A versatile synthesis of [2,3,4-13C3]isoflavones

Article abstract of DOI:10.1002/jlcr.1732

A flexible synthetic method is presented, which allows all the key isoflavones (daidzein, genistein, glycitein, formononetin and biochanin A) to be prepared in ¹³C-labelled form via the same route, involving the thallium(III)-mediated oxidative

Full text of DOI:10.1002/jlcr.1732

Research Article
Received 31 August 2009,
Revised 27 October 2009,
Accepted 11 November 2009
Published online 4 January 2010 in Wiley Interscience
(www.interscience.wiley.com) DOI: 10.1002/jlcr.1732
A versatile synthesis of [2,3,4-13C3]isoflavones
Ã
Nawaf Al-Maharik and Nigel P. Botting
A flexible synthetic method is presented, which allows all the key isoflavones (daidzein, genistein, glycitein, formononetin
and biochanin A) to be prepared in 13C-labelled form via the same route, involving the thallium(III)-mediated oxidative
rearrangement of a key chalcone intermediate. This method results in the incorporation of 13C atoms at the 2, 3 and
4 positions of the isoflavone skeleton. We also report the first syntheses of 13C-labelled versions of the daidzein metabolites,
equol and ODMA.
Keywords: daidzein; genistein; isoflavones; phytoestrogens; polyphenols
analysis.13–16,20,21 The only problem with this work has been that
Introduction
different routes have been employed for each isoflavone. In
Isoflavone phytoestrogens are polyphenolic compounds with order to simplify the production of the 13C-labelled isoflavones
weak oestrogenic and antiestrogenic activity,1,2 which are and harmonize the labelling pattern, we herein report the
present in the human diet where the main source is soybeans development of a synthetic method which allows all the key
and products derived from soybeans.3,4 The most common isoflavones to be prepared via the same route resulting in 13C
isoflavones are daidzein (1a), formononetin (1b), genistein (1c), labelling at the same positions in the isoflavone skeleton. We
biochanin A (1d) and glycitein (2) and, of these, genistein also report the first syntheses of 13C-labelled versions of the
appears to be the most biologically active.5 In epidemiological daidzein metabolites, equol22 and ODMA.23
studies, consumption of an isoflavone rich diet has been shown
to be correlated with a decrease in the incidence of hormone- Result and discussion
related cancers, including breast6,7 and prostate cancer.8 Other
The most efficient route employed in our previous work on
health-promoting activities have also been associated with the
13C-labelled isoflavones was that for the synthesis of [2,3,4-
13C3]glycitein.19 In this synthesis, the isoflavone framework was
constructed via oxidative rearrangement of a chalcone inter-
mediate. This gave the target compound in 57% overall yield
in only eight steps from [13C2]acetyl chloride, with the third
13C atom coming from potassium [13C]cyanide.19 It was thus
decided to modify this route for daidzein, genistein, formono-
netin and biochanin A.
isoflavones, including lessening of menopausal symptoms,9,10
and chemoprevention of osteoporosis11 and cardiovascular
disease (Scheme 1).12
Accurate analysis of isoflavones is highly important for
establishing the dietary exposure of the population to the soy
isoflavones and for subsequent epidemiological studies aimed
to investigate the associations between isoflavone exposure and
disease.13 In recent years, mass spectrometry-based methods,
such as LC-MS14,15 and GC-MS,16 have become the most popular
for isoflavone analysis. The choice of internal standard is vital if
one wishes to obtain optimum accuracy and reproducibility
from these methods and the best internal standard is a pure,
stable, isotopically labelled analogue of the analyte, with a
sufficiently large enough mass difference to nullify the effect of
natural abundance heavy isotopes in the analyte. The mass
difference required between standard and analyte depends on
the molecular weight of the analyte and whether any
heteroatoms are present. An extra 3 mass units is sufficient for
molecules of the size of isoflavones. This ensures that with 99%
enrichment at each position there is less than 1 ppm residual
unlabelled analyte in the internal standard and that the M31 ion
due to natural 13C in the analyte will be at less than 1%
abundance, giving minimal overlap.
Initially studies concentrated on the synthesis of daidzein and
genistein, using methyl ether protection for the hydroxyl
groups. The key chalcone intermediate was prepared via
condensation of two 13C-labelled components, an acetophe-
none derivative and 4-methoxy-[carbonyl-13C]benzaldehyde.
BCl3-assisted Friedel-Crafts acetylation of 3-methoxyphenol 5a
and 3,5-dimethoxyphenol 5b with [13C2]acetyl chloride in
anhydrous CH2Cl2 provided the two acetophenones required,
2-hydroxy-4-methoxy-[10,20-13C2]acetophenone 6a and 2-hydro-
xy-4,6-dimethoxy-[10,20-13C2]acetophenone 6b, in 77 and 87%
yield, respectively. The 4-methoxy-[carbonyl-13C]benzaldehyde
9, was prepared via Pd(OAc)2-catalysed cyanation17,24 of
4-methoxyiodobenzene 7 using potassium [13C]cyanide in the
School of Chemistry, University of St. Andrews, St. Andrews, Fife, KY16 9ST, UK
We have previously synthesized
a series of multiply
13C-labelled isoflavones, namely [3,4,8-13C3]daidzein,17 [3,4,10-
13C3]genistein18 and [2,3,4-13C3]glycitein.19 These compounds
*Correspondence to: Nigel P. Botting, School of Chemistry, University of
St. Andrews, St. Andrews, Fife, KY16 9ST, UK.
have all been successfully employed as internal standards for E-mail: npb@st-andrews.ac.uk
J. Label Compd. Radiopharm 2010, 53 95–103
Copyright r 2009 John Wiley & Sons, Ltd.

N. Al-Maharik and N. P. Botting
HO
O
O
HO
OH
O
HO
O
O
H3CO
OH
R1
3
OR2
2
1a: R1 = R2 = H
HO
OH
1
2
1b
: R = H, R = Me
1c: R1 = OH, R2 = H
1d: R1 = OH, R2 = Me
R1
O
OH
4a: R1=H
4b: R1=OH
Scheme 1. Isoflavone phytoestrogens and their metabolites.
MeO
OH
MeO
OH
a
13CH3
13C
O
R
R
5a R = H
6a R = H
MeO
OAc
H
6b R = OMe
d
5b R = OMe
OMe
13C
13C
O
H
13C
H
13C
13CN
I
R
O
b
c
10a R = H
10b R = OMe
OMe
OMe
OMe
e
8
9
7
MeO
O
HO
O
13CH
13CH
f
13C
13C
13C
13C
OH
O
R
O
OH
OMe
11a R = H
11b R = OMe
1c
Scheme 2. Synthesis of [2,3,4-13C3] genistein. Reagents and conditions: (a) (i) [13C2]Acetyl chloride, 1 M BCl3 in CH2Cl2, CH2Cl2, 01C-reflux, 3 h; (b) K13CN, Ca(OH)2,
Pd(OAc)2, DMF, reflux, 16 h; (c) DIBAL-H, THF, rt, overnight; (d) i) KOH, MeOH, reflux, 6 h, ii) Ac2O, Py, rt, 24 h; (e) Tl(NO3)3.3H2O, HC(OMe)3, MeOH, rt, 20 h; then 10% HCl,
MeOH, reflux, 2 h; and (f) 55% HI, AcOH, 1201C, 48 h.
presence of Ca(OH)2 in DMF, followed by DIBAL-H-mediated ethers in 11b was successfully achieved with 55% HI in AcOH
reduction of the nitrile 8. An aldol condensation of 9 with either under reflux temperature to afford [2,3,4-13C3]genistein 1c in
fragment 6a or 6b then gave the corresponding 20-hydro- 83% yield, with an overall yield of 49% in four steps from the
xychalcones, which were treated with acetic anhydride in [13C2]acetyl chloride (Scheme 2).
pyridine to afford the fully protected chalcones, 10a and 10b.
In order to overcome the demethylation problems, it was
The key oxidative rearrangement19,25 of 10a and 10b with decided to use an alternative O-benzyl protection strategy.
