound 7 (2.03 g, 89%) as
a white solid: mp 40–41 8C (Lit21 mp 39–41 8C); 1H NMR
(CDCl3, 300 MHz): d 2.56 (dd, J¼128.1, 6.3 Hz, 3H, H-2),
3.84 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 6.44 (dd, J¼2.4,
1.5 Hz, 1H, H-30), 6.51 (dd,0 J¼8.7, 2.4 Hz, 1H, H-50), 7.82
(dd, J¼8.7, 4.2 Hz, 1H, H-6 ); 13C NMR (CDCl3, 75 MHz):
d 31.8 (d, J¼168.7 Hz, C-2), 55.4 (OCH3), 55.5 (OCH3),
98.2 (d, J¼10.8 Hz, C-30), 105.0 (d, J0¼15.3 Hz, C-50), 120.5
(d, J¼132.9 Hz, C-10), 131.8 (s, C-6 ), 161.0 (d, J¼8.7 Hz,
C-20), 164.5 (C-40), 197.7 (d, J¼168.7 Hz, C-1); m/z (EI)
182.0852 (Mþ, C813C2H12O3 requires 182.0853), 182
(32%), 166 (100), 151 (6) and 122 (7).
(d, J¼8.7 Hz, 1H, H-60), 7.93 (d, J¼8.7 Hz, 1H, H-600); 13
C
NMR (CDCl3, 75 MHz): d 24.5 (SiCH3), 24.0 (SiCH3),
18.1 (SiC(CH3)3), 25.6 (SiC(CH3)3), 52.1 (CO2CH3), 55.16
(OCH3), 55.21 (OCH3), 55.5 (OCH3), 57.4 (C-2), 98.1
(C-3000), 105.2 (C-30), 105.5 (C-5000), 105.6 (C-50), 117.9
(C-10), 119.7 (C-100), 130.5 (C-6 ), 133.7 00(C-600), 154.1
(C-2 ), 159.7 (C-40), 160.7 (C-400), 164.9 (C-2 ), 170.8 (C-1),
192.7 (C-3); m/z (EI) 475.2165 (Mþ, C25H35O7Si requires
475.2152), 475 (2), 385 (6) and 165 (100).
3.1.4. Methyl 2-(2-hydroxy-4-methoxyphenyl)-3-(2,4-
dimethoxyphenyl)-3-oxopropanoate (4d). Under a N2-
atmosphere, a solution of TBAF (1 M in THF, 3.76 mL,
3.75 mmol) was added to a solution of 4c (0.80 g,
1.69 mmol) in THF (7 mL) at room temperature. After
15 min, the green solution was poured into water (20 mL),
and extracted with ethyl acetate (3£30 mL). The combined
extracts were dried over MgSO4, and the solvent was
evaporated. The residue was subjected to silica gel
chromatography (CH2Cl2/EtOAc 95:5) to give the title
compound 4d (0.55 g, 90%) as a white solid: mp 55–56 8C;
nmax (nujol)/cm21 3375 (OH), 1735 (CO2Me), 1654
(CvO), 1598; 1H NMR (CDCl3, 300 MHz): d 3.75 (s,
3H, OCH3), 3.76 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.92 (s,
3H, OCH3), 5.73 (s, 1H, C-2), 6.42 (d, J¼2.4 Hz, 1H, H-300),
6.42 (dd, J¼8.7, 2.4 Hz, 1H, H-50), 6.52 (d, J¼00 2.4 Hz, 1H,
H-30), 6.54 (dd, J¼8.7, 2.4 Hz, 1H, H-5 ), 7.00 (d,
J¼8.7 Hz,0 1H, H-60), 7.83 (d, J¼8.7 Hz, 1H, H-600), 8.29
(s, 1H, 2 -OH); 13C NMR (CDCl3, 75 MHz): d 52.8
(COOCH3), 55.2 (OCH3), 55.5 (OCH3), 55.6 (OCH3),
61.10 (C-2), 98.3 (C-300), 103.8 (C-30), 105.7 (C-5000), 106.8
(C-500), 113.6 (C-100), 119.5 (C-100), 132.7 (C-60), 133.9
(C-600), 156.9 (C-2 ), 160.6 (C-400), 161.0 (C-4 ), 165.6
(C-2 ), 170.7 (C-1), 196.7 (C-3); m/z (EI) 360.120822 (Mþ,
C19H20O7 requires 360.120903), 360 (3%), 342 (9) and 165
(100).
