The synthesis of [3,4,1′-13C3]genistein

Article abstract of DOI:10.1002/jlcr.1460

A facile synthesis is described for [3,4,1′-¹³C ₃]genistein for use as an internal standard in isoflavone analysis by mass spectrometric methods. Ethyl 4-hydroxy[1-¹³C]benzoate was first prepared from the reaction of dieth

Full text of DOI:10.1002/jlcr.1460

Journal of Labelled Compounds and Radiopharmaceuticals
J Label Compd Radiopharm 2007; 50: 1266–1271.
Published online 4 October 2007 in Wiley InterScience
(www.interscience.wiley.com). DOI: 10.1002/jlcr.1460
JLCR
Research Article
The synthesis of [3,4,10-13C3]genistein
MARK F. OLDFIELD, LIRONG CHEN and NIGEL P. BOTTING*
School of Chemistry, University of St. Andrews, St. Andrews, Fife, KY16 9ST, UK
Received 16 July 2007; Revised 14 August 2007; Accepted 16 August 2007
Abstract: A facile synthesis is described for [3,4,10-13C3]genistein for use as an internal standard in isoflavone
analysis by mass spectrometric methods. Ethyl 4-hydroxy[1-13C]benzoate was first prepared from the reaction of
diethyl [2-13C]malonate and 4H-pyran-4-one. Two further 13C atoms were incorporated using potassium
[
13C]cyanide as the source to give 40-benzyloxy-[1,2,10-13C3]phenylacetonitrile. [3,4,10-13C3]Genistein was then
constructed through coupling of the isotopically labelled phenylacetonitrile with phloroglucinol under Hoesch
conditions, followed by formylation and cyclization. Copyright # 2007 John Wiley & Sons, Ltd.
Keywords: genistein; isoflavones; phytoestrogens; polyphenols
Introduction
exposure and disease.14 To improve the accuracy and
reproducibility the choice of internal standard is highly
important. The optimum internal standard for LC-MS
and GC-MS is a pure, stable, isotopically labelled
analogue of the analyte, which must have a mass
difference large enough to nullify the effect of natural
abundance heavy isotopes in the analyte. This mass
difference will depend on the molecular weight of the
analyte and the presence of heteroatoms. For mole-
cules of the size of isoflavones an extra three mass
units is sufficient, as 99% enrichment at each position
results in less than 1 ppm residual unlabelled analyte
in the internal standard and also the Mþ3 ion due to
natural 13C in the analyte will be at less than 1%
abundance giving minimal overlap. In previous studies
we synthesized [3,4,8-13C3]daidzein (1)15 and
[2,3,4-13C3]glycitein (2)16 for use as internal standards.
Herein we report the synthesis of [3,4,10-13C3]genistein
(3), which completes the set of internal standards for
the three most common isoflavones.
Isoflavone phytoestrogens, such as daidzein, genistein
and glycitein, are a group of polyphenolic compounds
with weak estrogenic and antiestrogenic activity, pre-
sent in the human diet,1,2 particularly in soybeans and
soy-derived products.3,4 Epidemiological studies have
shown that the consumption of an isoflavone-rich diet
is associated with a decrease in the incidence of
hormone-related cancers, such as breast and prostate
cancer.5,6 It has also been suggested that isoflavones
may possess other health-promoting activities, includ-
ing chemoprevention of osteoporosis7 and cardiovas-
cular disease8 and lessening of menopausal
symptoms.7 However, although many studies have
been carried out, there are still questions to be
answered concerning the absorption, metabolism and
bioavailability of isoflavonoids.9,10
Analytical chemistry has played a key role in under-
standing the significance of the biological effects of
phytoestrogens, in particular, mass spectrometry-
based methods, i.e. LC-MS11,12 and GC-MS.13 Accurate
analysis has been important in attempts to establish
the exposure of the population to the soy isoflavones
through their diet and also in epidemiological studies to
investigate the associations between isoflavone
Results and discussion
Previously, [3,4,8-13C3]daidzein (1)15 was prepared
from [2-13C]resorcinol (4) and 40-hydroxy[1,2-13C2]phe-
nylacetic acid (5) (Scheme 1) and it was initially
intended to use a similar strategy for the synthesis of
13C-labelled genistein. However, this route requires the
synthesis of 13C-labelled phloroglucinol and this
proved a much more challenging target than the
[2-13C]resorcinol. Introduction of the extra oxygen
*Correspondence to: Nigel P. Botting, School of Chemistry, University of
St. Andrews, St. Andrews, Fife, KY16 9ST, UK.
