trans-resveratrol-d4, a molecular tracer of the wild-type phytoalexin; synthesis and spectroscopic properties

Article abstract of DOI:10.1055/s-2008-1067239

A convenient, six-step synthesis of the so far unknown trans-resveratrol- d₄, (E)-3′,4,5′-trihydroxy-2,3,5,6-tetradeuterostilbene, starting from commercially available phenol-d₆, with an overall yield of 25%, is described. The final

Full text of DOI:10.1055/s-2008-1067239

PAPER
2953
trans-Resveratrol-d4, a Molecular Tracer of the Wild-Type Phytoalexin;
Synthesis and Spectroscopic Properties
S
ynthesis of tran
a
-
Resverat r
r
ol-
d
4
tolo Gabriele,*a Hicham Benabdelkamel,b Pierluigi Plastina,b Alessia Fazio,a Giovanni Sindona,b
Leonardo Di Donnab
a
Dipartimento di Scienze Farmaceutiche, Università della Calabria, 87036 Arcavacata di Rende (Cosenza), Italy
Fax +39(984)492044; E-mail: b.gabriele@unical.it
Dipartimento di Chimica, Università della Calabria, 87036 Arcavacata di Rende (Cosenza), Italy
b
Received 28 May 2008; revised 5 June 2008
tive, starting from commercially available phenol-d6, with
an overall yield of 25% over six steps.
Abstract: A convenient, six-step synthesis of the so far unknown
trans-resveratrol-d4, (E)-3¢,4,5¢-trihydroxy-2,3,5,6-tetradeuterostil-
bene, starting from commercially available phenol-d6, with an over-
all yield of 25%, is described. The final labeled resveratrol was fully
(E)-3¢,4,5¢-Trihydroxy-2,3,5,6-tetradeuterostilbene (trans-
resveratrol-d4, 7) has been prepared according to the con-
vergent synthetic strategy shown in Scheme 1.
1
characterized by MS, IR, and H and 13C NMR spectroscopy. The
isotopic distribution of the final product, determined by high reso-
Commercially available phenol-d6 (1) was converted into
2,3,4,5,6-pentadeuteroanisole (2) in 90% yield by depro-
tonation with sodium hydride in tetrahydrofuran at 0 °C
followed by quenching with iodomethane. Formylation/
oxidation of 2 with formaldehyde–sulfuric acid-d2/2,3-
dichloro-5,6-dicyano-1,4-benzoquinone in acetonitrile at
80 °C led to a mixture of 3,4,5,6-tetradeutero-o-anisalde-
hyde (20% yield) and 2,4,6,7-tetradeutero-p-anisaldehyde
(3, 60% yield), which could be easily separated by column
chromatography. On the other hand, commercially avail-
able 3,5-dimethoxybenzyl bromide (4) was easily con-
verted into (3,5-dimethoxybenzyl)triphenylphosphonium
bromide (5) in 92% yield by the reaction with an excess of
triphenylphosphine in acetonitrile at room temperature.
The key step of the synthesis then consisted of the ole-
fination reaction between 3 and 5, to give 3¢,4,5¢-tri-
methoxy-2,3,5,6-tetradeuterostilbene (6) as a E/Z mixture
in 70% yield based on 3. Double bond isomerization with
a catalytic amount of diphenyl disulfide in refluxing tet-
rahydrofuran to give the trans-isomer (E)-6 followed by
deprotection (BBr3, CH2Cl2, –20 °C) eventually led to the
desired deuterated resveratrol 7 in 47% yield based on
deuterated aldehyde 3. The overall yield of 7 based on
starting 1 was 25% over six steps.
lution mass spectrometry, was as follows: d4, 96%; d3, 4%.
