Facile synthesis of deuterated and [14C]labeled analogs of vanillin and curcumin for use as mechanistic and analytical tools

Article abstract of DOI:10.1002/jlcr.3102

Curcumin is a dietary diphenol with antioxidant, antinflammatory, and antitumor activity. We describe facile procedures for the synthesis of [ ¹⁴C₂]curcumin (4 mCi/mmol), [d₆]curcumin, [d₃]curcumin, [¹³C₅]curcumin, and [d ₆]bicyclopentadione, the major oxidative metabolite of curcumin. We also describe synthesis of the labeled building blocks [¹⁴C]vanillin, [d₃]vanillin, and [¹³C₅]acetylacetone. The overall molar yields of the labeled products were 52 ([¹⁴C]) and 47% ([d₃]) for vanillin and 25 ([¹⁴C₂]) and 27% ([d₆]) for curcumin. The compounds can be used as radiotracers in biotransformation studies and as isotopic standards for mass spectrometry-based quantification in pharmacokinetic analyses. Copyright 2013 John Wiley & Sons, Ltd. Curcumin is readily synthesized by fusing two vanillin moieties with acetylacetone. [¹⁴C] and [²H]isotopic labels are introduced by modification of the methoxy group of vanillin, which is accessible by selective methylation of the meta position of 3,4-dihydroxybenzaldehyde in basic conditions. A [¹³C₅]analog is synthesized using [¹³C₅]acetylacetone, which is prepared from [ ¹³C₃]acetone and [¹³C₄]acetic anhydride. Copyright

Full text of DOI:10.1002/jlcr.3102

Research Article
Received 26 April 2013,
Revised 5 June 2013,
Accepted 28 June 2013
Published online 23 July 2013 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3102
14
Facile synthesis of deuterated and [ C]labeled
analogs of vanillin and curcumin for use as
mechanistic and analytical tools
*
Odaine N. Gordon, Leigh A. Graham, and Claus Schneider
Curcumin is a dietary diphenol with antioxidant, antinflammatory, and antitumor activity. We describe facile procedures for the
synthesis of [14C2]curcumin (4mCi/mmol), [d6]curcumin, [d3]curcumin, [13C5]curcumin, and [d6]bicyclopentadione, the major
oxidative metabolite of curcumin. We also describe synthesis of the labeled building blocks [14C]vanillin, [d3]vanillin, and
[
13C5]acetylacetone. The overall molar yields of the labeled products were 52 ([14C]) and 47% ([d3]) for vanillin and 25 ([14C2])
and 27% ([d6]) for curcumin. The compounds can be used as radiotracers in biotransformation studies and as isotopic standards
for mass spectrometry-based quantification in pharmacokinetic analyses.
Keywords: bicyclopentadione; methyliodide; acetylacetone; polyphenol; oxidative metabolism; biotransformation; dietary
labeled, [13C]labeled, and deuterated curcumin to use in
mechanistic studies and as isotopic standards for quantification
using liquid chromatography–mass spectrometry (LC-MS).