E-mail: npb@st-andrews.ac.uk
Contract/grant sponsor: FSA; contract/grant numbers: T05001,
T05023
Copyright # 2007 John Wiley & Sons, Ltd.

THE SYNTHESIS OF [3,4,10-13C3]GENISTEIN 1267
substituent, either as a protected alcohol or carbonyl
group, caused severe problems and made the previous
synthetic route untenable.
using LiAlH4 and brominated using PBr3. A simple
nucleophilic substitution using potassium [13C]cyanide
in acetonitrile with 18-crown-6 gave the key triply
13C-labelled precursor, 40-benzyloxy-[1,2,10-13C3]pheny-
lacetonitrile (12).
Therefore, it was decided to incorporate all three 13C-
atoms into the phenylacetic acid fragment, although this
was less efficient as it requires more steps and is not a
convergent synthesis. Ethyl 4-hydroxy[1-13C]benzoate
(6) was first prepared from the reaction of diethyl
[2-13C]malonate and 4H-pyran-4-one (7), using chem-
istry originally developed by Steglich (Scheme 2),17 to
incorporate the first 13C-atom into the benzene ring,
which will become the B-ring of the isoflavone .Hydro-
lysis of the ethyl ester under basic conditions was then
followed by copper-catalysed decarboxylation to give
[4-13C]phenol (8). Selective iodination at the para
position was achieved under basic conditions and then
the hydroxyl group was protected by benzylation to give
For the synthesis of daidzein, the nitrile was hydrolysed
to the acid, which was then coupled with resorcinol to give
the deoxybenzoin.15 However, with genistein better yields
are obtained if the nitrile is used directly. Thus, coupling of
the 40-benzyloxy-[1,2,10-13C3]phenylacetonitrile (12) and
phloroglucinol was carried out under modified Hoesch
reaction conditions,19 using catalytic zinc chloride,20 to
give the genistein deoxybenzoin. Finally cyclization and
formylation provided the final product, [3,4,10-13C]genis-
tein (3). The [3,4,10-13C3]genistein (3) was found to be
identical to genistein in all respects except for the expected
increase in mass and changes to the NMR spectra. The
three 13C atoms were observed at 182.3 (C4), 125.0 (C3)
and123.3 ppm (C10) in the 13C NMR spectrum. The purity
of the compound was confirmed by reverse-phase HPLC
(Kingsorb 3m C18 Column (150 ꢀ 4.6 mm)) giving a reten-
tion time of 7.85 min with a mobile phase of acetonitrile:-
water (1:1) and a 0.5 mL minꢁ1 flow rate. This gave a single
peak at the identical retention time to unlabelled genistein.
Furthermore, the UV spectrum was measured giving a
4-benzyloxy-[1-13C]iodobenzene (9). The two further 13
C
atoms were derived from two moles of [13C]cyanide ion,
which were added consecutively. Although it may also
be possible to add both 13C atoms in one go using
[1,2-13C2]acetyl chloride, it was decided to use potas-
sium [13C]cyanide as a cheaper source of isotopic label.