Tl(NO3)3.3H2O in a mixed solvent of dry MeOH and CH(OCH3)2 AlCl3-mediated acetylation of resorcinol 12 with [13C2]acetyl
(3:2 v/v) led to the corresponding acetal, which was hydrolysed chloride in anhydrous nitrobenzene according to a literature
in situ and cyclized using 10% HCl at reflux temperature to give procedure26 gave 2,4-dihydroxy-[10,20-13C2]acetophenone 13a,
7,40-dimethoxy-[2,3,4-13C3]isoflavone 11a and 5,7,40-trimethoxy- which was then O-benzylated, without further purification, using
[2,3,4-13C3]isoflavone 11b. The presence of the three 13C atoms benzyl bromide in acetone in the presence of excess anhydrous
was confirmed by the three enhanced signals in the 13C NMR K2CO3 and a catalytic amount of 18-crown-6 to give 2,4-O-
spectra and the 3 mass unit increase in the molecular ion dibenzyloxy-[10,20-13C2]acetophenone 14a (Scheme 3). 2,4,6-O-
compared with the unlabelled compounds. Unexpectedly, the Tribenzyloxy-[10,20-13C2]acetophenone 14b was prepared via a
O-demethylation of 11a was very problematic and treatment of BBr3-mediated O-demethylation of 6b, followed by O-benzyla-
11a with 1 M BBr3-CH2Cl2 solution for 3 days gave an intractable tion of the crude 2,4,6-trihydroxy-[10,20-13C2]acetophenone
mixture. Various other demethylation methods were attempted 13b, prepared previously (Scheme 3). Three chalcones were
with 11a without success; however, cleavage of the methyl then prepared via the aldol condensation of 14a with
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Copyright r 2009 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2010, 53 95–103

N. Al-Maharik and N. P. Botting
HO
OH
13CH3
BnO
OBn
13CH3
HO
OH
a
b
13C
13C
O
O
12
14a
13a
BnO
OBn
13CH3
MeO
OH
13CH3
HO
OH
13CH3
c
b
13C
13C
13C
OBn O
OMe O
OH
13b
O
6b
14b
Scheme 3. Synthesis of benzyloxy substituted acetophenones (14a and 14b). Reagents and conditions: (a) [13C2]Acetyl chloride, AlCl3, nitrobenzene, rt, 15 h; (b) BnBr,
K2CO3, 18-crown-6, acetone, reflux, 8 h; and (c) 1 M BBr3 in CH2Cl2, CH2Cl2, 24 h.
13CHO
BnO
OBn
13C
BnO
OBn
13CH3
H
b
OR1
a
+
13C
13C
13C
H
R
O
R
O
OR
R = Me
14a R = H
14b R = OBn
9
16a R = H, R1 = Bn
1
15 R = Bn
16b
R = H, R = Me
16c R = OBn, R1 = Me
OMe
OMe
H
HO
OH
HO
O
BnO
OBn
13C
13C
13C
13C
13C
OMe
H
13C
d
OMe
H
c
13C
13C
13C
H
H
R
O
R
O
R
O
OR1
OR1
OR
1a R = H, R1 = H
17a R = H, R1 = Bn
18a R = H, R1 = H
1
1
1
1b
17b
R = H, R = Me
R = H, R = Me
18b
R = H, R = Me
1d R = OH, R1 = Me
17c R = OBn, R1 = Me
18c R = OH, R1 = Me
Scheme 4. Synthesis of [2,3,4-13C3] isoflavones. Reagents and conditions: (a) KOH, MeOH, reflux, 7 h; (b) Tl(NO3)3.3H2O, HC(OMe)3, MeOH, rt, 20 h; (c) H2, 5% Pd/C, MeOH,
acetone, overnight; and (d) 10% HCl, MeOH, reflux, 6 h.
4-benzyloxy-[carbonyl-13C]benzaldehyde19 15 and 4-methoxy- (Scheme 5). [1,2,3-13C3]ODMA 4a was prepared as outlined in
[carbonyl-13C]benzaldehyde 9, and the aldol condensation of Scheme 5. Selective mono-C-methylation of 4-benzyloxyphenyl-
14b with 9, so that the three target isoflavones were all [1,2-13C2]acetonitrile 19 with 1 eq. of [13C]methyl iodide in
accessible (Scheme 3). The chalcones were subjected to the presence of 1 eq. of lithium i-propylcyclohexylamide
oxidative rearrangement with Tl(NO3)3.3H2O, as before, to give afforded ethyl 2-(4-benzyloxyphenyl)-[1,2,3-13C3]propionitrile
the corresponding substituted acetals 17a, 17b and 17c in 89%, 20 in 73% yield. Basic hydrolysis of the nitrile 20 with 1.5 eq.
82% and 40% yields, respectively. O-Debenzylation of 17a-17c of NaOH in a 1:1 mixture of ethanol and water at reflux
using 5% Pd/C-catalyzed hydrogenolysis proceeded smoothly to temperature afforded 2-(4-benzyloxyphenyl)-[1,2,3-13C3]propio-
afford the hydroxyl substituted 1,2-diphenyl-3,3-dimethoxy-1- nic acid 21 in almost quantitative yield, which was then
[1,2,3-13C3]propanones 18a, 18b and 18c, which on heating to subjected to O-debenzylation by means of 5% Pd/C-catalyzed
reflux with 10% HCl in MeOH gave the desired [2,3,4-13C3]- hydrogenolysis to give 2-(4-hydroxyphenyl)-[1,2,3-13C3]propio-
daidzein 1a, [2,3,4-13C3]formononetin 1b and [2,3,4-13C3]biochanin nic acid 22. Condensation of the crude acid 22 with a two-fold
A 1d, in excellent yields and purity. The presence of the three 13
C
excess of resorcinol in the presence of BF3.Et2O and ZnCl2.Et2O
1
atoms was confirmed by means of H and 13C NMR spectroscopy at reflux temperature for 15 min afforded [1,2,3-13C3]ODMA 4a
and mass spectrometry (Scheme 4).
in 80% yield.
Two daidzein metabolites were also required in 13C-labelled
The thallium(III)-mediated oxidative rearrangement of appro-
form, equol and ODMA. [2,3,4-13C3]Equol 3 was synthesized by priate chalcone intermediates, derived from 13C-labelled aceto-
catalytic hydrogenation of [2,3,4-13C3]daidzein 1a, using 10% phenones and benzaldehyde precursors, therefore offers a short,
palladium on charcoal in 95% EtOH at 3 bar, in 71% yield efficient method for the synthesis of [2,3,4-13C3]isoflavones.
J. Label Compd. Radiopharm 2010, 53 95–103
Copyright r 2009 John Wiley & Sons, Ltd.
www.jlcr.org

N. Al-Maharik and N. P. Botting
H
HO
O
HO
O
13C
13C
13CH2
13CH
a
13C
13C
H
H
O
OH
OH
3
1a
13CN
13CH2
13CO2H
13CH
13CO2H
13CH
H313C
13CN
13CH
H313C
H313C
c
b
d
e
OBn
OBn
20
OH
OBn
21
19
22
HO
OH13
CH3
13CH
13C
O
OH
4a
Scheme 5. Synthesis of [2,3,4-13C3] equol (3) and [13C3]ODMA (4). Reagents and conditions: (a) Pd/C, H2(g), EtOH; (b) n-BuLi, i-propylcyclohexylamine, 13CH3I, THF, ꢀ781C
for 1 h, then 3 h at rt; (c) aq. NaOH, EtOH, reflux, 1.5 h; (d) H2, 5% Pd/C, EtOH:acetone (2:1); and (e) resorcinol, BF3.Et2O, ZnCl2, 15 min, reflux.
J = 2.3, 1.4 Hz, H-3), 6.06 (1H, d, J = 2.2 Hz, H-5); dC (75 MHz,
CDCl3) 31.2 (d, enhanced,. J = 42.7 Hz, CH3), 55.51, 55.52
(2 ꢁ OCH3), 203.6 (d, enhanced, J = 42.7 Hz, CO).
Experimental
4-Methoxy-2-hydroxy-[1’,2’-13C]acetophenone 6a
A solution of methoxyphenol 5a (1.85 g, 14.9 mmol) in dry
CH2Cl2 (10 ml) was slowly added to a solution of BCl3 (12.5 mL,
1 M in CH2Cl2, 12.42 mmol) at ꢀ101C. After 5 min stirring [1,2,3-13C3]prop-2-en-1-one 10a
at ꢀ101C,
solution [13C2]acetyl choride (1 g, 0.906 ml,
1-(2-Acetoxy-4-methoxyphenyl)-3-(4-methoxyphenyl)-
a
A mixture of the acetophenone 6a (1.75 g, 10.42 mmol),
benzaldehyde (1.7 g, 12.41 mmol) and KOH (5.83 g,
12.4 mmol) in CH2Cl2 (5 mL) was added over 10 min. The
reaction mixture was heated under reflux for 3 h before being
carefully quenched with excess 1 M HCl (50 mL). After stirring for
an hour at room temperature, the aqueous phase was extracted
with CH2Cl2 (3 ꢁ 50 mL), dried over MgSO4, and the solvent was
removed at reduced pressure. Column chromatography on silica
gel with petroleum ether (bp 40–60):diethyl ether (4:1) as eluant
afforded 6a (1.63 g, 78%) as a white solid: mp 48–501C (Lit.27mp.