3.1.7. 20,40-Dimethoxy[1-13C1]acetophenone (6). The title
compound was prepared according to the procedure
described for 7, using [1-13C1]acetyl chloride to give the
product as white solid (2.1 g, 88%): 40–41 8C (Lit21 mp
39–41 8C); 1H NMR (CDCl3, 300 MHz): d 2.56 (d,
J¼6.3 Hz, 3H, H-2), 3.84 (s, 3H, OCH3), 3.88 (s, 3H,
OCH3), 6.44 (dd, J¼2.4, 1.5 Hz, 1H, H-30), 6.51 (dd, J¼8.7,
2.4 Hz, 1H, H-50), 7.82 (dd, J¼8.7, 3.9 Hz, 1H, H-60); 13C
NMR (CDCl3, 75 MHz): d 31.8 (d, J¼169.5 Hz, C-1), 55.4
(OCH3), 55.5 (OCH3), 98.2 (C-30), 105.0 (d, J¼13.8 Hz,
C-50), 120.5 (d,0 J¼132.9 Hz, C-10), 132.6 (C-60), 161.0 (d,
J¼8.7 Hz, C-2 ), 164.5 (C-40), 197.7 (C-1); m/z (EI)
181.0818 (Mþ, C913C1H12O3 requires 181.0819), 181
(29%), 166 (100), 151 (8) and 122 (6).
3.1.5. Coumestrol (1). An excess of 1 M BBr3/CH2Cl2
(13.34 mL, 13.34 mmol) was added with stirring to a
solution of 4a (0.5 g, 1.34 mmol) in CH2Cl2 (5 mL) at
room temperature under Ar. The mixture was stirred for
72 h, then water was added and CH2Cl2 was evaporated at
reduced pressure. The mixture was refluxed for 3 h. After
cooling to room temperature the yellow precipitate was
3.1.8. 2,4-Dimethoxy[carboxy-13C]benzoic acid (9). A
solution of the acetophenone 6 (1.0 g, 5.52 mmol),
Mn(NO3)3 (0.064 g, 0.22 mmol), and Co(NO3)3 (0.064 g,
0.22 mmol) glacial acetic acid (10 mL) was stirred at 110 8C
for 24 h under an O2-atmosphere. The solvent was
evaporated at reduced pressure, and the residue was

N. Al-Maharik, N. P. Botting / Tetrahedron 60 (2004) 1637–1642
1641
dissolved in 2 N NaHCO3 (15 mL) at 60 8C, which was
extracted with ethyl acetate (3£30 mL). The aqueous
solution was acidified with concentrated H2SO4, and
extracted with ethyl acetate (3£30 mL). The combined
organic layers were washed with brine (50 mL), dried over
MgSO4, and the solvent was evaporated under reduced
pressure. The resulting yellow solid was subjected to silica
gel chromatography (CH2Cl2/EtOAc 97:3) to furnish the
title compound 9 (0.76 g, 75%) as a white solid: 105–
3.1.11. Methyl 2,3-bis(2,4-dimethoxyphenyl)-3-
[1,2,3-13C3]oxopropanoate (12). Condensation of benzoyl
chloride 10 (0.50 g, 2.49 mmol) and 13C-labelled acetate 11
(0.44 g, 2.07 mmol) as described for the preparation of 4a,
afforded the title compound 12 (0.65 g, 83%) as a light
yellow solid: mp 106–107 8C; nmax (nujol)/cm21 1700
(CO2Me), 1623 (CvO), 1591; 1H NMR (CDCl3,
300 MHz): d 3.73 (s, 3H, OCH3), 3.74 (s, 3H, OCH3),
3.77 (s, 6H, OCH3), 3.82 (s, 3H, OCH3), 5.95 (ddd,
J¼133.2, 8.7, 6.3 Hz, 1H, H-2), 6.37 (q, J¼02.4 Hz, 1H,
H-300), 6.43 (dd, J¼9.0, 2.4 Hz, 1H, H-5 ), 6.43 (d,
J¼2.4 Hz, 1H, H-30), 6.52 (dd, J¼8.7, 2.4 Hz, 1H, H-500),
7.04 (dd0, J¼8.7, 4.2 Hz, 1H, H-6), 7.91 (dd, J¼8.7, 4.2 Hz,
1H, H-6 ); 13C NMR (CDCl3, 75 MHz): 52.2 (OCH3), 55.2
(OCH3), 55.3 (OCH3), 55.5 (2£OCH3), 57.2 (dd, J¼235.6,
162.5 Hz, H-2), 98.1 (d, J¼11.7 Hz, C-3000), 98.6 (d,
J¼8.7 Hz, C-30), 104.2 (d, J¼13.8 Hz, C-5 ), 105.5 (0d,
J¼13.8 Hz, C-500), 115.70 (C-10), 119.7 (C-100), 130.3 (C-6 ),
133.6 (C-600), 157. 8 (C-2 ), 160.3 (C-40), 160.6 (C-400), 164.8
(C-200), 170.8 (dd, J¼235.5, 8.7 Hz, C-1), 193.0 (dd,
J¼162.5, 8.7 Hz, C-3); m/z (EI) 377.147475 (Mþ,
C1713C3H22O7 requires 377.146618), 377 (4%), 166 (100);
Anal. calcd for C1713C3H20O7: C, 63.47; H, 5.66. Found: C,
63.65; H, 5.89.