Palladium-catalysed cyanation of the 4-benzyloxy-
[1-13C]iodobenzene, as employed in the synthesis of
[3,4,8-13C3]daidzein (1),15,18 gave the doubly labelled
product, in very good 87% yield. The nitrile was then
hydrolysed to the carboxylic acid, reduced to the alcohol
lmax (EtOH) of 262 nm and e¼ 35460 dm3 molꢁ1 cmꢁ1
,
compared with literature value21 of e¼ 35842 dm3
molꢁ1 cmꢁ1, implying 98% purity within experimental
OH
HO 13C
O
HO 13C
OH
a,b
OH
13C
+
13C
13C
O
13C
O
4
5
OH
1
Scheme 1 (Reagents and conditions: (a) BF3 ꢂ Et2O (55%) and (b) DMF ꢂ (OMe)2, DMF (80%))
CO2Et
13C
13CO2H
13C
I
13C
13C
O
b.c
a
d,e
f,g
O
OH
OH
OBn
OBn
6
7
8
9
10
h,i
13CH213CN
13C
13CH2Br
13C
HO
O
j
k,l
13C
13C 13C
O
OH
OH
OBn
OBn
3
12
11
Scheme 2 (Reagents and conditions: (a) diethyl [2-13C]malonate, tBuOH, tBuOK (85%); (b) NaOH, H2O, reflux, 3 h (91%);
(c) quinoline, copper bronze, reflux, 4 h (80%); (d) NaOH, MeOH, I2 (65%); (e) BnBr, K2CO3, acetone (80%);
(f) K 13CN, Pd(OAc)2, Ca(OH)2, DMF (87%); (g) 2 N NaOH, MeOH (86%); (h) LiAlH4, THF (82%); (i) PBr3, Et2O (90%); (j) K13CN,
18-crown-6, MeCN (88%); (k) phloroglucinol, ZnCl2, Et2O (51%); (l) BF3 ꢂ Et2O, DMF, PCl5 (52%))
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 1266–1271
DOI: 10.1002.jlcr

1268 M. F. OLDFIELD ET AL.
error. The compound has since been successfully em-
ployed as an internal standard in both GC-MS13 and
LC-MS11,12 using isotope dilution methods.
(2H, q, J 7 Hz, –CH2–), 1.39 (3H, t, J 7 Hz, –CH3); dC
(75.45 MHz, CDCl3) 167.4 (d, J 77 Hz, –CO–), 160.6 (d,
J
9 Hz, C-4), 132.3 (d, J 60 Hz, C-2, 6), 122.9
(enhanced, C-1), 115.6 (C-3, 5), 61.4 (–CH2–), 14.7
(–CH3); m/z (EI) 167 (Mþ, 2%), 139 [ðM2EtÞþ, 20], 122
[ðM2OEtÞþ, 100], 94 [ðM2COOEtÞþ, 15]; m/z (ESꢁ) 166
[(M–H)ꢁ, 100%].
Experimental
General
4-Hydroxy-[1-13C]benzoic acid
NMR spectra were recorded on a Varian Gemini 2000
(1H 300 MHz, 13C 75.45 MHz) or a Bruker Avance 300
(1H 300 MHz, 13C 75.45 MHz) spectrometer. Chemical
shifts (d ) in ppm are given relative to Me4Si, coupling
constants (J) in Hz. Elemental analyses were carried
out in the departmental microanalytical laboratory. IR
spectra were recorded on a Perkin-Elmer series 1420
FT IR spectrophotometer. The samples were prepared
as Nujol mulls or thin films between sodium chloride
discs and recorded in cmꢁ1. EI and CI mass spectra
were recorded on a VG Autospec. Electrospray mass
spectra were recorded on a Micromass LC-T UV spectra
were recorded on a Kontron Uvikon 930 spectrometer.
Melting points were recorded on an Electrothermal
melting point apparatus and are uncorrected. Analy-
tical thin layer chromotography (TLC) was carried out
on Merck 5785 Kieselgel 60F254 fluorescent plates.
Flash chromatography was performed according to the
procedure of Still22 using silica gel of 35–70 mm particle
size. Dimethylformamide (DMF) was distilled from
magnesium sulfphate. Diethyl ether and tetrahydro-
furan (THF) were distilled from sodium metal and
benzophenone.