Introduction
The dietary diphenol curcumin is widely studied for use as an anti-
inflammatory and anticancer agent.1 A plethora of cellular targets
of curcumin have been identified,2 and its ‘polypharmacology’ of
Results and discussion
affecting diverse cellular processes make curcumin a potentially
powerful therapeutic agent.3 Application of curcumin in clinical
[
14C]- and [d3]Vanillin
studies has been hampered by its low bioavailability and rapid
metabolism.4,5 A large number of medicinal chemistry approaches Synthesis of [14C]- as well as [d3]vanillin was accomplished by
have focused on improving biological activity or bioavailability of following the method by Schneider and Rolando with some
curcumin.6–10
modifications (Figure 3). The authors employed a strong base
The labeled curcumin derivatives synthesized are shown in in ethanol to direct methylation in the meta position of the
Figure 1. A straightforward synthesis of curcumin was originally aromatic ring of 3,4-dihydroxybenzaldehyde in moderate
developed by Pabon.11 The procedure is a two-step reaction yield (38%).23 Methylation of the meta position of 3,4-
with the formation of a boron complex of acetylacetone (‘paste’) dihydroxybenzaldehyde allows introduction of label ([d3] or
and its butylamine-catalyzed reaction with two molar [14C]) into vanillin in a single step and therefore was preferred
equivalents of vanillin (Figure 2). Slight modifications of this over a six-step alternative for [14C]labeling of the carbonyl.14
approach have been reported.12–16 A readily accessible position
We found that 3,4-dihydroxybenzaldehyde had only
for introduction of label into curcumin is the methoxy group of limited solubility in ethanol, which was overcome by
vanillin that can accommodate a [14C]radiolabel and three using methanol instead. Addition of four volumes of
deuterium atoms, respectively. The apparent stability of the dimethylacetamide as
methoxy group during chemical and metabolic transformation reduced the reaction time.24 Methyl iodide ([14C] and [d3],
of curcumin is a further advantage.5,17
respectively) was added as a dilution in 100 μl toluene
a co-solvent increased yield and
We are interested in defining the mechanism, products, and without any apparent influence of toluene on yield or
biological consequences of oxidative transformation of positional specificity. For synthesis of [14C]vanillin, a twofold
curcumin in vitro and in vivo. Oxidative transformation of molar excess of 3,4-dihydroxybenzaldehyde relative to [14C]
curcumin is a spontaneous reaction in buffer of physiological methyl iodide was used to enhance consumption of the
pH.18 Reactive intermediates from the early stages of oxidative
transformation of curcumin—rather than curcumin itself—
Division of Clinical Pharmacology, Department of Pharmacology, Vanderbilt
Institute of Chemical Biology, Vanderbilt University Medical School, Nashville,
are responsible for poisoning of human topoisomerase.19,20
TN 37232, USA
The major end product of oxidative transformation is a
dioxygenated bicyclopentadione.21 In addition, formation of a
* Correspondence to: Claus Schneider, Department of Pharmacology, Vanderbilt
number of less abundant products has been observed.18,22 In
University Medical School, RRB 572, 23rd Ave. S. at Pierce, Nashville, TN 37232-
order to identify all possible transformation products and to
6602, USA.
E-mail: claus.schneider@vanderbilt.edu
analyze their formation in vivo, we decided to prepare [14C]
J. Label Compd. Radiopharm 2013, 56 696–699
Copyright © 2013 John Wiley & Sons, Ltd.

O. N. Gordon et al.
Figure 1. Curcumin and its [14C2]-, [d6]-, [d3]-, and [13C5]-labeled derivatives.
Figure 2. Synthesis of curcumin from vanillin and acetylacetone.11
expensive reagent. The isolated yields for [14C]- and [d3] curcumin, and [d6]curcumin, when starting with a mixture of
vanillin were 52 and 47%, respectively. The isotopic vanillin and [d3]vanillin. Chromatographic resolution of the [d0],
incorporation of [d3] into vanillin exceeded 99.9%.
[d3], and [d6]isotopologues by RP-HPLC is expected to be tedious
if not impossible. Therefore, we devised an alternative strategy in
order to enhance the difference in polarity of [d3]curcumin relative
to the unwanted [d0] and [d6]isomers. Using a 1:1 mixture of [d3]
vanillin and acetylvanillin, the products were a mixture of
[d6]curcumin, [d3]acetylcurcumin (the desired product), and
diacetylcurcumin in 1:2:1 ratio (Figure 4). [d3]Acetylcurcumin was
readily resolved from the other products using RP-HPLC, and [d3]
curcumin was obtained by hydrolysis with K2CO3 in ethyl acetate.
[
14C2]- and [d6]Curcumin
We found that the yield of curcumin largely depended on the
reaction time used to form the boron oxide-acetylacetone paste.
When the paste was allowed to form overnight (16 h), the yield
of curcumin detected by HPLC increased to >80% in pilot
reactions of unlabeled reagents (100 mg vanillin).
