A convenient synthesis of 14C-labelled resveratrol

Article abstract of DOI:10.1002/jlcr.806

Resveratrol (trans-3,4′,5-trihydroxystilbene) is a naturally occurring phytoalexin and polyphenol existing in grapes and various plants. It shows remarkable beneficial bioactivities in the prevention of cancer, inflammation and platelet aggregation, etc. This paper reports the synthesis of [β-¹⁴C]-trans-resveratrol using ¹⁴C-formic acid (exchanged with sodium ¹⁴C-formate) and 3,5-dihydroxybenzoic acid as the starting materials. [¹⁴C-formyl]-4-methoxybenzaldehyde and diethyl 3,5-dimethoxy benzylphosphonate reacted following the Wittig-Horner reaction to give trans-3,4′,5-[β-¹⁴C]-trimethoxystilbene. The final product was obtained through the demethylation of trans-3,4′ ,5-[β-¹⁴C]-trimethoxystilbene and identified by TLC and UV spectroscopy. Adoption of the whole procedure provided ¹⁴C-resveratrol with a specific radioactivity of 40.8 μCi/mmol, chemical yield of 15.3% and radiochemical yield of 12.5%. Copyright

Full text of DOI:10.1002/jlcr.806

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2004; 47: 167–174.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.806
Research Article
14
A convenient synthesis of C-labelled resveratrol
Dongli Zeng, Qixi Mi, Hongfang Sun and Haifang Wang*
Department of Chemical Biology, College of Chemistry and Molecular Engineering,
Peking University, Beijing 100871, China
Summary
Resveratrol (trans-3,40,5-trihydroxystilbene) is a naturally occurring phytoalexin and
polyphenol existing in grapes and various plants. It shows remarkable beneficial
bioactivities in the prevention of cancer, inflammation and platelet aggregation, etc.
This paper reports the synthesis of [b-14C]-trans-resveratrol using 14C-formic acid
(exchanged with sodium 14C-formate) and 3,5-dihydroxybenzoic acid as the starting
materials. [14C-formyl]-4-methoxybenzaldehyde and diethyl 3,5-dimethoxy benzyl-
phosphonate reacted following the Wittig–Horner reaction to give trans-3,40,5-[b-14C]-
trimethoxystilbene. The final product was obtained through the demethylation
of trans-3,40,5-[b-14C]-trimethoxystilbene and identified by TLC and UV spectro-
scopy. Adoption of the whole procedure provided 14C-resveratrol with a specific
radioactivity of 40.8 mCi/mmol, chemical yield of 15.3% and radiochemical yield
of 12.5%. Copyright # 2004 John Wiley & Sons, Ltd.
Key Words: 14C-labelling; anisaldehyde; trans-resveratrol; natural product synthesis
Introduction
Resveratrol (trans-3,40,5-trihydroxystilbene) 1, a naturally occurring phenolic
antioxidant and phytoalexin, has become a compound of intense interest
because of its suggested health promoting roles. It can inhibit the low-density
lipoprotein (LDL) oxidation and platelet aggregation and is a factor
associated with lowering the risk of coronary heart disease (CHD).[1,2]
Recently, its potential cancer chemopreventive properties have been recog-
nized owing to its capacity in inhibiting diverse cellular events associated with
the three major stages of carcinogenesis: initiation, promotion and prolifera-
tion.[3] It also has a direct antiproliferative effect on human breast epithelial
cells and consequently is a potential chemopreventive agent for breast
cancer.[4] Although resveratrol was reported to suppress the growth of cancer
*Correspondence to: H. Wang, Department of Chemical Biology, College of Chemistry and Molecular
Engineering, Peking University, Beijing 100871, China. E-mail: raindrop@pku.edu.cn
Copyright # 2004 John Wiley & Sons, Ltd.
Received 10 September 2003
Revised 10 November 2003
Accepted 13 November 2003

168
D. ZENG ET AL.
cell lines and decrease the tumor growth in animal models, the mechanism of
its antiproliferative activity has not been clearly established.
In view of these effects, resveratrol may have a wide range of target organs.