Ethyl
4-hydroxy[1-13C]benzoate
(7)
(2.30 g,
13.85 mmol) was mixed with aqueous sodium hydro-
xide (2 N, 250 mL) and the mixture was heated under
reflux for 3 h. The resulting mixture was acidified to pH
1 with concentrated hydrochloric acid and extracted
with diethyl ether:methanol (4:1) (6 ꢀ 40 mL). The
organic layers were then combined, washed with brine
(2 ꢀ 40 mL) and dried (MgSO4). After removal of the
solvent, the product was obtained as a pale white solid
(1.75 g, 91%) m.p. 215–2188C (Lit24 215–2168C);
(Found C, 60.67, H, 4.20%. Calc. for 12C6 13CH6O3 C,
61.15, H, 4.35%); dH (300 MHz, Methanol-d4) 7.84 (2 H,
d, J 8.3 Hz, H-2, 6), 6.76 (2H, dd, J 8.9, 7.5 Hz, H-3, 5);
dC (75.45 MHz, Methanol-d4) 174.2 (d,
J 74 Hz,
–COOH), 166.6 (C-4), 137.0 (d, J 59 Hz, C-2, 6), 126.3
(enhanced, C-1), 120.0 (C-3, 5); m/z (CI) 140 [ðMHÞþ,
100%], 122 [ðM2OHÞþ, 10].
[4-13C]Phenol (8)
A mixture of 4-hydroxy-[1-13C]benzoic acid (1.65 g,
11.86 mmol), quinoline (20 mL) and copper bronze
(1.38 g, 21.63 mmol) was heated under reflux for 4 h,
then cooled and filtered to remove the copper bronze.
The filtrate collected was poured into hydrochloric acid
(10%, 300 mL) and stirred at room temperature. The
resulting mixture was then extracted with diethyl ether
(4 ꢀ 50 mL), washed with brine (2 ꢀ 30 mL) and dried
over MgSO4. The crude product obtained was purified
by column chromatography on silica, eluting with
hexane:ethyl acetate (1:1). After removal of the solvent
at reduced pressure, a light yellow solid was obtained
(900 mg, 80%) m.p. 35–398C (Lit25 41–428C); dH
(300 MHz, CDCl3) 7.16 (2H, m, H-3, 5), 6.88 (1H, dtt,
J 174, 7.4, 1.0 Hz, H-1), 6.76 (2H, m, H-2, 6), 5.06
(1H,brs, –OH); dC (75.45 MHz, CDCl3) 121.4 (enhanced,
C-4); m/z (EI) 95 (Mþ, 100%), 67 [ðM2COÞþ, 15].
Ethyl 4-hydroxy[1-13C]benzoate (7)
A solution of (4H)-pyran-4-one (6) (2.85 g, 29.80 mmol)
and diethyl [2-13C]malonate (3.00 g, 2.85 mL,
18.60 mmol) in dry t-butanol (60 mL) was heated to
reflux, then potassium t-butoxide (freshly dried, 2.76 g,
24.5 mmol) in dry t-butanol (120 mL) was added
dropwise under nitrogen. The resulting mixture was
heated under reflux for 3 h, then hydrochloric acid (1 N,
60 mL) was added. After a further 0.5 h heating at
reflux, the solvent was removed under reduced pres-
sure and water (150 mL) was added. The reaction
mixture was then extracted with diethyl ether
(3 ꢀ 100 mL) and the combined organic extracts
washed with water (2 ꢀ 100 mL), brine (100 mL) and
dried (MgSO4). Evaporation of the solvent at reduced
pressure gave the crude product obtained which was
purified by column (7) as a pale yellow solid (2.62 g,
85%) m.p. 114–118 (Lit23 112–1158C); Found C, 64.98,
H, 5.96%. Calc. for 12C8 13CH10O3 C, 65.26, H, 6.03%);
4-Iodo-[4-13C]phenol
To
a
solution of sodium hydroxide (635 mg,
15.90 mmol) in methanol (10 mL) was added
[4-13C]phenol (8) (755 mg, 7.94 mmol). The solution