49–501C for the unlabelled); dH (300 MHz, CDCl3) 2.57 (3H, dd,
J = 129.2, 6.2 Hz, 13CH3); 3.87 (3H, s, OCH3), 6.51 (1H, dd, J = 9.0,
2.5 Hz, H-5), 6.54 (1H, d, J = 2.5 Hz, H-3), 7.44 (1H, dd, J = 9.0,
2.5 Hz, H-6), 12.87 (1H, s, 2-OH); dC (75 MHz, CDCl3) 26.2
(d, enhanced, J = 44.5 Hz, CH3), 55.6 (OCH3), 100.9 (d, J = 2.5 Hz,
C-3), 107.7 (d, J = 4.5 Hz, C-5), 114 (dd, J = 2.4, 1.1 Hz, C-6), 132.3
(d, J = 4.5 Hz, C-1), 165 (d, J = 2.5 Hz, C-2), 166 (d, J = 0.5 Hz, C-4),
202.6 (d, enhanced, J = 44.5 Hz, CO).
9
104.16 mmol) in EtOH (70 mL) was heated to reflux under a
nitrogen-atmosphere for 5 h. After cooling down to room
temperature, the mixture was poured into ice-water, neutra-
lized with 1 N HCl, and the yellow precipitate was filtered
off and washed with 50% aqueous MeOH to afford the
1-(2-hydroxy-4-methoxyphenyl)-3-(4-methoxyphenyl)-[1,2,3-13C3]
prop-2-en-1-one (2.7 g, 91%) as yellow solid; mp 102–1041C
(Lit29 106–1081C for the unlabelled); dH (500MHz, CDCl3) 3.86,
3.87 (6H, 2 ꢁ s, 2 ꢁ OCH3), 6.47–6.50 (2H, m, H-30,50), 6.95 (2H, d,
J = 8.7 Hz, H-300,500), 7.46 (1H, dddd, J = 155, 15.5, 5.4, 1.5 Hz, H-2),
7.62 (2H, dd, J = 8.7, 4.6 Hz, H-200,600), 7.83 (1H, dd, J = 8.7, 3.6 Hz,
H-60), 7.87 (1H, dddd, J = 157, 15.4, 5.7, 2.3 Hz, H-3), 13.56
(1H, s, OH); dC (75 MHz, CDCl3) 118.0 (dd, J = 72.7, 58.3 Hz, C-2);
144.5 (d, enhanced, J = 72.7 Hz, C-3), 191.9 (d, enhanced,
J = 58.3 Hz, 13C-3). To a solution of the 2’-hydroxychalcone
(2.5 g, 8.71 mmol) in dry pyridine (20 mL) was added acetic
anhydride (3 mL) at room temperature, and the resulting
4,6-Dimethoxy-2-hydroxy-[1’,2’-13C]acetophenone 6b
As described for 6a, [13C2]acetyl choride (1 g, 0.906 ml, mixture was stirred overnight. The reaction was quenched
12.4 mmol) in CH2Cl2 (5 ml) with a solution of dimethoxyphenol with ice-water and extracted with CH2Cl2 (3 ꢁ 50 mL). The
5b (1.96 g, 12.74 mmol) BCl3 (1.49, 1 M in CH2Cl2 12.7 ml, combined extracts were washed with 1 N HCl, brine, water and
12.74 mmol). Column chromatography on silica with petroleum dried over MgSO4. The solvent was evaporated and the residue
ether (bp 40–60)/diethyl ether (4:1) as eluant afforded 6b (2.21 g, was purified by flash chromatography on silica gel using
90%) as a white solid: mp 79–801C (Lit.28mp. 771C for the CH2Cl2:EtOAc (98:2) as eluant to give the title compound 10a
unlabelled); dH (300 MHz, CDCl3) 2.61 (3H, dd, J = 129.2 Hz, (2.72 g, 91%) as a yellow semi-solid, which was used without
J = 6.3 Hz, 13CH3); 3.82, 3.85 (6H, 2s, 2 ꢁ OCH3), 5.92 (1H, dd, purification in the next step. dH (500MHz, CDCl3) 2.27 (3H, s,
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Copyright r 2009 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2010, 53 95–103

N. Al-Maharik and N. P. Botting
CH3CO) 3.85, 3.87 (6H, 2 ꢁ s, 2 ꢁ OCH3), 6.66 (1H, d, J = 2.5 Hz, concentrated under reduced pressure and poured into cold
H-30), 6.86 (1H, dd, J = 8.7, 2.5 Hz, H-50), 6.92 (2H, d, J = 9.0 Hz, water, extracted with CH2Cl2 (2 ꢁ 100 mL), dried (MgSO4),
H-300,500), 7.12 (1H, ddd, J = 157, 16.0, 2.5 Hz, CH-2), 7.54 (2H, dd, filtered and concentrated at reduced pressure. The brown solid
J = 9.0, 4.6 Hz, H-200,600), 7.64 (1H, dm, J = 150 Hz H-3), 7.76 (1H, dd, was purified by flash chromatography on silica gel with
J = 8.7, 3.9 Hz, H-60); dC (75 MHz, CDCl3) 122.7 (dd, J = 57.2, EtOAc:CH2Cl2 (95:5) as eluant to afford 11b (0.675 g, 77%) as a
70.6 Hz, C-2); 144.2 (d, enhanced, J = 70.6 Hz, C-3), 189.7 (d, light yellow solid, mp 160–1611C (Lit31 162–1631C for the
enhanced, J = 57.2 Hz, C-1).
unlabelled); dH (300 MHz, CDCl3) 3.83 (3H, s, OCH3), 3.89 (3H, s,
OCH3), 3.93 (3H, s, OCH3), 6.36 (1H, d, J = 1.6 Hz, H-8), 6.44 (1H,
dd, J = 2.3, 1.6 Hz, H-6), 6.93 (2H, d, J = 8.8 Hz, H-30,50), 7.48 (2H,
dd, J = 8.8, 3.4 Hz, H-20, 60), 7.76 (1H, dt, J = 194.3, 6.3 Hz, H-2); dC
(75 MHz, d6-CDCl3) 125.9 (dd, enhanced J = 70.8, 55.4 Hz, C-3),
150.0 (d, enhanced J = 70.8 Hz, C-2), 175.4 (d, enhanced
J = 55.4 Hz, C-4); HRMS (EI): C1513C3H16O5 requires 315.1098;
Found 315.1098.
3-(4-Methoxyphenyl)-1-[2-acetoxy-4,6-dimethoxyphenyl]-2-
[1,2,3-13C3]propen-1-one 10b
A
benzaldehyde (0.72 g, 5.25 mmol) and KOH (2.82 g,
mixture of the acetophenone 6b (1.0 g, 5.05 mmol),
9
50.50 mmol) in dry MeOH (70 mL) and THF (20 mL) was heated
to reflux under a nitrogen atmosphere for 6 h. The mixture was
cooled down to room temperature, poured into ice-water, the
yellow solid was filtered, washed with 50% aq. MeOH and dried
to afford the product (1.41 g, 88%) as a yellow solid which was
subjected to further reaction without purification. Acetic
anhydride (5 mL) was added to a solution of the yellow solid
(1.4 g, mmol) in dry pyridine (10 mL). After overnight stirring at
rt, the mixture was poured into ice-water, extracted with
CH2Cl2 (2 ꢁ 50 mL), the combined extracts were washed with
1 N HCl, brine, water and dried over MgSO4. The solvent was
evaporated at reduced pressure and the residue was purified
by chromatography on silica gel with CH2Cl2:EtOAc (98:2) as
eluant to yield the title compound 10b (1.46 g, 92%) as an
yellow semi solid; dH (500 MHz, CDCl3) 2.17 (3H, s, CH3CO), 3.79
(3H, s, OCH3), 3.85 (6H, s, 2 ꢁ OCH3), 6.29 (1H, d, J = 2.5 Hz, H-30),
6.41 (1H, dd, J = 8.7, 2.5 Hz, H-50), 6.87 (1H, ddd, J = 160, 16.0,
2.5 Hz, H-2), 6.89 (2H, d, J = 9.0 Hz, H-300,500), 7.38 (1H, dm,
J = 160 Hz, H-3), 7.49 (2H, dd, J = 9.0, 4.5 Hz, H-200,600); dC
(75 MHz, CDCl3) 126.0 (dd, enhanced, J = 58.3, 69.4 Hz, C-2);
144.2 (d, enhanced, J = 69.4 Hz, C-3), 191.9 (dd, enhanced,
J = 58.3 Hz, C-1); HRMS (EI): C1713C3H20O6 requires 359.1361;
Found 359.1355.