Key words: deuterated resveratrol, olefination, resveratrol, stable
isotope dilution assay
Resveratrol (3,4¢,5-trihydroxystilbene) is a polyphenolic
phytoalexin found in more than 70 plants and many foods,
including grapes, peanuts, berries, and red wine.1 Resver-
atrol has recently attracted considerable attention in view
of its significant biological activity, which include antiox-
idant and/or anti-inflammatory activity.2 Recent data sug-
gest that nutritional intake of resveratrol may contribute to
the so-called ‘French paradox’, that is, the unexpectedly
low incidence of coronary heart disease in the Mediterra-
nean population, in spite of a relatively high intake of sat-
urated fats. Resveratrol has been shown to inhibit LDL
oxidation and prevent the oxidative stress in general,
which results in an anti-aging effect.2,3 Moreover, resver-
atrol has been classified as a phytoestrogen, due to its abil-
ity to interact to estrogenic receptors, and has been shown
to exert antiproliferative and cancer-protective effects.4
Considering the potential beneficial effects on human
health of resveratrol, the development of new, reliable,
and sensitive techniques for its quantitative determination
in foods appears to be of primary importance. One of the
most promising methodologies for the highly sensitive
quantitative determination of microcomponents in food
products is currently represented by stable isotope dilu-
tion assay (SIDA).5 Thus, resveratrol could in principle be
detected and quantified in food samples by means of
SIDA using, for example, a deuterated derivative such as
(E)-3¢,4,5¢-trihydroxy-2,3,5,6-tetradeuterostilbene as in-
ternal standard, which, however, to our knowledge, has
not been reported in the literature so far. Here we wish to
present an easy and convenient synthesis for this deriva-
No H/D exchange and/or redistribution was observed in
the course of the synthesis, as confirmed by the spectro-
scopic characterization (MS, 1H NMR, 13C NMR, IR) of
the key intermediates 2, 3, and (E)-6. The final deuterated
resveratrol 7 was also isotopically stable and fully charac-
1
terized by MS, IR, and H and 13C NMR spectroscopy.
The isotopic distribution of 7 was determined by high res-
olution mass spectrometry, and gave the following result:
d4, 96%; d3, 4%. The characteristics of the new labeled
resveratrol 7 are therefore suitable in view of its utiliza-
tion as internal standard for the quantitative determination
of naturally occurring resveratrol in food samples.
In conclusion, a convenient synthesis of a new labeled res-
veratrol derivative, (E)-3¢,4,5¢-trihydroxy-2,3,5,6-tetra-
deuterostilbene (7), with an overall yield of 25% over six
SYNTHESIS 2008, No. 18, pp 2953–2956
Advanced online publication: 22.08.2008
DOI: 10.1055/s-2008-1067239; Art ID: Z12408SS
x
x.
x
x
.
2
0
0
8
© Georg Thieme Verlag Stuttgart · New York

2954
B. Gabriele et al.
PAPER
OD
D
D
D
D
D
D
D
D
OH
OH
HO
D
1
7 (68%)
NaH
MeI
THF
0 °C
CH2Cl2
–20 °C
BBr3
OMe
D
D
D
D
D
D
D
OMe
OMe
CH2Br
MeO
D
D
MeO
OMe
2 (90%)
(E)-6 (98%)
4
HCHO
DDQ
D2SO4
MeCN
80 °C
MeCN
25 °C
PPh3
THF
reflux
PhSSPh
OMe
CH2PPh3
Br–
D
D
OMe
D
D
D
D
t-BuOK
MeO
CH CH
+
THF
–10 °C
MeO
OMe
D
D
OMe
CHO
3 (60%)
5 (92%)
6 (Z/E mixture, 70%
based on starting 3)
Scheme 1 Synthesis of (E)-3¢,4,5¢-trihydroxy-2,3,5,6-tetradeuterostilbene (trans-resveratrol-d4, 7)
60 (Merck, 70–230 mesh). Evaporation refers to the removal of sol-
vent under reduced pressure.
steps and an isotopic purity of 96%, has been developed.