was cooled to ꢁ58C, then iodine (2.02 g, 7.94 mmol) in
d
H (300 MHz, CDCl3) 7.96 (2H, d, J 8.9 Hz, H-2, 6), 6.89
(2H, dd, J 8.9, 7.6 Hz, H-3, 5), 6.50 (1H, brs, –OH), 4.37
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 1266–1271
DOI: 10.1002.jlcr

THE SYNTHESIS OF [3,4,10-13C3]GENISTEIN 1269
methanol (15 mL) was added dropwise. After 1 h
stirring at ꢁ58C, the resulting mixture was acidified
with hydrochloric acid (0.5 N) to pH 2 and extracted
with diethyl ether (5 ꢀ 25 mL). The combined organic
layers were washed with sodium thiosulfate
(3 ꢀ 20 mL), then brine (2 ꢀ 20 mL) and dried over
MgSO4. Further purification was carried out by column
chromatography on silica, eluting with dichlorometha-
ne:diethyl ether (20:1). After removal of the solvent, a
white solid was obtained (1.15 g, 65%) m.p. 91–928C
(Lit26 93–938C); dC (300 MHz, CDCl3) 7.44 (2H, dd, J
8.9, 2.9 Hz, H-2, 6), 6.55 (2H, dd, J 10.3, 8.9 Hz, H-3,
5), 4.78 (1H, s, –OH); dC (75.45 MHz, CDCl3) 83.1
(enhanced, C-4); m/z (EI) 221 (Mþ, 100%), 94 [ðM2IÞþ,
30], 66 [ðM2I2COÞþ, 30]. HRMS (ESꢁ): 12C5 13CH5IO
requires 220.9416; Found 220.9419.
ether (40–608C) (3:10). After removal of the solvent, a
white solid was obtained. (653 mg, 87%) m.p. 95–978C
(Lit28 968C); dH (300 MHz, CDCl3) 7.50 (2H, dd, J 8.9,
5.0 Hz, H-2, 6), 7.36–7.25 (5H, m, H-20 to H-60), 6.94
(2H, dd, J 8.9, 8.3 Hz, H-3, 5), 5.03 (2H, s, CH2); dC
(75.45 MHz, CDCl3, ppm) 119.6 (enhanced, d, J 87 Hz,
C-1), 104.6 (enhanced, d, J 87 Hz, ꢁ13CN); m/z (CI) 212
[ðMHÞþ, 100%], 91 (C7Hþ7 , 11).
4-Benzyloxy-[1,carboxyꢁ13C2]benzoic acid (10)
4-Benzyloxy-[1,nitrile-13C2]benzonitrile (569 mg, 2.70
mmol) was mixed with sodium hydroxide (2 N, 25 mL)
and methanol (2.5 mL). The mixture was heated under
reflux until the starting material was consumed
according to TLC. The resulting mixture was cooled
and adjusted to pH 1 with concentrated hydrochloric
acid. The solid precipitated was collected by filtration
and heated under reflux with sodium hydroxide (2 N,
50 mL) for a further 5 h. The resulting mixture was
cooled and again adjusted to pH 1 with concentrated
hydrochloric acid, extracted with diethyl ether:metha-
nol (6:1, 3 ꢀ 20 mL). The organic fractions were
combined, washed with brine (2 ꢀ 20 mL) and dried over
MgSO4. After removal of the solvent at reduced pressure,
4-Benzyloxy-[1-13C]iodobenzene (9)
To
a
solution of 4-iodo-[4-13C]phenol (1.04 g,
4.71 mmol) in acetone (15 mL) was added potassium
carbonate (1.88 g, 14.13 mmol) and benzyl bromide 23
(0.56 mL, 806 mg, 4.71 mmol). The mixture was heated
under reflux for 5 h, then cooled and filtered to remove
the solid. The crude product obtained on removal of the
solvent was dissolved in diethyl ether (20 mL), washed
with water (3 ꢀ 10 mL), dried over MgSO4 and the
solvent removed at reduced pressure. Further purifica-
tion was carried out by column chromatography on
silica, eluting with ethyl acetate:petroleum ether (1:20).
