Synthesis and biological evaluation of caffeic acid 3,4-dihydroxyphenethyl ester

Article abstract of DOI:10.1021/np900519d

A high-yield synthesis of caffeic acid 3,4-dihydroxyphenethyl ester (1) has been achieved through Knoevenagel condensation of 3,4-dihydroxybenzaldehyde and 3,4-dihydroxyphenethyl monomalonate as the key step. Compound 1 was tested against a 56-cell-line cytotoxicity panel and for its free-radical-scavenging activity in the DPPH test.

Full text of DOI:10.1021/np900519d

252
J. Nat. Prod. 2010, 73, 252–254
Synthesis and Biological Evaluation of Caffeic Acid 3,4-Dihydroxyphenethyl Ester
Zhizhen Zhang,† Binghua Xiao,† Qi Chen,† and Xiao-Yuan Lian*,‡
Jiangxi Doctors Science and Technology R&D Co., Ltd., Nanchang 330029, People’s Republic of China, and College of Pharmaceutical
Sciences, Zhejiang UniVersity, Hangzhou 310058, People’s Republic of China
ReceiVed August 26, 2009
A high-yield synthesis of caffeic acid 3,4-dihydroxyphenethyl ester (1) has been achieved through Knoevenagel
condensation of 3,4-dihydroxybenzaldehyde and 3,4-dihydroxyphenethyl monomalonate as the key step. Compound 1
was tested against a 56-cell-line cytotoxicity panel and for its free-radical-scavenging activity in the DPPH test.
Phenolic compounds are prominent plant-derived components
of the human diet and many folk medicines, and include (-)-
epigallocatechin-3-gallate (EGCG), the major polyphenol in green
tea, caffeic acid phenethyl ester (CAPE), a phenolic acid ester from
propolis, and curcumin, a phenolic diarylheptanoid of curry powder
and the rhizomes of Curcuma longa, a traditional Chinese medicine.
These three compounds have received extensive attention because
of their broad spectrum of biological properties including anti-
inflammatory,1 antioxidative,2 neuroprotective,3 cardioprotective,4
and antitumor activities.5 Recent research has clarified that these
phenolic compounds exhibit antitumor effects by modulating
multiple cellular targets and signal pathways,5 in a fashion that
interrupts the carcinogenic process.5c,d,6a It has been proposed that
modulating multiple targets could be beneficial for the prevention
and treatment of complex human diseases such as cancer.5d,6b-d
Caffeic acid 3,4-dihydroxyphenethyl ester (1, CADPE) was
originally isolated from Teucrium pilosum as a substance named
teucrol in 2000.7 However, no activity was reported therein. In the
course of the investigation on the active ingredients of Sarcandra
glabra, a traditional Chinese medicine with reported antitumor
activity,8 our group found that 1 isolated from an active fraction
of this plant inhibited the growth of solid tumor S180 in mice after
administration of 1 by oral (35 mg/kg), intraperitoneal (5 mg/kg),
intramuscular (10 mg/kg), and tail intravenous (5 mg/kg) means
for 10 days.9 Jung et al. reported that 1 suppressed tumor growth
and angiogenesis by inhibiting the activity of signal transducers
and activated transcription 3 (STAT 3), the expression of hypoxia
inducible factor-1R (HIF-1R), and vascular endothelial growth factor
(VEGF), using a mouse xenograft model implanted with Caki-I
human renal carcinoma cells.10