A technical inconvenience in scaling down curcumin synthesis
to a low milligram scale was the preparation and handling of a
small amount of the paste. Therefore, an excess of the paste
[d6]Bicyclopentadione
was prepared, dissolved in ethyl acetate, and an aliquot added An aliquot of synthesized [d6]curcumin was subjected to
into the reaction with labeled vanillin. Both [14C2]- as well as autoxidation for preparation of [d6]bicyclopentadione. Autoxidation
[d6]curcumin were isolated in good overall yield (25 and 27%, of curcumin is a spontaneous and rapid reaction in slightly
respectively) using RP-HPLC. The specific activity of [14C2] alkaline buffer (pH 7.5–8) that can be monitored by following
curcumin was 4 mCi/mmol. The incorporation of deuterium into the disappearance of the curcumin chromophore at 430 nm in
[d6]curcumin was >99.9%.
a UV/Vis spectrophotometer.18 Products are recovered from
We have used [14C2]curcumin as a tracer for HPLC analyses of the reaction by using a C18 solid phase cartridge and purified
autoxidation products of curcumin. This approach has led to the using RP-HPLC.
identification of at least 10 different products formed by oxidative
[d6]Curcumin and [d6]bicyclopentadione will be used as
transformation of curcumin, including unstable reaction intermediates isotopic standards for quantitative LC-MS analysis of curcumin
and novel end products.
metabolism in cultured cells and in vivo. [d3]Curcumin will be
added to samples after collection in order to quantify artifactual
autoxidative transformation to [d3]bicyclopentadione during
sample work-up. [d6]Curcumin is also available commercially.
[d3]Curcumin
The approach of fusing two molar equivalents of vanillin with
acetylacetone can be modified for the preparation of curcumin
derivatives, for example, [d3]curcumin. For asymmetric derivatives,
[
13C5]Curcumin
one of the two vanillin equivalents is substituted with a desired The label of [13C5]curcumin is located in the five central carbons
analog. Thus, an equimolar mixture of vanillin and 3,4- of the heptadienone chain of curcumin. In this case, the label
dimethoxybenzaldehyde gives curcumin, 4′-methylcurcumin, and was introduced from [13C5]acetylacetone, which was synthesized
4′,4″-dimethylcurcumin in
a
1:2:1 ratio.18 For synthesis of by fusion of [13C3]acetone and [13C4]acetic anhydride in the
asymmetric [d3]curcumin, this approach will give curcumin, [d3] presence of BF3 (Figure 5). Separating [13C5]acetylacetone from
J. Label Compd. Radiopharm 2013, 56 696–699
Copyright © 2013 John Wiley & Sons, Ltd.
www.jlcr.org

O. N. Gordon et al.
[13C]content of the heptadienone chain in order to enhance signal
intensities for 13C NMR spectroscopic identification of oxidative
transformation products of curcumin.
Experimental
Materials
[
14C]Methyl iodide (2mCi/mmol) was from American Radiolabeled
Chemicals, Inc., MO, USA. [13C3]Acetone (99%) and [13C4]acetic anhydride
(99%) were from Cambridge Isotope Laboratories, Inc., MA, USA. [d3]Methyl
iodide (99.5%), acetylvanillin, 3,4-dihydroxybenzaldehyde, and other
reagents used for synthesis were from Sigma, St. Louis, MO, USA.
[
14C]Vanillin
Figure 3. Introduction of [14C]- or [d3]label into the methoxy group of vanillin.23
3,4-Dihydroxybenzaldehyde (15mg, 0.1mmol) was dissolved in 4 M
methanolic NaOH (50 μl) and diluted with dimethylacetamide (200μl).
[
14C]Methyl iodide (7mg, 0.05 mmol, 2 mCi/mmol) in toluene (100μl) was
added dropwise to the solution. The reaction was stirred for 2h, acidified
with 1 M HCl, and extracted three times with dichloromethane (500 μl).
The solvent was evaporated, and the product was isolated using RP-HPLC
(Method A) to yield 3.5mg [14C]vanillin (52μCi, 52% radiochemical yield;
purity 98% by HPLC). The identity of [14C]vanillin was confirmed by
comparison of its RP-HPLC retention time and UV spectra with an authentic
standard. LC-MS analysis was not performed in order to avoid
contamination of the instrument with radioactive material.