After removal of the solvent at reduced pressure, white
crystals were obtained (1.18 g, 80%) m.p. 59–618C
the
desired
product
was
obtained
as
a
white solid (536 g, 86%) m.p. 192–1948C (Lit29 191–
1928C); (Found C, 73.23, H, 5.22%. Calc. for
12
C
12
13C2H12O3 C, 73.90, H, 5.25%); dH (300 MHz, CDCl3)
7.95 (2H, dd, J 8.7, 3.9 Hz, H-2, 6), 7.39–7.28 (5H, m, H-20
to H-60), 6.95 (2H, dd, J 8.7, 7.7 Hz, H-3, 5), 5.08 (2H, s,
CH2); dC (75.45 MHz, CDCl3) 168.7 (enhanced, d, J 75 Hz,
13COOH), 122.9 (enhanced, d, J 75 Hz, C-1); m/z (CI) 231
[ðMHÞþ, 100%], 91 (C7Hþ7 , 12); HRMS(CI) 12C12 13C2H12O3
requires 231.0934, Found 231.0932.
(Lit27 61–628C); (Found C, 50.81, H, 3.45%. Calc. for
12
C
13CH11IO C,50.51, H, 3.56%); dH (300 MHz,
12
CDCl3) 7.48 (2H, dd, J 9.0, 2.9 Hz, H-2, 6), 7.36–7.23
(5H, m, H-20 to H-60), 6.68 (2H, dd, J 10.4, 9.0 Hz, H-3,
5), 4.96 (2H, s, H-7); dC (75.45 MHz, CDCl3) 83.4
(enhanced, C-1); m/z (EI) 311 (Mþ, 37%), 91 (C7H7þ, 100).
4-Benzyloxy-[1, methylene-13C2]benzyl alcohol
Under the protection of a nitrogen atmosphere, a
solution of 4-benzyloxy-[1,carboxy-13C2]benzoic acid
(10) (450 mg, 1.96 mmol) in dry THF (8 mL) was added
dropwise to the suspension of lithium aluminium
hydride (300 mg, 7.83 mmol) in dry THF (7 mL). The
mixture was stirred at room temperature overnight.
Sulphuric acid (10%, 20 mL) was then added carefully
to quench the reaction. The resulting mixture was
extracted with diethyl ether (4 ꢀ 20 mL), washed with
brine (2 ꢀ 10 mL) and dried over MgSO4. After removal
of the solvent at reduced pressure, the desired product
was obtained as a white solid (346 mg, 82%). m.p. 82–
858C (Lit28 84–858C); dH (300 MHz, CDCl3) 7.34–7.13
(7H, m, H-20 to H-60 and H-2, 6), 6.86 (2H, dd, J 8.7,
7.1 Hz, H-3, 5), 4.96 (2H, s, CH2), 4.41 (2H, dd, J 141,
3.8 Hz, 13CH2O); dC (75.45 MHz, CDCl3) 135.4
4-Benzyloxy-[1,nitrile-13C2]benzonitrile
To a solution of 4-benzyloxy-[1-13C]iodobenzene (9)
(1.10 g, 3.54 mmol) in dry DMF (20 mL), calcium
hydroxide (131 mg, 1.77 mmol), potassium [13C]cya-
nide (273 mg, 4.13 mmol) and palladium acetate
(122 mg, 0.54 mmol) were added. Under the protection
of a nitrogen atmosphere, the mixture was heated
under reflux for 5 h, then cooled and filtered through
celite. The DMF was removed under reduced pressure
and the residue was extracted with diethyl ether
(5 ꢀ 30 mL), washed with water (3 ꢀ 20 mL), dried over
MgSO4 and the solvent removed at reduced pressure.
Further purification was carried out by column chroma-
tography on silica, eluting with ethyl acetate:petroleum
Copyright # 2007 John Wiley & Sons, Ltd.