The new labeled resveratrol is isotopically stable and,
therefore, suitable for its utilization as internal standard
for the quantitative determination of resveratrol in food
samples by means of the stable isotope dilution assay
technique.
2,3,4,5,6-Pentadeuteroanisole (2)
The method of Kendall6 was employed. A soln of phenol-d6 (1, 1.0
g, 9.99 mmol) in anhyd THF (4 mL) was added dropwise over 1 h
under N2 to a cooled suspension (0 °C) of NaH (350.0 mg, 13.85
mmol) in anhyd THF (3 mL). The mixture was stirred at 0 °C for an
additional 10 min, MeI (4.6 g, 32.4 mmol) was added rapidly. The
mixture was allowed to warm up to r.t. and then refluxed for 19 h.
After cooling to r.t., the mixture was quenched with H2O (80 mL)
and extracted with hexane (3 × 50 mL). The combined organic lay-
ers were dried (Na2SO4), the solvent was removed by distillation at
atmospheric pressure, and the residue subjected to short path distil-
lation (bp 153–155 °C) to give 2 (1.02 g, 90%) as a colorless liquid.
The spectroscopic properties of the product were in agreement with
those previously reported.7
Melting points were taken on a Reichert Thermovar apparatus are
1
uncorrected. H and 13C NMR spectra were recorded on a Bruker
DPX Avance 300 spectrometer at 25 °C in CDCl3 or DMSO-d6
solns at 300 MHz and 75 MHz, respectively, with TMS as internal
standard. IR spectra were taken with a Perkin-Elmer Paragon 1000
PC FT-IR spectrophotometer. MS spectra were obtained using a
Shimadzu QP-2010 GC-MS apparatus at 70 eV ionization voltage.
The high resolution electrospray ionization (ESI) experiments were
carried out with a hybrid Q-Star Pulsar-i (PE SCIEX) mass spec-
trometer equipped with an ion spray ionization source. All samples
were acquired at the optimum ion spray voltage of 4800 V by direct
infusion (5 mL/min) of a soln containing the appropriate compound
dissolved in MeOH (10 pmol/mL). The N2 gas flow was set at 20 psi
and the declustering and the focusing potentials were kept at 50 and
220 V relative to ground, respectively. Commercially available
Renin substrate was used as calibration standard compound. The ac-
curacy of the measurement was within 5 ppm.
2,4,6,7-Tetradeutero-p-anisaldehyde (3)
The method of Neumann8 was employed. A mixture of 2 (1.0 g,
8.84 mmol), paraformaldehyde (2.4 g, 79.92 mmol), D2SO4 (0.25
mg, ca. 2.5 mmol), and DDQ (3.61 g, 15.90 mmol) in MeCN (10
mL) was stirred under N2 at 80 °C for 5 h. After cooling, EtOAc was
added, the mixture was filtered, and the solid washed with EtOAc.
The solvent used for washing the solid was added to the filtrate. The
collected EtOAc phases were evaporated and the residue was puri-
fied by column chromatography (silica gel, hexane–EtOAc, 97:3) to
give pure 3,4,5,6-tetradeutero-o-anisaldehyde (colorless oil, 245.2
mg, 20%) and 2,4,6,7-tetradeutero-p-anisaldehyde (3) (colorless
oil, 746.1 mg, 60%) in this order.
Phenol-d6 (1) (99% atom D), NaH (anhyd, 95%), MeI, paraformal-
dehyde, DDQ, D2SO4 (96–98 wt% in D2O, 99.5 atom% D) 3,5-
dimethoxybenzyl bromide (4), Ph3P, (PhS)2, and BBr3 were com-
mercially available (Aldrich) and were used as received.
IR (film): 1689, 1580, 1571, 1236 cm–1.
1H NMR (300 MHz, CDCl3): d = 3.88 (s, 3 H, OCH3), 9.88 (s, 1 H,
CHO).