[2,3,4-13C3]Genistein 1c
A mixture of 11b (1 g, 3.17 mmol) and 55% HI in AcOH (100 mL)
was stirred at 1201C for 48 h. The mixture was cooled down to rt,
poured into ice-water, the precipitate was filtered and
recrystallized from 80% MeOH to afford 1c (0.72 g, 83%) as
light yellow solid; mp 297–3001C (Lit32 301–3021C for the
unlabelled); dH (300 MHz, d6-DMSO) 6.22 (1H, dd, J = 2.1, 0.9 Hz,
H-8), 6.39 (1H, dd, J = 2.1, 1.5 Hz, H-6), 6.81 (2H, d, J = 8.6 Hz,
H-30,50), 7.37 (2H, d, J = 8.6, 3.5 Hz, H-20, 60), 8.31 (1H, dt, J = 199.3,
6.8 Hz, H-2), 9.59 (1H, s, 40-OH), 10.88 (1H, s, 7-OH), 12.95 (1H, s,
5-OH); dC (75 MHz, d6-DMSO) 122.13 (dd, enhanced J = 71.5,
54.6 Hz, C-3), 153.90 (d, enhanced J = 71.5 Hz, C-2), 170.11 (d,
enhanced J = 54.6 Hz, C-4); HRMS (EI): C1213C3H11O5 requires
274.0707; Found 274.0708.
2,4-[10,20-13C3]Dibenzyloxyacetophenone 14a
AlCl3 (2.15.2 g, 16.14 mmol) followed by a solution of
[
13C2]acetyl choride (1 g, 0.906 ml, 12.4 mmol) in nitrobenzene
(2 mL) were added to an ice-cold solution of resorcinol 12
(1.77 g, 16.09 mmol) in dry nitrobenzene (8 mL) under a
nitrogen atmosphere. After 15 h stirring at room temperature,
the reaction was quenched with an excess of 1 M HCl (50 mL).
The aqueous phase was extracted with EtOAc (3 ꢁ 50 mL) and
dried over MgSO4. Removal of the solvent under reduced
pressure gave a brown solid, which on column chromatogra-
phy on silica gel with CH2Cl2:MeOH (98:2) as eluant, afforded
14a as a white solid (1.8 g, 95%); mp 142–1441C (Lit.33 1431C
for the unlabelled); dH (300 MHz, d6-acetone) 2.53 (3H, dd,
J = 127.9, 5.8 Hz, CH3), 6.31 (1H, dd, J = 2.4, 0.9 Hz, H-3), 6.43
(1H, ddd, J = 8.8, 2.4, 0.6 Hz, H-5), 7.78 (1H, dd, J = 8.8, 4.1 Hz,
H-6), 9.43 (1H, s, 4-OH), 12.74 (1H, d, 2-OH). To a solution
of the acetophenone 13a (1.8 g, 11.69 mmol) in acetone
(100 mL) were added anhydrous K2CO3 (19.35 g, 140.24 mmol),
18-crown-6 (0.309 g, 1.17 mmol) and benzyl bromide (3.13 mL,
26.3 mmol) at room temperature. The mixture was heated
under reflux for 8 h, cooled and concentrated under reduced
pressure to give a slurry, which was partitioned between H2O
(100 mL) and CH2Cl2 (3 ꢁ 100 mL). The combined extracts were
dried over MgSO4 and concentrated at reduced pressure to
7,40-Dimethoxy-[2,3,4-13C3]isoflavone 11a
The chalcone 10a (2.75 g, 8.36 mmol) was dissolved in a mixture
of MeOH (30 mL) and CH(OMe)3 (20 mL), followed by the
addition of Tl(NO3)3.3H2O (4.87 g, 10.96 mmol). After 24 h stirring
at room temperature, a 10% HCl solution (40 mL) was added and
the reaction mixture was stirred under reflux for another 3 h. The
mixture was cooled in an ice bath, and precipitates were
removed by filtration. The filtrate was concentrated under
reduced pressure and poured into ice cold water. After 24 h the
precipitates were filtered and recrystallized from MeOH to afford
11a (1.98 g, 83%) as light yellow solid, mp 160–1611C (Lit.30
161–1621C for the unlabelled); dH (300 MHz, CDCl3) 3.79, 3.81
(6H, 2 ꢁ s, OCH3), 6.86 (1H, dd, J = 2.3, 1.6 Hz, H-8), 6.95 (2H, d,
J = 8.8 Hz, H-30,50), 6.99 (1H, dd, J = 8.9, 2.5 Hz, H-6), 7.49 (2H, d,
J = 8.8, 3.4 Hz, H-2’, 6’), 7.94 (1H, dt, J = 191.3, 6.4 Hz, H-2), 8.21
(1H, dd, J = 8.9, 5.3 Hz, H-5).
5,7,40-Trimethoxyisoflavone 11b
Tl(NO3)3.3H2O (1.97 g, 4.43 mmol) was added to a solution of the give an oil, which was purified on column chromatography to
chalcone 10b (1 g, 2,78 mmol) in a mixture of MeOH (30 mL) and afford 13a (3.2 g, 82%) as a white semi solid: dH (300 MHz,
CH(OMe)3 (20 mL). After 24 h stirring at room temperature, 10% CDCl3) 2.58 (3H, dd, J = 128.1, 6.3 Hz, CH3), 5.09, 5.12 (4H, 2 ꢁ s,
HCl (40 mL) was added and the reaction mixture stirred under PhCH2), 6.61–6.65 (2H, m, H-3, 5), 7.32–7.47 (10H, m, 2 ꢁ Ph),
reflux for a further 3 h. The mixture was cooled in an ice bath, 7.87 (1H, dd, J = 9.4, 4.1 Hz, H-6); dC (75 MHz, d6-CDCl3) 32.2 (d,
and precipitates were removed by filtration. The filtrate was enhanced, J = 42.3 Hz, CH3), 70.2, 70.7 (C-PhCH2); 197.4 (d,
J. Label Compd. Radiopharm 2010, 53 95–103
Copyright r 2009 John Wiley & Sons, Ltd.
www.jlcr.org

N. Al-Maharik and N. P. Botting
enhanced, J = 42.3 Hz, C-2); Spectroscopic data in agreement
with previous literature.34
3-(4-Methoxyphenyl)-1-[2,4,6-tris(benzyloxy)phenyl]-[1,2,3-
13C3]-2-propenone 16c
A described for 10a using [1’,20-13C]-2,4,6-tribenzyloxyacetophe-
none 14b (1.9 g, 4.32 mmol) with benzaldehyde 9 (0.65 g,
2,4,6-[10,20-13C3]Tribenzyloxyacetophenone 14b
An excess of 1 M BBr3/CH2Cl2 (24 mL, 24.24 mmol) was added 4.78 mmol). After 8 h stirring at reflux temperature, the mixture
slowly to a solution of 6b (1.2 g, 6.66 mmol) in CH2Cl2 (10 mL) was poured into ice-water and extracted with CH2Cl2
under Ar at rt. Ater 24 h stirring at rt, water (100 mL) was added (3 ꢁ 100 mL). The combined extracts were washed with brine
to the mixture which was heated under reflux for 3 h. The CH2Cl2 (100 mL), water (100 mL), and dried (MgSO4). The solvent was
was removed at reduced pressure and the residue was extracted evaporated under reduced pressure and the residue was
with EtOAc (3 ꢁ 50 mL). The combined extracts were washed purified by flash chromatography on silica with petroleum
with water, dried (MgSO4), and concentrated to give an orange ether:CH2Cl2 (7:3) as eluant to afford the desired the chalcone
solid that was dried and reacted without further purification. To 16c (1.85 g, 76%) as a yellow semi solid, which was used without
a solution of the acetophenone 13b in acetone (100 mL), benzyl further purification. dH (300 MHz, CDCl3) 3.74 (3H, s, OCH3), 4.92,
bromide (2.88 mL, 24.24 mmol), 18-crown-6 (100 mg) and K2CO3 4.97 (4H, 2 ꢁ s, OCH2Ph), 6.18 (2H, d, J = 0.95 Hz, H-30, 50), 6.75
(8.11 g, 58.8 mmol) were added. After 8 h stirring at 601C, the (1H, ddd, J = 146, 16.1, 2.1 Hz, H-2), 6.88 (2H, d, J = 8.8 Hz,
mixture was cooled, poured into water and extracted with H-300,500), 7.07–7.28 (15.5H, m, PhH and 1/2 H-3), 7.31 (2H, dd,
EtOAc (3 ꢁ 50 mL). The combined extracts were washed with J = 8.8, 4.5 Hz, H-200,600), 7.41–7.49 (1/2H, m, 1/2 H-3); dC (75 MHz,
brine, water, dried (MgSO4) and the solvent removed at reduced CDCl3) 127.29 (dd, enhanced, J = 70.5, 56.5 Hz, C-2), 144.5 (d,
pressure. The residue was purified by flash chromatography on enhanced, J = 70.5 Hz, C-3), 194.4 (d, enhanced, J = 58.5 Hz, C-1);
silica with CH2Cl2:petroleum ether (9:1 to 7:3) as eluant to afford HRMS (ES) C3413C3H32O5Na requires 582.2248; Found 582.2256.