Thus, its absorption, distribution, metabolism and excretion in vivo are
considered to be primarily selective issues. Soleas et al. reported that oral
administration of resveratrol led to an important absorption resulting in a
significant plasma bioavailability in rat.[5] Whether resveratrol is metabolized
to other compounds is not however known. In order to carry out our
accelerator mass spectrometry (AMS) study on the biodistribution and
pharmacokinetics of resveratrol, a 14C-labelled compound is required. In 2003,
Vitrac et al. reported a biosynthesis of 14C labelled resveratrol.[6] However, this
method required professional skills in biology cell culture and some specific
equipment. It consumed a large amount of radioactive material and the
radiochemical yield was very low due to the early introduction of the labelled
atom and multitudinous steps. Here we present a total chemical synthesis of
14C-resveratrol that is convenient and easy to perform. Only a small amount
of labelled agent is required and the product was obtained in fairly good
radiochemical yield.
Results and discussion
To ensure the retention of resveratrol during the metabolism processes, the
radioisotope 14C should be located in the alkene carbon. The majority of
previously published syntheses of unlabelled resveratrol involved the
condensation of aldehyde and phosphonate via the Wittig–Horner reaction.
We planned to synthesize the hydroxystilbene by demethylation of the
corresponding methoxystilbene 4, which was prepared by the Witting–Horner
reaction of methoxybenzaldehyde with methoxybenzylphosphonate (Scheme 1).
Incorporation of the radioisotope in the aldehyde component rather than in
the phosphonate was selected since (a) the appropriate intermediate could be
synthesized from a one-carbon unit in very high yield, (b) the labelled
intermediate p-anisaldehyde may also be of use in other biochemical studies
and (c) the starting labelled material, formic acid, could be obtained by
exchanging the formic anion between unlabelled anhydrous formic acid and
14CHO
HO
HO
H3CO
H3CO
CH2PO(OEt)2
CH
CH
a
b
+
H3CO
OCH3
H14
C
OH
H14
C
OCH3
H3CO
2
3
4
1
Scheme 1. (a) NaH, then 3, in dry THF, 94%; (b) BBr3, dry CH2Cl2, 98%
(crude), 37% (recrystalized)
Copyright # 2004 John Wiley & Sons, Ltd.
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A CONVENIENT SYNTHESIS
169
H3C 14 CHO
N
14CHO
H3C
NH
c
d
H14COOH
+
H3CO
5
2
Scheme 2. (c) DCC, dry CH2Cl2, 100%; (d) p-MeOC6H4MgBr, then saturated
NH4Cl solution, in dry THF, 44%
labelled sodium formate, the latter being one of the most common labelled
organics and readily available.
p-Anisaldehyde (4-methoxybenzaldehyde) 2 was chosen as an important
intermediate reagent (Scheme 2). Synthesis of 14C-labelled anisaldehyde has
not been previously reported. Several methods can be adopted to synthesize
anisaldehyde, however, most of them preclude the incorporation of 14C from a
one-carbon precursor. Besides the Blanc & Sommelet reactions and Vilsmeier–
Haack reaction, the Bouveault reaction provides an alternative route to
introduce a labelled carbon atom into the molecular skeleton. In preliminary
optimization experiments, we have tried all three mentioned routes. In the
Blanc chloromethylation reaction and successive Sommelet reaction, a
chloromethyl group was first introduced into the aromatic ring of anisole by
treatment with formaldehyde and hydrogen chloride, then anisaldehyde was
prepared from the above alkyl halide by treatment with hexamethylenete-
tramine to yield a quaternary salt and followed by mild hydrolysis. When the
procedure was optimized for yield based on formaldehyde, 1.0 to 2.0 molar
equivalents of anisole in benzene at various reaction temperatures (ꢀ15–708C)
by varying the bubbling time of dry hydrogen chloride gas (30 min to 6 h),
under the catalysis of ZnCl2 or without the catalyst, followed by appropriate
work-up, gave a maximum yield of benzyl chloride of 25%. And after the
Sommelet reaction (with an average yield of 80–90%), the total yield of
anisaldehyde is less than 20%. The inefficiency of this process, coupled with
difficulties in purification of the unstable benzyl chloride, indicated that
another method should be used.