1
106 8C (Lit22 mp 106 8C); H NMR (CDCl3, 300 MHz): d
3.87 (s, 3H, OCH3), 4.04 (s, 3H, OCH3), 6.53 (dd, J¼2.4,
1.8 Hz, 1H, H-30), 6.64 (dd,0 J¼9.0, 2.4 Hz, 1H, H-50), 8.12
(dd, J¼9.0, 2.4 Hz, 1H, H-6 ), 10.45 (br s, 1H, COOH); 13
C
NMR (CDCl3, 75 MHz): d 55.7 (OCH3), 56.6 (OCH3), 98.6
(d, J¼12.9 Hz, C-3), 106.5 (d, J¼17.1 Hz, C-5), 111.3 (d,
J¼302.1 Hz, C-1), 135.5 (d, J¼9.0 Hz, C-6), 159.5 (d,
J¼8.7 Hz, C-2), 165.1 (C-4), 165.2 (s, COOH); m/z (EI)
183.0617 (Mþ, C813C1H10O4 requires 183.0612), 183
(98%), 166 (100), 154 (26), 136 (58) and 122 (5).
3.1.9. 2,4-Dimethoxy[carboxy-13C]benzoyl chloride (10).
Oxalyl chloride (1.26 g, 0.86 mL, 9.93 mmol) was added to
a solution of the carboxylic acid 9 (0.90 g, 4.92 mmol) in
dry CH2Cl2 (20 mL) containing a drop of dry DMF under
an Ar atmosphere. The solution was stirred at room
temperature under light exclusion for 24 h. The solvent
was then evaporated at reduced pressure and the yellow
orange solid was washed with dry hexane (2£30 mL).
Drying in vacuo gave the title compound 10 (0.98 g, 99%)
as light yellow solid, which was pure enough (purity <90%,
3.1.12. [6,6a,11a-13C3]Coumestrol (5). The title compound
was prepared according to the procedure described for 1,
using 12 to give the product as white solid (0.283 g, 80%):
1
mp 360–365 8C (dec); H NMR (DMSO-d6, 300 MHz): d
6.99–7.06 (m, 3H, H-2,4,8), 7.25 (d, J¼2.1 Hz, 1H, H-10),
7.78 (d, J¼8.7, 2.7 Hz, 1H, H-7), 7.93 (d, J¼8.7, 4.2 Hz,
1H, H-1), 10.11 (br s, 1H, 9-OH), 10.77 (br s, 1H, 3-OH);
13C NMR (CDCl3, 75 MHz): 98.6 (C-10), 102.0 (dd,
J¼348, 251.7 Hz, C-6a), 103.0 (C-4), 104.1 (C-1a), 113.7
(C-2), 113.9 (C-10), 114.5 (C-7a), 120.6 (C-7), 122.6 (C-1),
154.6 (C-4a), 155.9 (C-9), 156.9 (C-10a), 157.5 (dd, J¼348,
22.8 Hz, C-6), 159.4 (dd, J¼252, 22.8 Hz, C-6), 161.1
(C-3); m/z (EI) 271.047053 (Mþ, C1213C3H8O5 requires
271.047238), 132 (44) and 117 (9).
1
as indicated by H NMR spectroscopy) to be used in the
next step. 1H NMR (CDCl3, 300 MHz): d 3.90 (s, 3H,
OCH3), 3.91 (s, 3H, OCH3), 6.46 (t, J¼2.4 Hz, 1H, H-3),
6.55 (dd, J¼9.0, 2.4 Hz, 1H, H-5), 8.16 (dd, J¼9.0, 6.6 Hz,
1H, H-6).