the title compound 14b (1.72 g, 65%) as a white solid, mp
81–831C (Lit.35 82–841C for the unlabelled); dH (300 MHz, CDCl3)
2-(4-Benzyloxyphenyl)-1-(2,4-bis(benzyloxyphenyl)-3,3-
dimethoxy-1-[1,2,3-13C3]propanone 17a
2.38 (3H, dd, J = 128.1, 6.3 Hz, CH3), 4.91 (2H, s, OCH2Ph), 4.96
(4H, s, 2 ꢁ OCH2Ph), 6.16 (2H, d, J = 1.0 Hz, H-3, 5), 7.17–7.31
Tl(NO3)3.3H2O (2.17 g, 4.89 mmol) was added to a suspension of
the chalcone 16a (2.15 g, 4.06 mmol) in a mixture of MeOH
J = 42.5 Hz, CH3), 70.2, (PhCH2); 70.6 (2 ꢁ PhCH2), 201.5 (d,
(15H, m, 3 ꢁ Ph); dC (75 MHz, d6-CDCl3) 32.6 (enhanced d,
13
enhanced, J = 42.5 Hz, C-2); HRMS (ES) C C2H26O4Na requires
463.1796; Found 463.1801.
(20 mL) and CH(OMe)3 (15 mL). The mixture was stirred at rt for
8 h, poured into ice-cold saturated NaHCO3 and extracted with
CH2Cl2 (3 ꢁ 50 mL). The combined extracts were washed with
brine, water and dried over MgSO4. Removal of the solvent
27
2-(4-Benzyloxyphenyl)-1-[2,4-bis(benzyloxy)phenyl]-2-
under reduced pressure gave an orange oil that was purified by
[1,2,3-13C3]propenone 16a
chromatography on silica gel with PhCH3:EtOAc (8:2) as eluant
A described for 10a using [10,20-13C2]-2,4-dibenzyloxyacetophe- to give 17a (2.15 g, 89%) as a white semi solid, dH (300 MHz,
none 14a (1.5 g, 4.49 mmol) with 4-benzyloxy-[carbonyl-13C]- CDCl3) 3.04, 3.36 (6H, 2 ꢁ d, J = 4.5 Hz, CH(OCH3)2), 4.74 (1/2H, d,
benzaldehyde 15 (1.15 g, 5.4 mmol) as pale yellow precipitate. J = 8.7 Hz, 1/2H-3), 4.83–4.87 (1/2H, m, 1/2H-2), 4.98 (2H, s,
The precipitate was washed with water and MeOH to offer CH2Ph), 5.03 (2H, s, CH2Ph), 5.05 (1H, d, J = 10.8 Hz, CHbPh-20),
compound 16a (2.24 g, 93%) as a light yellow precipitate, which 5.12 (1H, d, J = 10.8 Hz, CHaPh-20), 5.28–5.30 (1H, m, 1/2H-2
was used without further purification. dH (300 MHz, CDCl3) 5.10, and 1/2 H3), 6.52 (1H, dd, J = 2.1, 1.1 Hz, H-30), 6.56 (1H, dd,
5.12, 5.14 (6H, 3 ꢁ s, OCH2Ph), 6.66–6.70 (2H, m, H-30, 50), 6.87 J = 8.7, 2.1 Hz, H-50), 6.78 (2H, d, J = 8.7 Hz, H-300, 500), 7.08 (2H,
(2H, d, J = 8.8 Hz, H-300,500), 7.23–7.83 (19H, m, 3 ꢁ Ph and H-200, dd, J = 8.7, 3.2 Hz, H-200,600), 7.31–7.41 (15H, m, 3 ꢁ Ph), 7.78 (1H,
600, H-2 and H-3), 7.89 (1H, dd, J = 9.2, 4.1 Hz, H-6’); dC (75 MHz, dd, J = 8.7, 4.2 Hz, H-6’); dC (75 MHz, CDCl3) 58.5 (dd, enhanced,
CDCl3) 125.0 (dd, enhanced, J = 70.8, 56.5 Hz, C-2), 141.8 (d, J = 47.5, 42.5 Hz, C-2), 106.8 (d, enhanced, J = 47.5 Hz, C-3), 198.0
enhanced, J = 70.8 Hz, C-3), 189.72 (d, enhanced, J = 56.5 Hz, C-1); (d, enhanced, J= 42.5 Hz, C-1), HRMS (ES1) C3513C3H36O6 requires
HRMS (EI) C3313C3H30O4 requires 529.2245; Found 529.2242.
614.2510; Found 614.2513.
3-(4-Methoxyoxyphenyl)-1-[2,4-bis(benzyloxy)phenyl]-2-
[1,2,3-13C3]propen-1-one 16b
2-(4-Methoxyphenyl)-1-(2,4-bis(benzyloxyphenyl)-3,3-
dimethoxy-1-[1,2,3-13C3]propanone 17b
A mixture of the acetophenone 14a (1.45 g, 4.34 mmol), 4- As described for 17a, using the chalcone 16b (1.225 g,
methoxy-[carbonyl-13C]benzaldehyde 9 (0.65 g, 4.78 mmol) and 2.70 mmol) with Tl(NO3)3.3H2O (1.44 g, 3.24 mmol), gave the
KOH (2.92 g, 52.14 mmol) in dry MeOH (70 mL) and THF (20 mL) title compound 17b (1.19 g, 82%) as a white semi solid; dH
was heated to reflux under a nitrogen atmosphere for 6 h. The (300 MHz, CDCl3) 3.05, 3.38 (6H, 2 ꢁ d, J = 4.5 Hz, CH(OCH3)2),
mixture was cooled in an ice bath, the yellow solid was filtered 3.76 (3H, s, OCH3), 4.76 (1/2H, d, J = 8.7 Hz, 1/2H-3), 4.84–4.90
off, washed with cold MeOH and water to give the desired (1/2iH, m, 1/2H-2), 5.04 (2H, s, CH2Ph), 5.08 (1H, d, J = 11.6 Hz,
chalcone 16b (1.78 g, 90%) as yellow oil which was used without CHbPh-20), 5.12 (1H, d, J = 11.6 Hz, CHbPh-20), 5.26–5.32 (1H, m,
further purification. dH (300 MHz, CDCl3) 3.84 (3H, s, OCH3), 5.12, 1/2H-2 and 1/2H-3), 6.53 (1H, dd, J = 2.2, 1.1 Hz, H-3’), 6.58 (1H,
5.13 (4H, 2 ꢁ s, 2 ꢁ PhCH2), 6.66–6.69 (2H, m, H-30,50), 6.80 (2H, d, dd, J = 8.7, 2.2 Hz, H-50), 6.72 (2H, d, J = 8.8 Hz, H-300, 500), 7.10
J = 8.8 Hz, H-300,500), 7.22–7.47 (13.5H, m, 2 ꢁ Ph, H-2, H-200,600 and (2H, dd, J = 8.8, 3.2 Hz, H-200,600), 7.31–7.41 (10H, m, 2 ꢁ Ph), 7.77
1/2H-3), 7.75–7.82 (1/2H, ddd, J = 15.8, 4.8, 2.8 Hz, 1/2H-3), 7.88 (1H, dd, J = 8.7, 4.1 Hz, H-60); dC (75 MHz, CDCl3) 58.4 (dd,
(1H, dd, J = 9.2, 4.1 Hz, H-60), dC (75 MHz, CDCl3) 126.2 (d, J = 71.6, enhanced, J = 47.5, 42.7 Hz, C-2), 106.7 (d, enhanced, J = 47.5 Hz,
56.3 Hz, C-2); 141.88 (d, enhanced, J = 71.6 Hz, C-3), 189.8 (d, C-3), 198.0 (d, enhanced, J = 42.7 Hz, C-1), HRMS (ES1)
enhanced, J = 56.3 Hz, C-1).