[d3]Vanillin
[d3]Vanillin was prepared as described for [14C]vanillin starting from 3,4-
dihydroxybenzaldehyde (50 mg, 0.35 mmol) and [d3]methyl iodide
(50 mg, 0.35 mmol) in toluene (100 μl). The product was isolated using
RP-HPLC (Method B) to yield [d3]vanillin (26 mg; 47% yield; purity 91%
by HPLC). LC-MS: 154.1 ([M À H]À). 1H NMR (CD3OD, 600 MHz): δ = 5.28
(s, 1H), 6.77 (d, 1H, J = 8.1 Hz) , 6.86 (dd, 1H, J = 8.1; 1.8 Hz) , 6.98 (d, 1H,
J = 1.8 Hz), 9.75 (s, 1H) ppm.
[
14C2]Curcumin
Boron oxide (20 mg, 0.3 mmol) and acetylacetone (40 μl, 0.4 mmol) were
stirred for 16 h to form a white paste. An aliquot (20 μl) of the paste
dissolved in ethyl acetate (360 μl) was added to 3.5 mg 14C-vanillin
(0.03 mmol, 52 μCi) dissolved in tributyl-borate (21 μl). The mixture was
stirred for 5 min after which a 10% dilution of butylamine in ethyl acetate
(2 μl) was added every 10 min for 40 min. The reaction was stirred for 16 h
overnight. The next day, 0.4 M HCl (100 μl; heated to 60°C) was added to
the reaction and stirred for 1 h. [14C2]Curcumin was extracted into ethyl
acetate (4 × 200 μl) and purified using RP-HPLC (Method A) to yield
1.2 mg purified [14C2]curcumin (13 μCi; 4 mCi/mmol; 25% radiochemical
yield; purity 97% by HPLC). 1H NMR (CD3OD, 600 MHz): δ = 3.91 (s, 6H),
5.97 (s, 1H), 6.64 (d, 2H, J = 15.0 Hz), 6.82 (d, 2H, J = 7.9 Hz), 7.1 (d, 2H,
J = 7.5 Hz), 7.22 (s, 2H), 7.57 (d, 2H, J = 15.0 Hz) ppm.
Figure 4. Synthesis of [d3]acetylcurcumin.
Figure 5. Synthesis of [13C5]acetylacetone.
[d6]Curcumin
the solvent used for extraction (dichloromethane) was challenging
because distillation was not feasible because of small volumes.
Instead, we decided to concentrate the dichloromethane extract
(≈1 ml volume) under a gentle stream of nitrogen to about 120 μl
and to use this solution directly in the next step. The small amount
of remaining dichloromethane (≈100 μl) did not interfere with the
formation of the acetylacetone-B2O3 paste although in pilot
experiments a larger amount of solvent (>500 μl) was inhibitory.
[13C5]Curcumin was prepared anticipating two potential
applications. It can serve as a standard or tracer in MS experiments
where loss of the methoxy group during metabolic transformation
[d6]Curcumin was prepared as described for [14C2]curcumin starting from
boron oxide (30 mg, 0.45 mmol), acetyl acetone (60 μl, 0.6 mmol) and
100 mg [d3]vanillin. [d6]Curcumin was purified using semi-preparative
RP-HPLC (Method C) to yield 30 mg (27%) [d6]curcumin (purity 98% by
HPLC). LC-MS: 373.2 ([M À H]À). 1H NMR (CD3OD, 600 MHz): δ = 5.97
(s, 1H), 6.64 (d, 2H, J = 15.7 Hz) , 6.82 (d, 2H, J = 8.1 Hz), 7.10 (dd, 2H,
J = 7.5; 1.1 Hz), 7.22 (d, 2H, J = 1.1 Hz), 7.58 (d, 2H, J = 15.7 Hz) ppm.
[d3]Curcumin
[d3]Curcumin was prepared as described for [14C2]curcumin starting from
is expected or a concern. Our main incentive was to enrich the boron oxide (320 mg, 4.60 mmol) and acetylacetone (660 mg, 6.60 mmol).
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O. N. Gordon et al.