All reactions were analyzed by TLC on silica gel 60 F254 and by
GLC using a Shimadzu GC-2010 gas chromatograph and capillary
columns with polymethylsilicone + 5% phenylsilicone as the sta-
tionary phase. Column chromatography was performed on silica gel
Synthesis 2008, No. 18, 2953–2956 © Thieme Stuttgart · New York

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Synthesis of trans-Resveratrol-d4
2955
13C NMR (75 MHz, CDCl3): d = 55.6 (OCH3), 114.0 (t, J = 23.8
Hz, C3, C5), 129.9 (C1), 131.7 (t, J = 23.8 Hz, C2, C6), 164.6 (C4),
190.9 (CHO).
GC-MS (EI, 70 eV): m/z (%) = 140 (73, [M+]), 139 (100), 111 (24),
96 (24), 81 (45), 80 (18), 69 (19), 68 (20), 66 (17).
ane–EtOAc, 6:4), to give pure 7 (105 mg, 68%) as a colorless solid;
mp 254–256 °C.
IR (KBr): 3294, 1590, 1407, 1345, 1155 cm–1.
1H NMR (300 MHz, DMSO-d6): d = 6.12 (s, 1 H, H4¢), 6.38 (s, 2 H,
H2¢, H6¢), 6.81 (distorted d, J = 16.5 Hz, 1 H, HC=CH), 6.93 (dis-
torted d, J = 16.5 Hz, 1 H, HC=CH), 9.18 (s, 2 H, C3¢-OH, C5¢-OH),
9.53 (s, 1 H, C4-OH).
13C NMR (75 MHz, DMSO-d6): d = 101.7 (C4¢), 104.2 (C2¢, C6¢),
115.0 (t, J = 19.0 Hz, C3, C5), 125.5 (HC=CH), 127.3 (t, J = 22.2
Hz, C2, C6), 127.7 (HC=CH), 127.8 (C1), 139.2 (C1¢), 157.0 (C4),
158.4 (C3¢, C5¢).
(3,5-Dimethoxybenzyl)triphenylphosphonium Bromide (4)
3,5-Dimethoxybenzyl bromide (2.0 g, 8.65 mmol) was added to a
stirred soln of Ph3P (3.0 g, 11.44 mmol) in MeCN (30 mL). The re-
sulting mixture was stirred at r.t. for 72 h. After removal of the sol-
vent by evaporation, the residue was solubilized in CH2Cl2 (30 mL).
Et2O (10 mL) was slowly added without stirring, leading to the pre-
cipitation of pure 4 (3.92 g, 92%) as colorless crystals; mp 273–274
°C. The 1H NMR data of the product were in good agreement with
those previously reported.9
HRMS (ES+): m/z [(M-d4 + H)+] calcd for C14H9D4O3: 233.1112;
found: 233.1105 (96%); m/z [(M-d3 + H)+] calcd for C14H10D3O3:
232.1050; found: 232.1043 (4%).
IR (KBr): 1607, 1582, 1438, 1155 cm–1.
1H NMR (300 MHz, DMSO-d6): d = 3.52 (s, 6 H, 2 OMe), 5.25 (d,
J = 15.4 Hz, 2 H, CH2P), 6.22 (t, J = 2.2 Hz, 2 H, H2, H6), 6.44 (q,
J = 2.2 Hz, 1 H, H4), 7.71–7.82 (m, 12 HPh), 7.88–7.98 (m, 3 HPh).
13C NMR (75 MHz, DMSO-d6): d = 28.3 (d, J = 46.3 Hz, CH2P),
55.0 (2 OCH3), 100.1 (d, J = 3.8 Hz, C43,5-MeO2C6H3), 108.9 (d,
J = 6.3 Hz, C23,5-MeO2C6H3, C63,5-MeO2C6H3), 117.8 (d, J = 86.3 Hz,
C1Ph), 129.9 (d, J = 8.8 Hz, C13,5-MeO2C6H3), 130.0 (d, J = 12.5 Hz,
C2Ph, C6Ph), 134.0 (d, J = 10.0, C3Ph, C5Ph), 135.0 (d, J = 3.8 Hz,
C4Ph), 160.2 (d, J = 2.5 Hz, C33,5-MeO2C6H3, C53,5-MeO2C6H3).