Scheme 1. Synthetic Route Leading to Caffeic Acid
3,4-Dihydroxylphenethyl Ester (1)
on 56 cell lines from nine different human cancers and its free-
radical-scavenging activity.
The methodology for preparation of 1 is outlined in Scheme 1.
The preparation started from 3,4-dihydroxybenzoic acid (67.26 g),
which was treated with SOCl2 in MeOH to furnish 3,4-dihydroxy-
phenylacetic acid methyl ester 1a (66.46 g, 91.2% yield). The
phenolic alcohol 1b (49.91 g, 92.5% yield) was obtained by
reduction of 1a (63.76 g) with LiAlH4 in anhydrous THF at room
temperature, and then 1b (46.25 g) was refluxed with Meldrum’s
acid in dioxane at 95-110 °C to form malonic acid phenolic alcohol
monoester 1c (62.48 g, 86.7% yield). Finally, 3,4-dihydroxyben-
zaldehyde (41.44 g) was reacted with 1c (48.04 g) in pyridine
catalyzed by piperidine (Knoevenagel condensation)11d,12 to afford
1 (54.72 g, 86.5% yield) at a purity of 97.6%, as determined by
HPLC. The identity of 1 was confirmed on the basis of comparison
of its NMR spectroscopic and HRESIMS data with literature
values.7
Caffeic acid 3,4-dihydroxyphenethyl ester (1) is a phenolic
alcohol with a phenolic acid ester motif. While this class of
compounds is structurally uncomplicated, their reported syntheses
have had poor yields11a,c,d and are not easily amenable to the
preparation of derivatives,11a,b because of the use of harsh reaction
conditions, the heavy burden of protecting groups needed, and the
large excess of one of the reactants required.11b Previously, we
synthesized 1 from 3,4-dihydroxyphenethyl alcohol and caffeic acid
with excess phenolic alcohol using N,N′-dicyclohexylcarbodiimide
(DCC) as a coupling reagent.9 However, this method had a very
low yield, and the target product was not easily purified by
chromatography.
Caffeic acid phenethyl ester, an analogue of 1, has been
synthesized by different methods such as acid-catalyzed esterifi-
cation, transesterification, DCC coupling reactions, esterification
via acyl chlorides, and Witting reactions.11 However, these reported
methods have some disadvantages.11 Previously, we prepared 1 in
a very low yield from 3,4-dihydroxyphenethyl alcohol and caffeic
acid through DCC coupling reactions.9 Unfortunately, attempted
alternative methods such as esterification via acyl chlorides and
acid-catalyzed esterification did not afford the expected product,
1. The Knoevenagel condensation of malonic acid 3,4-dihydroxy-
phenethyl monoester and 3,4-dihydroxybenzaldehyde presented in
this study is an efficient method to prepare sufficient quantities of
1 to facilitate an ongoing preclinical investigation on its potential
for the treatment of cancer.
In the present study, we present an efficient method through
Knoevenagel condensation of 3,4-dihydroxybenzaldehyde and 3,4-
dihydroxyphenethyl monomalonate as the key step to generate 1
in high yield, and we report the cytotoxic activity of this compound
* Corresponding author. Tel: (01186)-571-88208432. Fax: (01186)-571-
88208432. E-mail: xylian@zju.edu.cn.
† Jiangxi Doctors Science and Technology R&D Co., Ltd.
‡ Zhejiang University.
10.1021/np900519d  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/21/2010