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DOI: 10.1002.jlcr

1270 M. F. OLDFIELD ET AL.
(enhanced, d, J 52 Hz, C-1), 65.4 (enhanced, d, J 52 Hz,
13CH2O); m/z (EI) 216 (Mþ, 20%), 91 (C7Hþ7 , 100).
mixture was saturated with HCl gas at 08C for 6 h, then
warmed to room temperature and stirred overnight.
The diethyl ether was removed, and the resulting solid
was washed with diethyl ether (2 ꢀ 5 mL), then hydro-
lysed with hydrochloric acid (0.1 N, 20 mL) at reflux for
5 h. The reaction mixture was then extracted with
diethyl ether (4 ꢀ 10 mL), washed with brine (2 ꢀ 10 mL)
and dried over MgSO4. After removal of the solvent at
reduced pressure a pink gum was obtained. Further
purification was carried out by column chromatogra-
phy on silica, eluting with diethyl ether:petroleum ether
(40608C) (2:1 then 4:1). After removal of the solvent at
reduced pressure, the desired product was obtained as
a pale yellow solid (98 mg, 51%) m.p. >2008C (dec.)
(Lit31 189–1918C); dH (300 MHz, Methanol-d4) 7.00 (2H,
dd, J 8.4, 3.9 Hz, H-20, 60), 6.59 (2H, dd, J 8.4, 7.7 Hz,
H-30, 50), 5.69 (2H, d, J 1.0 Hz, H-4, 6), 4.16 (2H, dt, J
130, 6.2 Hz, H-8); dC (75.45 MHz, Methanol-d4) 205.5
(enhanced, d, J 40 Hz, C-7), 128.5 (enhanced, d, J
51 Hz, C-10), 50.0 (enhanced, d, J 83 Hz, C-8); m/z
(ESꢁ) 262 [(MH)ꢁ, 100%]; HRMS (ESꢁ) 12C11 13C3H12O5
requires 262.0709; Found 262.0711.
4-Benzyloxy-[1,methylene-13C2]benzyl bromide (11)
To a solution of 4-benzyloxy-[1, methylene-13C2]benzyl
alcohol (320 mg, 1.48 mmol) in dry diethyl ether (6 mL)
was introduced phosphorus tribromide (0.4 mL,
4.5 mmol, 1.15 g). The resultant solution was stirred
at room temperature overnight. The pale yellow solu-
tion formed was then poured into ice water (25 mL),
extracted with diethyl ether (3 ꢀ 15 mL) and dried over
MgSO4. After removal of the solvent at reduced
pressure, a white solid was obtained (373 mg, 90%)
m.p. 85–888C (Lit28 83–858C); dH (300 MHz, CDCl3)
7.33 (5H, m, H-20 to H-60), 7.25 (2H, dd, J 8.7, 5.1 Hz,
H-2, 6), 6.87 (2H, dd, J 8.7, 7.7 Hz, H-3, 5), 5.00 (2H, s,
CH2), 4.43 (2H, dd,
J
153, 3.6 Hz, 13CH2Br); dC
(75.45 MHz, CDCl3), 130.6 (enhanced, d, J 51 Hz, C-
1), 34.3 (enhanced, d, J 51 Hz, 13CH2Br); m/z (EI) 278/
280 (1:1, Mþ, 3%), 199 [ðM2BrÞþ, 55], 91 (C7Hþ7 , 100).
40-Benzyloxy-[1,2,10-13C3]phenylacetonitrile (12)
[3, 4,10-13C3]Genistein (3)
4-Benzyloxy-[1,methylene-13C2]benzyl bromide (11)
(370 mg, 1.33 mmol) was dissolved in acetonitrile
(10 mL), then 18-crown-6 (352 mg, 1.33 mmol) and
A
mixture of [10,7,8- 13C3]genistein deoxybenzoin
(86 mg, 0.33 mmol) and boron trifluoride diethyl ethe-
rate (0.13 mL, 142 mg, 1.00 mmol) was cooled to 108C,
then dry DMF (0.6 mL) was added dropwise to give
solution A. In another flask, dry DMF (0.9 mL) was
cooled to 108C, then phosphorus pentachloride
(104 mg, 0.50 mmol) was added in small portions. The
mixture was kept at 558C for 20 min to give solution B.