References
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(E)-3¢,4,5¢-Trimethoxy-2,3,5,6-tetradeuterostilbene [(E)-6]
The method of Tsuda10 was employed. To a cooled (–10 °C), stirred
mixture of (3,5-dimethoxybenzyl)triphenylphosphonium bromide
(1.68 g, 3.41 mmol) and t-BuOK (381.5 mg, 3.40 mmol) in anhyd
THF (50 mL) under N2 was added 3 (400 mg, 2.85 mmol) dropwise.
After additional stirring at –10 °C under N2 for 1 h, the mixture was
allowed to warm up to r.t. and then it was poured into H2O (ca. 100
mL) and neutralized with 1 M HCl. The resulting mixture was ex-
tracted with Et2O (3 × 30 mL) and the combined organic phases
were dried (Na2SO4). After filtration, the solvent was evaporated
and the residue purified by column chromatography (silica gel, hex-
ane–EtOAc, 8:2), to give 6 (544 mg, 70%) as a E/Z mixture. A mix-
ture of the latter (400 mg, 1.46 mmol) and (PhS)2 (60 mg, 0.27
mmol) in anhyd THF (50 mL) was refluxed under N2 for 2 h. After
cooling, the solvent was evaporated, and the residue was purified by
column chromatography (silica gel, hexane–EtOAc, 8:2), to give
pure (E)-6 (390 mg, 98%) as a colorless solid; mp 54–55 °C.
(4) See, for example: (a) Nicotra, G.; Peracchio, C.; Castino, R.;
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IR (KBr): 1591, 1456, 1204, 1151 cm–1.
1H NMR (300 MHz, CDCl3): d = 3.83 (s, 9 H, 3 OMe), 6.36–6.42
(m, 1 H, H4¢), 6.64–6.68 (m, 2 H, H2¢, H6¢), 6.89 (distorted d,
J = 16.5 Hz, 1 H, HC=CH), 7.04 (distorted d, J = 16.5 Hz, 1 H,
HC=CH).
13C NMR (75 MHz, CDCl3): d = 55.4 (2 OCH3), 100.0 (C4¢), 104.8
(C2¢, C6¢), 114.0 (t, J = 23.4 Hz, C3, C5), 126.9 (HC=CH), 127.5 (t,
J = 23.4 Hz, C2, C6), 128.9 (HC=CH), 129.5 (C1), 140.0 (C1¢),
159.6 (C4), 161.3 (C3¢, C5¢).
GC-MS (EI, 70 eV): m/z (%) = 274 (100, [M+]), 273 (8), 259 (4),
243 (8), 228 (6), 216 (5), 200 (6), 185 (4), 173 (5), 157 (7), 156 (6),
155 (5), 145 (6), 137 (6), 119 (4).
(E)-3¢,4,5¢-Trihydroxy-2,3,5,6-tetradeuterostilbene (trans-Res-
veratrol-d4, 7)
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To a cooled (–20 °C), stirred soln of (E)-6 (180.0 mg, 0.66 mmol)
in anhyd CH2Cl2 (20 mL) under N2 was added dropwise BBr3 (1.30
g, 5.19 mmol). The mixture was allowed to warm up to r.t., then it
was poured into ice-water, and extracted with EtOAc. The organic
layer was washed with brine and dried (Na2SO4). After filtration,
the residue was purified by column chromatography (silica gel, hex-
Synthesis 2008, No. 18, 2953–2956 © Thieme Stuttgart · New York

2956
B. Gabriele et al.
PAPER
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Synthesis 2008, No. 18, 2953–2956 © Thieme Stuttgart · New York