Notes
Compound 1 was tested for its activity against 56 cell lines from
nine different human cancers including leukemia, breast, CNS,
colon, ovarian, melanoma, lung, renal, and prostate, by the National
Cancer Institute (Bethesda, MD).13 The results (Figures S1 and S2,
and Tables S1 and S2, Supporting Information) indicated that 1
exhibited cytotoxic activities for the tested cell lines with most
GI50’s at 10-7 to 10-6 M.
Compound 1 was also evaluated for its free-radical-scavenging
activity using the DPPH scavenging assay.14 The results showed
that 1 has potent free-radical-scavenging activity with an EC50 of
4.7 ( 0.2 µg/mL. It is well known that oxidative stress produced
by free radicals plays an important role in the carcinogenesis
process.15 Compound 1 has a strong free-radical-scavenging activity
and may be effective against oxidative stress and therefore has the
potential for the prevention and treatment of cancer.
Journal of Natural Products, 2010, Vol. 73, No. 2 253
C-10), 67.7 (CH2, C-8), 117.5 (CH, C-5), 118.0 (CH, C-2), 122.5 (CH,
C-6), 132.2 (C, C-1), 144.1 (C, C-4), 145.5 (C, C-3), 172.6 (C, C-9),
175.0 (C, C-11); HRESIMS m/z 263.05293 [M + Na]+ (calcd for
C11H12NaO6, 263.05316).
Caffeic Acid 3,4-Dihydroxyphenethyl Ester (1). Piperidine (20 mL)
was added to a mixture of 1c (48.04 g, 0.2 mol) and 3,4-dihydroxy-
benzaldehyde (41.44 g, 0.3 mol) in pyridine (200 mL). The mixture
was stirred at room temperature until the reaction completely finished
using TLC to monitor the reaction. The reaction mixture was
concentrated under vacuum to produce a residue, which was dissolved
in EtOAc (200 mL) and then washed with 5% HCl (50 mL × 2) and
distilled water (50 mL × 2). The EtOAc extract was applied to a column
of Diaion HP-20, eluting with 65% methanol, to furnish 1 (54.72 g,
86.5% yield) as a white solid: mp 110-111 °C; UV(MeOH) λmax (log
ε) 223 (4.45), 246 (sh), 278 (4.40), 326 (4.46) nm; HRESIMS m/z
339.08408 [M + Na]+ (calcd for C17H16NaO6, 339.08446); 13C and 1H
NMR data see Supporting Information (Table S3). The purity of 1 was
determined by HPLC as 97.6%.
Experimental Section
Cytotoxicity Assay. The cytotoxicity of 1 was assayed by the
National Cancer Institute (Bethesda, MD) using the methodology of
the 60-cell-line cancer screen.13
General Experimental Procedures. Melting points were measured
with a Thomas-Hoover capillary melting point apparatus and are
uncorrected. UV spectra were recorded in MeOH with a Hewlett-
Packard 8435 spectrometer. NMR experiments were performed on a
Bruker 600 MHz NMR instrument, and NMR data are reported as δ
(ppm) values referenced to the solvent used. HRESIMS were acquired
on an electrospray instrument (MDS Sciex Pulsar Qstar). Silica gel
(60-200 mesh) and Diaion HP-20 for open column chromatography
were obtained from Sigma-Aldrich. TLC was conducted on precoated
silica gel F254 plates (Merck) with detection under UV light at 254
nm. HPLC analysis was performed on an Agilent 1100 HPLC system
with an Agilent 1100 diode array detector using a Hypersil ODS column
(column A, 250 × 4.6 mm, 5 µm, Supelco). 3,4-Dihydroxyphenylacetic
acid, 3,4-dihydroxybenzaldehyde, Meldrum’s acid, thionyl chloride
(SOCl2), lithium aluminum hydride (LiAlH4), pyridine, piperidine,
anhydrous tetrahydrofuran (THF), anhydrous dioxane, anhydrous
methanol (MeOH), and R,R-diphenyl-ꢀ-picrylhydrazyl (DPPH) were
purchased from Sigma-Aldrich.
Free-Radical-Scavenging Activity. The R,R-diphenyl-ꢀ-picrylhy-
drazyl (DPPH) scavenging assay was carried out according to a
procedure described previously.14 Briefly, different concentrations of
1 (100 µL) were mixed with 900 µL of a 0.04 mg/mL methanolic
solution of DPPH. The mixture was kept at room temperature for 20
min, and then the UV absorbance at 517 nm was measured. The
inhibition percentage was calculated using the following equation: I )
[(Ac - As)/Ac] × 100, where I is the inhibition percentage, Ac is the
absorbance of the negative control (containing 100 µL of methanol
instead of the test samples), and As is the absorbance of the samples.
The inhibition percentage was plotted against the concentration of 1,
and the EC50 value (mean ( SD) was determined by linear regression
analysis of three determinations.
Acknowledgment. This study was supported by the Ministry of
Science and Technology of the People’s Republic of China (X.Y.L.).
The authors would like to thank Dr. V. Narayanan at the National
Cancer Institute (Bethesda, MD) for the cytotoxicity testing.
3,4-Dihydroxyphenylacetic Acid Methyl Ester (1a). A quantity
of SOCl2 (34.8 mL, 0.48 mol) was added dropwise to a stirred
solution of 3,4-dihydroxyphenylacetic acid (67.26 g, 0.4 mol) in
anhydrous methanol (800 mL) at 0 °C. The mixture was stirred at
room temperature for 7-8 h. After removal of the solvent under
reduced pressure, the residue was subjected to column chromatog-
raphy on silica gel eluting with hexane-EtOAc (4:1) to afford the
Supporting Information Available: The cytotoxic activity of 1;
NMR data and NMR spectra of 1. This material is available free of
charge via the Internet at http://pubs.acs.org.
1
desired compound 1a (66.46 g, 91.2% yield) as a colorless oil: H
References and Notes
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254 Journal of Natural Products, 2010, Vol. 73, No. 2
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NP900519D