Besides formaldehyde, another compound, N-methylformanilide (MFA) 5
was taken into consideration. [14C-formyl]-N-methylformanilide could be used
in the Vilsmeier–Haack reaction and Bouveault reaction as an intermediate for
introducing the labelled aldehyde group into reactive aromatics. MFA was
available by simple synthesis from formic acid in very high yield. In the
Vilsmeier–Haack reaction, anisole was formylated by MFA and phosphorous
oxychloride. In the preliminary investigations, 1 mmol of MFA was used.
However, we still could not obtain a satisfactory yield by varying the
Copyright # 2004 John Wiley & Sons, Ltd.
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170
D. ZENG ET AL.
experimental conditions including the quantity of phosphorous oxychloride
(from 1.0 to 2.0 equivalent moles) and anisole (equivalent or as solvent),
reaction temperatures (ꢀ20–808C), holding periods (30 min to 18 h) and
reaction modes (by thermal heating or ultrasonic irradiation).[7] One of the
by-products, o-anisaldehyde (2-methoxysaldehyde) also caused difficulties and
this route was therefore given up.
In the Bouveault reaction, substituted formylamine reacted with the
Grignard reagent, and following acid hydrolysis afforded anisaldehyde. For
incorporation of isotope in the aldehyde from a one-carbon unit, this
procedure must be optimized for yield based on the substituted formylamine,
which was MFA in our scheme, rather than bromoanisole. The optimization
study showed that the MFA would not be used to completion unless excess
Grignard reagent was prepared. Further optimization experiments gave the
best proportion of these two reagents (shown in Table 1). Reactions in THF
gave higher yields than in Et2O, because the intermediate adduct was only
soluble in the former. Entries 1–3 showed that at least 1.5 equiv. Grignard
reagent was required. With MFA fixed at 1.0, entries 4–6 confirm the optimum
proportion of Bouveault reaction as 1.55:1, giving a maximum yield (45%) of
anisaldehyde from MFA.
MFA was prepared from formic acid and N-methylaniline. Applying N,N0-
dicyclohexylcarbondiimide (DCC, 1.0 equiv.) as dehydration reagent in
CH2Cl2 provided a much better yield (>95%) than traditional azeotropic
drying methods (5 70%). Exchange between the labelled sodium formate and
unlabelled formic acid would introduce the 14C atom into the reaction system.
Starting from Na14COOH with these optimized conditions, p-anisaldehyde 3
was prepared in good yield (44% from formic acid).
Dimethyl 3,5-dimethoxybenzylphosphonate 3 was prepared from 3,5-
dihydroxybenzoic acid as outlined in Scheme 3. This synthesis was as reported
in the literature.[8] We used methyl as protecting groups for phenols instead of
Table 1. Optimization of yield of unlabelled p-anisaldehyde
In THF
In Et2O
Entry Mg
BrPh
equiv. equiv. equiv. MFA (%)
MFA Yield from Residual
status
(Yes/No)
Yield from Residual
MFA (%)
status
(Yes/No)
1
2
3
4
5
6
7
8
1.00
1.20
1.40
1.50
1.550
1.60
1.80
2.00
1.00
1.20
1.40
1.50
1.550
1.60
1.90
2.20
1.00
1.00
1.00
1.00
1.00
1.00
1.00
1.00
510
12
25
38
45
40
36
35
Yes
Yes
Yes
No
No
No
No
No
510
10
20
25
25
25
25
25
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
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A CONVENIENT SYNTHESIS
171
COOCH3
COOH
CH2 OH
CH2 Br
CH2PO(OEt)2
e, f
h
g
i
H3CO
HO
OH
H3CO
OCH3
H3CO
OCH3 H3CO
OCH3
OCH3
6
7
8
3
Scheme 3. (e) Me2SO4, NaOH; (f) SOCl2, then MeOH, 80%; (g) LiAlH4, dry
ethyl ether, 92%; (h) PBr3, dry CH2Cl2, 94%; (i) P(OEt)3, 1308C, 95%
original benzyl groups and obtained a higher total yield (> 60%) than that
reported in the literature.