3.1.10. Methyl 20,40-dimethoxy[1,2-13C2]phenyl acetate
(11). A solution of the acetophenone 7 (1.0 g, 5.49 mmol) in
methanol (10 mL) was dropwise added to a well stirred
solution of TTN (2.68 g, 6.04 mmol) in methanol (10 mL)
containing perchloric acid (5 mL, 70% w/w) under Ar. The
reaction mixture was stirred at room temperature for 2 h,
and the resulted white precipitate thallium(I) nitrate was
removed by filtration and the filtrate was carefully poured
into 2 N aq. NaHCO3 (100 mL). The aqueous solution was
extracted with CH2Cl2 (3£50 mL), washed with brine
(2£50 mL) and dried (MgSO4). The solvent was removed
at reduced pressure, and the orange oily residue was purified
by silica gel chromatography (CH2Cl2/hexane 4:1) to afford
the title compound 11 (0.93 g, 80%) as a light yellow solid:
48–49 8C (Lit20 mp 50 8C); nmax (nujol)/cm21 1698
(CO2Me), 1617, 1590; 1H NMR (CDCl3, 300 MHz): d
3.56 (dd, J¼129.9, 8.1 Hz, 2H, H-2), 3.68 (d, J¼3.9 Hz, 3H,
OCH3), 3.795 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 6.45 (dd,
J¼9.0, 2.4 Hz, 1H, H-50), 6.50 (d, J¼2.4 Hz, 1H, H-30), 7.08
(dd, J¼9.0, 2.4 Hz, 1H, H-60); 13C NMR (CDCl3, 75 MHz):
d 35.0 (d, J¼231.5 Hz, C-2), 51.50 (d, J¼13.8 Hz,
CO2CH3), 55.4 (OCH3), 55.5 (OCH3), 98.7 (d, J¼9.0 Hz,
C-30), 104.1 (d, J¼13.2 Hz, C-50), 115.4 (dd, J¼187.6,
11.7 Hz, C-10), 131.1 (t, J¼9.0 Hz, C-60), 158.9 (d,
J¼8.7 Hz, C-20), 160.2 (C-40), 172.6 (d, J¼231.5 Hz,
C-1); m/z (EI) 212.0960 (Mþ, C913C2H14O4 requires
212.0959), 212 (34%), 152 (100) and 122 (26).
Acknowledgements
This work was funded by the Food Standards Agency (FSA)
under contract T05023.
References and notes
1. Boland, G. M.; Donnelly, D. M. X. Nat. Prod. Rep. 1995,
321–338.
2. Donnelly, D. M. X.; Boland, G. M. Nat. Prod. Rep. 1998,
241–260.
3. Chansakaow, S.; Ishikawa, T.; Sekine, K.; Okada, M.;
Higuchi, Y.; Kudo, M.; Chaichantipyuth, C. Planta Med.
2000, 66, 572–575.
4. Le-Van, N. G. O. Phytochemistry 1984, 23, 1204–1205.
5. Chang, L. C.; Gerhaeuser, C.; Song, L.; Farnsworth, N. R.;
Pezzuto, J. M.; Kinghorn, A. D. J. Nat. Prod. 1997, 60,
869–873.
6. O’Neill, M. J.; Adesanya, S. A.; Roberts, M. F. Phyto-
chemistry 1984, 23, 2704–2705.

1642
N. Al-Maharik, N. P. Botting / Tetrahedron 60 (2004) 1637–1642
7. O’Neill, M.; Adesanya, S. A.; Roberts, M. F.; Pantry, I. R.
Phytochemistry 1986, 25, 1315–1322.
15. Hiroya, K.; Suzuki, N.; Yasuhara, A.; Yuya, E.; Kasano, A.;
Sakamoto, T. J. Chem. Soc., Perkin Trans. 1 2000, 24,
4339–4346.
8. Reinli, K.; Block, G. Nutr. Cancer 1996, 26, 123–148.
9. Draper, C. R.; Edel, M. J.; Dick, I. M.; Randall, A. G.; Martin,
G. B.; Prince, R. L. J. Nutr. 1997, 127, 1795–1799.
10. Shemesh, M.; Lindner, H. R.; Ayolan, N. J. Reprod. Fertil.
1972, 29, 1–9.
16. Emerson, O. H.; Bickoff, E. M. J. Am. Chem. Soc. 1958, 80,
4381–4388.
17. Kawase, Y. Bull. Chem. Soc. Jpn 1959, 32, 690–696.
18. Jurd, L. J. Org. Chem. 1964, 29, 3036–3038.
19. Alesso, E. N.; Aguirre, J. M.; Iglesias, G. Y. M. Org. Prep.
Proced. Int. 1992, 24, 690–692.
11. Clarke, D. B.; Lloyd, A. S.; Oldfield, M. F.; Botting, N. P.;
Needs, P. W.; Wiseman, H. Anal. Chem. 2002, 309, 158–172.
12. Grace, P. B.; Taylor, J. I.; Botting, N. P.; Fryatt, T.; Oldfield,
M. F.; Bingham, S. A. Anal. Biochem. 2003, 315, 114–121.
13. 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.
20. Theunis, G. A.; Rensburg, H.; Ferreira, D. Tetrahedron 2001,
57, 7113–7126.
21. Moon, M. W.; Wade, R. A. J. Org. Chem. 1984, 49,
2663–2669.
22. Chakravarti, D.; Saha, M. J. Indian Chem. Soc. 1971, 48,
209–216.
14. Kraus, A. G.; Zhang, N. J. Org. Chem. 2000, 65, 5644–5646.