C2913C3H32O6Na, requires 538.2197; Found 538.2181.
www.jlcr.org
Copyright r 2009 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2010, 53 95–103

N. Al-Maharik and N. P. Botting
stirred under reflux for 4 h. After cooling to rt, water (10 mL) was
added, and the precipitates were filtered, washed water and
recrystalliZed from 70% EtOH to yield 1a (0.88 g, 92%) as a white
solid, mp 214–2161C (Lit.32 212–2141C for the unlabelled);
dH (500 MHz, d6-DMSO) 6.80 (2H, d, J = 8.7 Hz, H-30,50), 6.86 (1H,
dd, J = 2.2, 1.8 Hz, H-8), 6.93 (1H, dd, J = 8.7, 2.2 Hz, H-6), 7.38 (2H,
d, J = 8.7, 3.4 Hz, H-20, 60), 7.96 (1H, d, J = 8.7, 3.5 Hz, H-5), 8.26
(1H, dt, J = 197, 6.5 Hz, H-2), 9.53 (1H, s, 40-OH), 10.79 (1H, s, 7-
OH); dC (75 MHz, d6-DMSO) 123.4 (dd, enhanced, J = 72.6, 54.2 Hz,
C-3), 152.8 (d, enhanced, J = 72.6 Hz, C-2), 174.3 (d, enhanced,
J = 54.6 Hz, C-4), HRMS (EI) C1213C3H11O4 requires 258.0758;
Found 258.0755.
1-(2,4-dihydroxyphenyl)-2-(4-hydroxyphenyl)-3,3-dimethoxy-1-
[1,2,3-13C3]propanone 18a
Compound 17a (2.15 g, 3.64 mmol) was hydrogenated over 5%
Pd/C (1.0 g) in a mixture of MeOH (65 ml) and acetone (65 ml).
The mixture was stirred overnight at room temperature and
filtered through a pad of Celite. Removal of the solvent under
reduced pressure afforded the desired compound 18a (1.08 g,
93%) as white foam; dH (300 MHz, d6-acetone) 3.18, 3.36 (6H,
2 ꢁ d, J = 4.5 Hz, CH(OCH3)2), 4.96 (1 H, dddd, J = 131.7, 13.0, 8.7,
4.4 Hz, H-2), 5.11 (1H, ddd, J = 166.5, 8.7, 0.7 Hz, H-3), 6.29 (1H,
dd, J = 2.4, 0.9 Hz, H-30), 6.42 (1H, dd, J = 8.9, 2.4 Hz, H-50), 6.79
(2H, d, J = 8.6 Hz, H-300, 500), 7.35 (2H, dd, J = 8.6, 3.3 Hz, H-200,600),
8.05 (1H, dd, J = 8.9, 3.9 Hz, H-60); dC (75 MHz, d6-acetone) 55.0
(dd, enhanced, J = 47.3, 41.4 Hz, C-2), 107.10 (d, enhanced,
[2,3,4-13C3]Formononetin 1b
J = 47.3 Hz, C-3), 203.4 (d, enhanced, J = 41.4 Hz, C-1); HRMS (ES1) As described for 1a, the hydroxyacetal 18c (1.1 g, 3.28 mmol)
C1413C3H18NaO6 requires 344.1102; Found 344.1098.
gave [2,3,4-13C3]formononetin 1b (0.78 g, 88%) as a white
solid, mp 255–2561C (Lit.32 256–2571C for the unlabelled);
dH (500 MHz, d6-DMSO) 3.78 (3H, s, OCH3), 6.87 (1H, dd,
J = 1.8 Hz, H-8), 6.94 (1H, dd, J = 8.8, 2.3 Hz, H-6), 6.99 (2H, d,
J = 8.8 Hz, H-30,50), 7.50 (2H, d, J = 8.8, 3.4 Hz, H-20, 60), 7.97 (1H,
dd, J = 8.7, 3.6 Hz, H-5), 8.33 (1H, dt, J = 197.1, 6.5 Hz, H-2), 10.80
(1H, s, 7-OH); dC (75 MHz, d6-DMSO) 123.0 (dd, enhanced,
J = 72.5, 55.6 Hz, C-3), 153.1 (d, enhanced, J = 72.5 Hz, C-2), 174.5
(d, enhanced, J = 55.6 Hz, C-4), HRMS (EI) C1313C3H13O4 requires
272.0914; Found 272.0912.
1-(2,4-Dihydroxyphenyl)-2-(4-methoxyphenyl)-3,3-dimethoxy-1-
[1,2,3-13C3]propanone 18b
As described for 18a, using the chalcone 17a (1.1 g, 2.13 mmol),
gave the title compound (0.615 g, 86%) as a white foam. dH
(300 MHz, acetone-d6) 3.22, 3.47 (6H, 2 ꢁ d, J = 4.7 Hz, CH(OCH3)2),
3.76 (3H, s, OCH3), 4.73 (1H, ddt, J = 129.2, 8.3, 4.6 Hz, H-2), 5.10 (1H,
dd, J = 166.7, 8.3 Hz, H-3), 6.26–6.29 (2H, m, H-30,50), 6.86 (2H, d,
J = 8.8 Hz, H-300,500), 7.33 (2H, d, J = 8.8, 3.4 Hz, H-200, 600), 7.67 (2H,
dd, J = 8.8, 3.8 Hz, H-60), 12.73 (1H, s, OH); dC (75 MHz, CDCl3) 58.4
(dd, enhanced, J = 47.5, 42.7 Hz, C-2), 106.7 (d, enhanced,
J = 47.5 Hz, C-3), 198.0 (d, enhanced, J = 42.7 Hz, C-1), HRMS (Cl1)
C1513C3H21O6 requires 336.1439; Found 336.1444.
[2,3,4-13C3]Biochanin A 1d
As for 1a, the hydroxyacetal 18c (0.3 g, 0.855 mmol) gave [2,3,4-
13C3] biochanin 1d (0.214 g, 87%) as a white solid, mp 319–322
dec 1C (Lit.32 322–3231C for the unlabelled); dH (300 MHz, MeOD)
3.84 (3H, s, 4-OCH3), 6.24 (1H, d, J = 1.8 Hz, H-8), 6.35 (1H, d,
J = 1.8 Hz, H-6), 7.00 (2H, d, J = 8.7 Hz, H-30,50), 7.48 (2H, dd,
J = 8.7, 3.3 Hz, H-20, 60), 8.09 (1H, dt, J = 197.1, 6.7 Hz, H-2); dC NMR
(75 MHz, MeOD) 124.58 (dd, enhanced, J = 71.3, 54.7 Hz, C-3),
154.98 (d, enhanced, J = 71.3 Hz, C-2), 182.15 (d, enhanced,
J = 54.7 Hz, C-4); HRMS (EI) C1313C3H13O5 requires 288.0864;
Found 288.0853.