Solution B was introduced into solution A slowly, the
temperature was kept below 278C during the addition.
The resulting mixture was stirred at room temperature
for 20 min, then poured into aqueous sodium acetate
(12.5%, 30 mL), stirred for 2 h and left overnight. The
solid formed was collected by filtration and washed
with water (3 ꢀ 5 mL). Further purification was carried
out by HPLC (Kingsorb 3m C18 Column, acetonitrile:-
water as eluant). After removal of the solvent at reduced
pressure, the desired product was obtained as a white
solid (47 mg, 52%) m.p. 3078C (Lit32 301–3028C); dH
(300 MHz, acetone-d6) 6.28 (1H, dd, J6,8 2.2, J4,8
0.8 Hz, H-8), 6.42 (1H, d, J6,8 2.2, J4,6 1.5 Hz, H-6), 6.9
potassium
[
13C]cyanide (88 mg, 1.33 mmol) were
added. The mixture was heated under reflux for 4 h,
then cooled and the solvent was removed at reduced
pressure. The pale white residue was then extracted
with diethyl ether (3 ꢀ 20 mL), washed with water
(2 ꢀ 10 mL), brine (10 mL) and dried over MgSO4. The
crude product was purified by column chromatography
on silica, eluting with petroleum ether (40–608C):ethyl
acetate (5:3). After removal of the solvent at reduced
pressure, the desired product was obtained as white
crystals (264 mg, 88%) m.p. 67–708C (Lit15 66–698C);
(Found C, 80.61, H, 5.77, N, 6.11%. Calc. for
12
C
13C3H13NO. C, 80.95, H, 5.79, N, 6.19%); dH
12
(300 MHz, Methanol-d4) 7.34–7.20 (5H, m, H-20 to H-
60), 7.16 (2H, dd, J 8.9, 4.8 Hz, H-2, 6), 6.90 (2H, dd, J
8.4, 8.3 Hz, H-3, 5), 4.97 (2H, s, CH2), 3.69 (2H, ddd, J
136, 10.8, 6.9 Hz, –13CH2); dC (75.45 MHz, Methanol-
d4) 124.8 (enhanced, d, J 52 Hz, C1), 120.3 (d, J 61 Hz,
–
13CN), 23.0 (d, J 102 Hz, –13CH2–); m/z (EI) 226 (Mþ,
10%), 91 (C7Hþ7 , 100); (ESþ) 249 [ðMNaÞþ, 100%], 227
[ðMHÞþ;15].
(2H, dt, J2;3 ¼ J5 ;6 8.5, J1;3 ¼ J1;5 1.9 Hz, H-30,50), 7.45
0
0
0
0
(2H, dd, J2 ;3 ¼ J5 ;6 8.5, J1 ;2 ¼ J1 ;6 3 Hz, H-20,60), 8.15
(1H, m, H-2), 8.5 (1H, s, OH-4), 9.7 (1H, s, OH-7); dC
(75.45 MHz, Methanol-d4, ppm) 182.3 (enhanced, dd,
J 52, 4 Hz, C-4), 125.0 (enhanced, dd, J 60, 52 Hz, C-3),
123.3 (enhanced, dd, J 60, 4 Hz, C-10); m/z (CI) 274
0
0
0
0
0
0
0
0
[7,8,10-13C3]Genistein deoxybenzoin
Under vigorous stirring, freshly fused zinc chloride
(15 mg, 0.11 mmol) was added to the solution of (12)
(150 mg, 0.66 mmol) in dry diethyl ether (6 mL). The
(MHþ,
100%)
(HRMS
C12 13C3H11O5
requires
274.070713. Found 274.069965).
Copyright # 2007 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2007; 50: 1266–1271
DOI: 10.1002.jlcr

THE SYNTHESIS OF [3,4,10-13C3]GENISTEIN 1271
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DOI: 10.1002.jlcr