An aliquot (5%) of the boron complex (paste) was reacted with [d3] using a Waters Symmetry Shield C18 3.5-μm column (2.1 × 100 mm)
vanillin (60 mg, 0.40 mmol), vanillin acetate (77 mg, 0.40 mmol), tributyl
eluted with a gradient of MeCN/H2O (5/95, by volume, containing
borate (213 μl, 7.90 mmol), and butylamine (2 μl) in ethyl acetate (1 ml). 10 mM NH4OAc) to MeCN/H2O (95/5, by volume, containing 10 mM
The products were separated by semi-preparative RP-HPLC (Method D) NH4OAc) over 10 min followed by 3 min of isocratic elution and
to give 11 mg of purified [d3]acetylcurcumin (yield 18%). The acetate re-equilibration in the starting solvent (Method E).
group was hydrolyzed using a saturated solution of K2CO3 (25 mg) in
Nuclear magnetic resonance spectra were recorded using a Bruker
ethyl acetate (1 ml) at room temperature overnight (16 h) to release [d3] AV-II 600 MHz spectrometer equipped with a TCI cryoprobe. Chemical
curcumin (purity 95% by HPLC). 1H NMR (CD3OD, 600 MHz): δ = 3.91 shifts are reported in ppm relative to the non-deuterated solvent peak
(s, 3H), 5.97 (s, 1H), 6.62 (d, 2H, J = 15.8 Hz), 6.82 (d, 2H, J = 8.3 Hz), 7.10 of methanol-d4 (δ = 3.34 ppm).
(d, 2, J = 8.3 Hz), 7.21 (s, 2H), 7.58 (d, 2H, J = 15.9 Hz) ppm.
Acknowledgements
[
13C5]Curcumin
13C3]Acetone (120 μl, 2 mmol) and 13C4]acetic anhydride (500 μl,
[
This work was supported awards CA159382 and AT006896 by
NCI and NCCAM, respectively, of the National Institutes of Health.
ONG acknowledges support by training grants 2 T32GM07628
and a pre-doctoral fellowship award (F31AT007287) from
NCCAM of the National Institutes of Health. The content is solely
the responsibility of the authors and does not necessarily
represent the official views of the National Institutes of Health.
[
5 mmol) were placed in a 1-ml reaction vial and cooled in an ice-salt bath.
Boron trifluoride diethyl etherate (400 μl, 3 mmol) was added slowly over
the course of 3 min. The reaction was stirred for 4 h and then poured into
3 ml of 10 M sodium acetate heated to 80 °C. The reaction was extracted
twice using 500 μl dichloromethane to yield 12 mg (0.1 mmol; 5% yield)
[
13C5]acetylacetone.
[
13C5]Curcumin was prepared as described for [14C2]curcumin starting
from boron oxide (47 mg, 0.7 mmol) and [13C5]acetylacetone (12 mg)
dissolved in dichloromethane (100 μl). Vanillin (250 mg), tributylborate
(800 μl) in 800 μl ethyl acetate, and butylamine (5 μl) were added. The
products were loaded onto a 2-g Supelco DSC-18 cartridge in 30%
acetonitrile and eluted using 100% acetonitrile. [13C5]curcumin was
purified using RP-HPLC (Method A; 51% yield; purity 92% by HPLC). LC-
Conflict of Interest
The authors did not report any conflict of interest.
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MS: 372.2 ([M À H]À). H NMR (CD3OD, 600 MHz): δ = 3.91 (s, 6H), 5.97 (s,
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(55/45/0.01, by vol; Method C) or MeOH/H2O/HOAc (75/25/0.01, by vol.;
Method D) at a flow rate of 10 ml/min. LC-MS was performed using a
Thermo LTQ ion trap instrument equipped with an electrospray
ionization interface. The instrument was operated in the negative ion
mode, and mass spectra were acquired at a rate of 2 s/scan. The settings
for the heated capillary (300 °C), spray voltage (4.0 kV), spray current
(0.22 μA), auxiliary (37 mTorr), and sheath gas (16 mTorr) were optimized
using direct infusion of a solution of curcumin (20 ng/μl) in MeCN/H2O
95/5, by volume, containing 10 mM NH4OAc. Samples were introduced
J. Label Compd. Radiopharm 2013, 56 696–699
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