1-(2,4,6-Trihydroxyphenyl)-2-(4-methoxyphenyl)-3,3-
dimethoxy-1-[1,2,3-13C3]propanone 18c
Tl(NO3)3.3H2O (1.52 g, 3.42 mmol) was added to a suspension
chalcone 16c (1.6 g, 2.857 mmol) in a mixture of MeOH (20 mL)
and CH(OMe)3 (15 mL). After 48 h stirring at rt, the white solid was
filtered, and the filtrate was poured into ice-cold saturated
NaHCO3 and then extracted with CH2Cl2 (3 ꢁ 100 mL). The
combined extracts were washed with brine, water and dried over
MgSO4. Removal of the solvent under reduced pressure gave the
crude acetal that was purified by chromatography on silica gel
with CH2Cl2:EtOAc (95:5) as eluant to give 17c (0.71 g, 40%) as
a yellow oil. The crude product was then hydrogenated over 5%
Pd/C(0.5 g) in amixture of MeOH(20 mL) and acetone (20 mL). The
mixture was stirred overnight at room temperature, and filtered
through a pad of Celite. After removal of solvents under reduced
pressure, the residue was purified by flash chromatography using
CH2Cl2:EtOAc (8:2) as eluant to give 18c (0.31 g, 77%) as a white
foam; dH (300 MHz, d6-acetone) 3.18, 3.36 (6H, 2 ꢁ d, J = 4.6 Hz,
CH(OCH3)2), 3.74 (3H, s, OCH3), 5.07 (1H, dd, J = 163.2, 8.7 Hz, H-3),
5.35–5.42 (1/2H, m, 1/2H-2), 5.78–587 (1/2H, m, 1/2H-2), 6.89 (2H, s,
H-30,50), 6.84 (2H, d, J = 8.9 Hz, H-300,500), 7.35 (2H, dd, J = 8.7, 3.3 Hz,
H-200,600), 9.38, 11.87, 11.95 (3 br s, 3 ꢁ OH); dC (75 MHz, d6-acetone)
59.1 (dd, enhanced, J = 47.6, 41.3 Hz, C-2), 108.5 (d, enhanced,
J = 47.6 Hz, C-3), 205.2 (d, enhanced, J = 41.3 Hz, C-1); HRMS (ES1)
C1513C3H20NaO7 requires 374.1207; Found 374.1198.
[2,3,4-13C3]Equol 3
Compound 1a (0.12 g, 0.467 mmol) was reduced with H2 over
10% Pd/C (0.05 g) in EtOH (10 ml) until no more hydrogen was
consumed. The reaction mixture was filtered through a pad of
Celite and the solvent was evaporated under reduced pressure
to yield
a light yellow semi-solid that was purified by
chromatography on silica gel with CH2Cl2:EtOAc (9:1 to 9:2) as
eluant to give 3 (0.084 g, 74%) as a white solid, mp 154–1561C
(Lit.36 155–1571C for the unlabelled); dH (300 MHz, d6-DMSO)
2.5–3.3 (3H, m, H-3 and CH2-4), 3.88 (1H, dm, J = 150 Hz, H-2a),
4.13 (1H, dm, J = 150 Hz, H-2b), 6.18 (1H, d, J = 2.4 Hz, H-8), 6.28
(1H, dd, J = 2.4 Hz, H-6), 6.71 (2H, d, J = 8.5 Hz, H-30and 50), 6.87
(1H, dd, J = 8.2, J 4.5 Hz, H-5), 7.11 (2H, dd, J = 8.5, 3.6 Hz, H-20and
60), 9.14 (1H, s, OH), 9.26 (1H, s, OH); dC (75 MHz, d6-DMSO) 70.4
(d, enhanced, J = 33 Hz, C-2), 37.1 (t, enhanced, = 33 Hz, C-3), 31.5
(d, enhanced, J = 33 Hz, C-4); m/z (CI) 246 (MH1, 100%); HRMS
C1213C3H15O3 requires 246.1122; Found 246.1124.
3-(4-Benzyloxyphenyl)-[1,2,3-13C3]propionitrile 20
[2,3,4-13C3]Daidzein 1a
A solution of n-BuLi in hexane (1.96 mL, 2.5 M, 4.89 mmol) was
added dropwise to a solution of i-propylcyclohexylamine
Conc. HCl (5 mL) was added to a solution of the hydroxyacetal
18b (1.2 g, 3.74 mmol) in MeOH (20 mL) and the mixture was
J. Label Compd. Radiopharm 2010, 53 95–103
Copyright r 2009 John Wiley & Sons, Ltd.
www.jlcr.org

N. Al-Maharik and N. P. Botting
(0.814 mL, 4.89 mmol) in dry THF (5 mL) under nitrogen atꢀ781C. unlabelled); dH (300 MHz, d6-acetone) 1.65 (3H, ddt, J = 129, 7.0,
After 10 min stirring atꢀ781C, a solution of 40-benzyloxyphenyl- 4.0 Hz, CHCH3), 4.75 (1H, dm, J = 125 Hz, CHCH3), 6.40 (1H, d,
[1,2-13C2]acetonitrile 19 (1 g, 4.44 mmol) was slowly added and J = 2.4 Hz, H-5’), 6.47 (1H, dd, J = 8.4, J 2.4 Hz, H-3’), 6.89 (2H, dt,
the resulting solution was stirred for 5 min. [13C]Methyl iodide J = 8.4, 2 Hz, H-3 and H-5), 7.22 (2H, dd, J = 8.4, 4 Hz, H-2 and H-6),
(0.306 mL, 4.89 mmol) was added, and the reaction mixture was 7.90 (1H, dd, J = 9, 4 Hz, H-6’), 8.2 (1H, br, OH), 9.4 (1H, br, OH);
stirred for 1 h atꢀ781C and then allowed to warm gradually to rt. dC (75.43 MHz, d6-acetone) 19.6 (d, enhanced, J = 37 Hz, CH3),
After 2 h at rt the solution was poured into 1 N HCl (20 mL) and 46.3 (dd, enhanced, J = 40, 37 Hz, CH), 108,6 (d, J = 4.5 Hz), 206.5
extracted with diethyl ether (3 ꢁ 30 mL). The combined organic (d enhanced,, J = 40 Hz, CO).
phases were then washed with brine and water, dried (MgSO4)
and the solvent removed at reduced pressure. Chromatography
on silica gel using hexane:diethyl ether (9:1) as eluant gave the
title compound 20 (0.78 g, 73%) as a white solid, mp 69–711C
Acknowledgements
(Lit.37 71–721C for the unlabelled); dH (300 MHz, CDCl3) 1.59 (3H,
We wish to thank Caroline Horsburgh for mass spectrometry
and Tomas Lebl and Melanja Smith for NMR spectroscopy. This
work was supported by FSA (Contracts T05023 and T05028).
dddd, J = 131.1, 10.2, 6.3, 4.2 Hz, CH3), 3.81 (1H, dm, J = 134.1 Hz,
CHCN), 5.07 (2H, s, CH2Ph), 6.98 (2H, d, J = 8.7 Hz, H-3,5), 7.27 (2H,
dd, J = 8.7, 4.2 Hz, H-2,6), 7.32–7.49 (5H, m, Ph); dC (75 MHz,
CDCl3) 21.5 (d, enhanced, J = 33.2 Hz, CH3), 30.50 (dd, enhanced,
J = 56.1, 33.2 Hz, C-2), 121.76 (d, enhanced, J = 56.1 Hz, CN);
C1313C3H15NO: calcd. C, 81.22; H, 6.29; N, 5.83; C. found, 79.79; H,
6.29; N = 5.56.
References
[1] J. T. Dwyer, B. R. Goldin, N. Saul, S. Gualfieri Barakat,
H. Adlercreutz, J. Am. Diet. Assoc. 1994, 94, 739–743.
[2] S. Hendrich, K-W. Lee, X. Xu, H-J. Wang, P. A. Murphy, J. Nutr.
1994, 124, 1789S.
[3] P. A. Murphy, T. Song, G. Buseman, K. Barua, J. Agric. Food. Chem.
1997, 45, 4635–4638.
2-(4-Benzyloxyphenyl)-[1,2,3-13C3]propionic acid 21
The nitrile 20 (0.41 g, 1.71 mmol) was stirred under reflux in
ethanol/2 N NaOH (20:10 mL) for 1.5 h. Half of the solvent was
removed, the residue was acidified with 2 N HCl and extracted
with diethyl ether (50 mL ꢁ 3). The combined organic phases
were washed with brine and water, dried (MgSO4), filtered and
evaporated to give the desired acid 21 (0.423 g, 96%) as a white
solid; mp 134–1381C (lit.38 mp 135–1371C for the unlabelled); dH
(CDCl3, 300 MHz) 1.50 (3H, dddd, J = 129.0, 11.4, 7.2, 4.8 Hz, CH3),
3.80 (1H, dm, J = 131 Hz, CHCO2H), 5.17 (2H, s, CH2Ph), 6.94 (2H,
d, J = 8.7 Hz, H-3,5), 7.24 (2H, dd, J = 8.7, 3.9 Hz, H-2,6), 7.32–7.44
[4] K. D. R. Setchell, Z-L. Nechemias, J. N. Cai, J. E. Heubi, Lancet 1997,
350, 23–27.