In the condensation of p-anisaldehyde 2 with dimethyl 3,5-dimethoxy-
benzylphosphonate 3, a slight excess of phosphonate 3 was used. When this
Wittig–Horner reaction was performed at relatively low temperature (08C) and
followed by proper work-up, only trans-3,40,5- trimethoxystilbene was
obtained (HPLC results). During the final deprotective step, the yield of
crude resveratrol was over 95% while the final product yield was much lower
(38%). This decrease was attributed to the loss in the micro-recrystalization
step.
Experimental
[14C] Sodium formate, specific activity 53 mCi/mmol, ethanol–water (7:3)
solution (0.1 mCi/ml), was purchased from Movarek Products Inc. and was
used without further purification. Tetrahydrofuran (THF), dichloromethane
(CH2Cl2) and diethyl ether (Et2O) were dried and redistilled before use.
Anhydrous formic acid and N-methylaniline were purified before use.
1H-NMR spectra were measured in CDCl3 or d6-DMSO solutions. Mass
spectra (MS) and high resolution mass spectra (HRMS) were obtained in
the electronic impact (EI) mode. The stationary phase of the thin layer
chromatograph (TLC) was silica gel 254. Some spectral data of the
intermediates were obtained by using unlabelled compounds in the cold
experiments. The final labelled resveratrol was characterized by TLC and UV
spectroscopy.
[14C-formyl]-N-methylformanilide 5.
500 ml of ethanol–water (7:3) solution of [14C] sodium formate (0.1 mCi/ml)
was placed in a 5-ml flask and evaporated to dryness by N2 bubbling leaving
solute on the wall of the flask. Dry CH2Cl2 (1 ml) and anhydrous formic
acid (as carrier, 38 ml, 1.0 mmol) were added. The mixture was sonicated
for 20 min in an ultrasonic cleaner at room temperature to dissolve the
labelled formate and promote the isotope exchange. Thereafter, freshly
distilled N-methylaniline (110 ml, 1.0 mmol) was added into the flask. DCC
(N,N’-dicyclohexylcarbondiimide) (206 mg, 1.0 mmol) in dry CH2Cl2 (1 ml)
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D. ZENG ET AL.
was added dropwise while stirring in an ice bath to prevent the decomposition
of formic acid. Then the isolated mixture was stirred at room temperature for
2 h, resulting in a milky suspension. The resulting mixture was filtered through
a fritted glass funnel and the precipitate washed with a little CH2Cl2; the
combined filtrates were first washed with 10% HCl (1 ml ꢁ 2) and then 1%
NaHCO3 (1 ml ꢁ 1), dried over anhydrous Na2SO4 and evaporated, giving
142 mg (100%) of a pale yellow oil, a crude product of [14C-formyl]-
N-methylformanilide (MFA).
1H NMR(BRUKER ARX400, CDCl3, dppm) 8.48 (s, 1 H, -CHO); 7.42
(t, 2 H, Ph–H); 7.29 (d, 1 H, Ph–H); 7.18 (d, 2 H, Ph–H); 3.33(s, 3 H, -CH3)
MS (EIMS): m/e calculated: 135, 106, 94 found: 135, 106, 94.
[14C-formyl]-4-methoxybenzaldehyde 2
Magnesium (Mg) turnings (37.2 mg, 1.55 mmol), p-methoxy bromobenzene
(195 ml, 1.55 mmol) and freshly dried THF (1.5 ml, as solvent) were placed into
a dry, 5 ml flask equipped with a condenser connected to a calcium chloride
tube and an additional funnel. The contents were stirred at room temperature
and the Grignard reaction commenced after ca 10 min. When all of magnesium
was dissolved, the mixture was cooled to 08C and MFA 5 (product of last step,
142 mg) in dry THF (1 ml) was added dropwise into the flask via the funnel.
Then the reaction mixture was stirred at room temperature for another hour,
and the reaction was quenched with 1.5 ml of saturated ammonium chloride
solution. The two phases were separated, and the aqueous phase was extracted
with ethyl ether (2 ml ꢁ 2). The organic phases were combined and condensed
to give a heavy yellow oil. After dissolving the oil in 0.5 ml of 95% ethanol,
0.2 g NaHSO3 was added in 6 drops of water into this solution and a slightly
white glossy precipitate formed. The precipitate was filtered, washed with ice-
cold ethanol, and then hydrolyzed using saturated NaHCO3 solution (3 ml).