[5] R. A. Dixon, D. Ferreira, Phytochemistry 2002, 60, 205–211.
[6] M. Verheus, van C. H. Gils, K-L. Boker, P. B. Grace,
S. A. Bingham, P. H. M. Peters, J. Clin. Oncol. 2007, 25, 648–655.
[7] L. S. Velentzis, J. V. Woodside, M. M. Cantwell, A. J. Leathem,
M. R. Keshtgar, Eur. J. Cancer 2008, 44, 1799–1806.
[8] F. G. E. Perabo, E. C. von Low, J. Ellinger, A. von Rucker,
S. C. Muller, P. J. Bastian, Prostate Cancer Prostatic Dis. 2008, 11,
6–12.
[9] G. Cheng, B. Wilczek, M. Warner, J-A. Gustafsson, B-M. Landgren,
Menopause 2007, 14, 468–473.
[10] L. G. Howes, J. B. Howes, D. C. Knight, Maturitas 2007, 55,
203–211.
¨
(5H, m, Ph); aC (75 MHz, d6-CDCl3) 18.2 (d, enhanced, J = 34.3 Hz,
CH3), 44.4 (dd, enhanced, J = 55.0, 34.3 Hz, C-2), 180.3 (d,
enhanced, J = 55.0 Hz, CN); C1313C3H16O: calcd. C, 75.29; H,
6.22; C. found, 74.22; H, 6.18.
[11] Y. Ikeda, M. Iki, A. Morita, E. Kajita, S. Kagamimori, Y. Kagawa,
H. Yoneshima, J. Nutr. 2006, 136, 1323–1328.
[12] F. M. Sacks, A. Lichtenstein, L. Van Horn, W. Harris, P. Kris-
Etherton, M. Winston, Circulation 2006, 113, 1034–1044.
[13] P. B. Grace, J. I. Taylor, Y-L. Low, R. N. Luben, A. A. Mulligan,
N. P. Botting, M. Dowsett, A. A. Welch, K-T. Khaw, N. J. Wareham,
N. E. Day, S. A. Bingham, Cancer Epidemiol Biomarkers Prev. 2004,
13, 698–708.
[14] D. B. Clarke, A. S. Lloyd, M. F. Oldfield, N. P. Botting, P. W. Needs,
H. Wiseman, Anal. Biochem. 2002, 309, 158–172.
[15] P. B. Grace, J. I. Taylor, N. P. Botting, T. Fryatt, M. F. Oldfield,
N. Al-Maharik, S. A. Bingham, Rapid Commun. Mass Spectrom.
2003, 17, 1350–1357.
[16] P. B. Grace, J. I. Taylor, N. P. Botting, T. Fryatt, M. F. Oldfield,
S. A. Bingham, Anal. Biochem. 2003, 315, 114–121.
[17] M. F. Oldfield, L. Chen, N. P. Botting, Tetrahedron 2004, 60,
1887–1893.
[18] M. F. Oldfield, L. Chen, N. P. Botting, J Label. Compd. Radiopharm.
2007, 50, 1266–1271.
[19] Q. Zhang, N. P. Botting, Tetrahedron 2004, 60, 12211–12216.
[20] G. G. C. Kuhnle, C. Dell’ Aquila, S. A. Runswick, S. A. Bingham, Food
Chem. 2009, 113, 1184–1187.
[21] G. G. C. Kuhnle, C. Dell’ Aquila, S. M. Aspinall, S. A. Runswick,
A. M. C. P. Joosen, A. A. Mulligan, S. A. Bingham, Food Chem.
2009, 116, 542–554.
[22] K. D. Setchell, N. M. Brown, E. Lydeking-Olsen, Eur. J. Nutr. 2002,
132, 3577–3584.
[23] C. Atkinson, C. L. Frankenfeld, J. W. Lampe, Exp. Biol. Med. 2005,
230, 155–170.
[1,2,3-13C3]-O-DemethylAngolensin, 1-(2,4-dihydroxyphenyl)-2-
(4-hydroxyphenyl)-[1,2,3-13C3]propan-1-one 4a
Compound 21 (0.40 g, 1.54 mmol) was reduced with H2 over 5%
Pd/C (0.10 g) in EtOH (10 ml) until no more hydrogen was
consumed. The reaction mixture was filtered through a pad of
Celite and the solvent was evaporated under reduced pressure
to give 2-(4-hydroxyphenyl)-[1,2,3-13C3] propionic acid 22
(0.25 g, 96%) as a light yellow solid; mp 127-1281C (Lit.39
126–1291C for the unlabelled); dH (300 MHz, CDCl3,) d 1.41 (3H,
dm, J = 128.4, 11.4, 7.2 Hz, CH3), 3.01 (1H, br s, phenol OH), 3.63
(1H, dm, CHCO2H)), 6.73 (2H, d, J = 8.1 Hz, H-3,5), 7.06 (2H, dd,
J = 8.1, 3.9 Hz, H-2,6).
A suspension of resorcinol (0.21 g,
1.91 mmol) and 22 (0.16 g, 0.946 mmol) in BF3.Et2O (0.355 mL,
2.89 mmol) was heated under reflux under a nitrogen atmo-
sphere. After 15 min the mixture was allowed to cool to rt and
saturated NaOAc solution (5 mL) and 10% aq. NaHCO3 (3 mL)
were added sequentially to the dark red mixture. The orange
oily precipitate was extracted with diethyl ether (3 ꢁ 10 mL). The
combined ethereal extracts were washed with water and brine,
dried (MgSO4) and the solvent removed at reduced pressure to
give an orange viscous oil. Chromatography on silica gel with
CH2Cl2:EtOAc (8:2) as eluant gave the title compound 4a as a
white solid (0.198 g, 80%); mp 99–1021C (lit40 1031C for the
[24] K. Takagi, T. Okamoto, Y. Sakakibara, A. Ohno, S. Oka, N. Hayama,
Bull. Chem. Soc. Jpn. 1975, 48, 3298–3301.
www.jlcr.org
Copyright r 2009 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2010, 53 95–103

N. Al-Maharik and N. P. Botting
[25] L. Farkas, A. Gottsegen, M. Nogradi, S. Antus, J. Chem. Soc. Perkin
Trans. 1 1974, 305–312.
[26] J. Beger, H. J. Binte, L. Brunne, R. Neumann, J. Prak. Chem. 1992,
334, 269–277.
[32] A. Pelter, S. Foot, Synthesis 1967, 326.
[33] K. G. Bauer, F. Schoder, Arch. Pharm. 1921, 259, 53–59.
[34] A. K. Chakraborti, S. V. Chankeshwara, J. Org. Chem. 2009, 74,
1367–1370.
[27] C. K. Lau, P. C. Belanger, C. Dufresne, J. Scheigetz, J. Org. Chem.
1987, 52, 1670–1673.
[35] M. L. Whitelaw, J. R. Daniel, J. Agric. Food Chem. 1991, 39, 44–51.
[36] A. Gupta, S. Ray, Synthesis 2008, 3783–3786.
[37] G. Chen, Z. Wang, J. Wu, K. Ding, Org. Lett. 2008, 10, 4573–4576.
[38] Y. G. Perron, W. F. Minor, C. T. Holdrege, W. J. Gottstein,
J. C. Godfrey, L. B. Crast, R. B. Babel, L. C. Cheney, J. Am. Chem.
Soc. 1960, 82, 3934–3938.
[39] F. Nerdel, L. Fischer, Chem. Ber. 1954, 87, 217–220.
[40] R. A. Micheli, A. N. Booth, A. L. Livingston, E. M. Bickoff, J. Med.
Phar. Chem. 1962, 5, 321.
[28] G. Bringmann, T. F. Noll, T. Gulder, M. Dreyer, M. Gru¨ne,
D. Moskau, J. Org. Chem. 2007, 72, 3247–3252.
[29] A. Maiti, M. Cuendet, V. L. Croy, D. C. Endringer, J. M. Pezzuto,
M. Cushman, J. Med. Chem. 2007, 50, 2799–2806.
[30] M. X. D. Donnelly, J-P. Finet, B. A. Rattigan, J. Chem. Soc. Perkin
Trans. 1 1993, 1729–1735.
[31] R. B. Bradbury, D. E. White, J. Chem. Soc. 1951, 3447–3449.
J. Label Compd. Radiopharm 2010, 53 95–103
Copyright r 2009 John Wiley & Sons, Ltd.
www.jlcr.org