The mixture was extracted with Et2O (2 ml ꢁ 3), then the combined organic
layers were dried over MgSO4 and the solvent evaporated off to give 59 mg
(44%) of p-anisaldehyde 2 as a light yellow oil with fragrant odour.
1H NMR (Varian 300 MHz, 258C, CDCl3, dppm): 9.89 (s, 1 H, -CHO); 7.85
(d, 2 H, Ph–H); 7.01 (d, 2 H, Ph–H); 3.90 (s, 3 H, -OCH3)
MS (EIMS): m/e calculated: 135, 111, 77 found: 135, 111, 77.
[b-14C]-3,40,5-trimethoxystilbene 4
Sodium hydride (0.2 g) was added to a well-stirred solution of phosphonate 3
(130 mg, 0.45 mmol) in dry THF (1 ml) at –108C. After 30 min, p-anisaldehyde
2 (0.44 mmol) in THF (1 ml) was added dropwise, and the reaction mixture
was stirred at 08C for over 10 h. The excess of sodium hydride was quenched
with several drops of ice cool water, followed by addition of 10% HCl to
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A CONVENIENT SYNTHESIS
173
adjust the pH to about 7. The product was extracted with ethyl acetate
(2 ml ꢁ 2) and the organic layer washed with a saturated solution of sodium
chloride (2 ml ꢁ 1). The ethyl acetate layer was concentrated to a yellow oil.
On adding methanol an indissolvable white oil was formed and after
discarding it the residue was crystallized in methanol–water and a white solid
precipitated at low temperature. On separation 110 mg (0.41 mmol, 94%) of
3,40,5-trimethoxystilbene 4 was obtained.
m.p.–55–578C, lit. 56–578C.
1H NMR (BRUKER ARX400, CDCl3, dppm) 7.46 (d, 2 H, Ph–H); 7.02
(s, 1 H, -CH=); 6.92 (s, 1 H, Ph–H); 6.90 (d, 2 H, Ph–H); 6.65 (s, 2 H, Ph–H),
6.37 (s, 1 H, -CH=); 3.83 (s, 9 H, -OCH3)
MS (EIMS): m/e calculated: 270 found: 270.
[b-14C]-3,40,5-trihydroxystilbene 1
Boron tribromide (3 mmol, 3 ml of 1 M solution in CH2Cl2) was slowly added
to a well stirred solution of [b-14C]-3,40,5-trimethoxystilbene 4 (110 mg,
0.41 mmol) in CH2Cl2 (7 ml) in an ice bath. The mixture was stirred at 08C for
30 min and left to stand at room temperature for another 30 min, and then
poured into 10 ml of ice-water, and the mixture stirred until the red color
disappeared. The mixture was centrifuged at 5000 rpm at 48C for 5 min.
The solid was separated and recrystallized in ethanol–water to give 35 mg of
resveratrol 1 (0.15 mmol, 38%).
m.p.– 254–257 8C, lit. 2568C, 260–2618C
1H NMR (Varian 300 MHz, d6-DMSO, dppm) 9.55 (s, 1 H); 9.19 (s, 2 H);
7.39 (d, 2 H, 20,60-H); 6.93 (d, 1 H, -CH=); 6.80 (d, 1 H, -CH=); 6.75 (d, 2 H,
30,50-H); 6.38 (s, 2 H, 2,6-H), 6.11 (s, 1 H, 4-H)
MS (EIMS): m/e calculated: 288.1 found: 288; (HRMS): m/e calculated:
228.07864 found: 228.07776.
Conclusion
A convenient and easily performed method for the chemical synthesis of 14C-
labelled resveratrol 1 has been developed. This has enabled the preparation of
resveratrol 1 starting from a simple and readily available one-carbon
precursor, H14COONa. The overall chemical yield from formic acid was
15.3%; the radiochemical yield from [14C] sodium formate was 12.5%. 6.3 mCi
(0.23 MBq) of 14C-labelled resveratrol was obtained with a specific activity of
40.8 mCi/mmol (1.5 MBq/mmol).
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