Synthesis and biological activity of a 3,4,5-trimethoxybenzoyl ester analogue of epicatechin-3-gallate

Article abstract of DOI:10.1021/jm701346h

Despite presenting bioavailability problems, tea catechins have emerged as promising chemopreventive agents because of their observed efficacy in various animal models. To improve the stability and cellular absorption of tea polyphenols, we developed a new catechin-derived compound, 3-O-(3,4,5- trimethoxybenzoyl)-(-)-epicatechin (TMECG), which has shown significant antiproliferative activity against several cancer cell lines, especially melanoma. The presence of methoxy groups in its ester-bound gallyl moiety drastically decreased its antioxidant and prooxidant properties without affecting its cell-antiproliferative effects, and the data indicated that the 3-gallyl moiety was essential for its biological activity. As regards its action mechanism, we demonstrated that TMECG binds efficiently to human dihydrofolate reductase and downregulates folate cycle gene expression in melanoma cells. Disruption of the folate cycle by TMECG is a plausible explanation for its observed biological effects and suggests that, like other antifolate compounds, TMECG could be of clinical value in cancer therapy.

Full text of DOI:10.1021/jm701346h

2018
J. Med. Chem. 2008, 51, 2018–2026
Synthesis and Biological Activity of a 3,4,5-Trimethoxybenzoyl Ester Analogue of
Epicatechin-3-gallate
Luís Sánchez-del-Campo,§ Francisco Otón,# Alberto Tárraga,# Juan Cabezas-Herrera,‡ Soledad Chazarra,§ and
José Neptuno Rodríguez-López*,§
Department of Biochemistry and Molecular Biology A, School of Biology, UniVersity of Murcia, Murcia, Spain, Department of Organic
Chemistry, Faculty of Chemistry, UniVersity of Murcia, Murcia, Spain, and Research Unit of Clinical Analysis SerVice, UniVersity Hospital
Virgen de la Arrixaca, Murcia, Spain
ReceiVed October 26, 2007
Despite presenting bioavailability problems, tea catechins have emerged as promising chemopreventive agents
because of their observed efficacy in various animal models. To improve the stability and cellular absorption
of tea polyphenols, we developed a new catechin-derived compound, 3-O-(3,4,5-trimethoxybenzoyl)-(-)-
epicatechin (TMECG), which has shown significant antiproliferative activity against several cancer cell
lines, especially melanoma. The presence of methoxy groups in its ester-bound gallyl moiety drastically
decreased its antioxidant and prooxidant properties without affecting its cell-antiproliferative effects, and
the data indicated that the 3-gallyl moiety was essential for its biological activity. As regards its action
mechanism, we demonstrated that TMECG binds efficiently to human dihydrofolate reductase and down-
regulates folate cycle gene expression in melanoma cells. Disruption of the folate cycle by TMECG is a
plausible explanation for its observed biological effects and suggests that, like other antifolate compounds,
TMECG could be of clinical value in cancer therapy.
Chart 1
Introduction
Green tea, one of the most popular and commonly consumed
beverages in the world, is an important source of flavonoids
called catechins (Chart 1). Among them, (-)-epigallocatechin-
3-gallate (EGCGa) is the most abundant (40–60%), followed
by (-)-epicatechin-3-gallate (ECG, 10–20%), (-)-epigallocat-
echin (EGC, 10–20%), and (-)-epicatechin (EC, 4–6%). In sum,
they contribute to more than 200 mg of catechin per cup of
green tea. Several in vivo experiments have shown the chemo-
preventive effects of EGCG against all stages of carcinogenesis
(initiation, promotion, and progression) in animal models of
breast, lung, skin, prostate, and colon cancers.1,2 There has been
extensive investigation into the ways by which EGCG might
act in cancer prevention, and recent attempts to define the
molecular action mechanism of green tea components have
found that EGCG affects several molecular targets and pathways
of carcinogenesis.2,3 The observed prooxidant nature of EGCG
appears to conflict with the generally recognized concept that
tea polyphenolics act as antioxidants.4 Several studies have
* To whom correspondence should be addressed. Phone: +34-968-
398284. Fax: +34-968-364147. E-mail: neptuno@um.es.
shown that the trihydroxyphenyl B-ring is the principal site for
prooxidant reactions, regardless of the presence of the 3-gallyl
moiety.5,6 However, the presence of hydroxyl groups in the
3-gallyl moiety may enhance the antioxidant potential of tea
catechins.5 The 3-gallyl moiety of catechins has also seen to
be essential for the inhibition of several of their proposed
enzymatic targets, such as the proteasome,7 5-cytosine DNA
methyltransferase (DNMT),8 and dihydrofolate reductase
(DHFR).9–11
On the basis of the observation that some tea polyphenols
possess similar chemical structures to classical and nonclassical
antifolates (Chart 2), we hypothesized that tea catechins could
inhibit DHFR activity. Recently, we reported that ester bound
gallate catechins isolated from green tea are potent inhibitors
of DHFR activity at concentrations found in the serum and
tissues of green tea drinkers (0.1–1.0 µM).9–11 DHFR (5,6,7,8-
§
Department of Biochemistry and Molecular Biology A, University of
Murcia.
#
Department of Organic Chemistry, University of Murcia.
University Hospital Virgen de la Arrixaca.
‡
a Abbreviations: EGCG, (-)-epigallocatechin-3-gallate; ECG, (-)-
epicatechin-3-gallate; EGC, (-)-epigallocatechin; EC, (-)-epicatechin;
DNMT, 5-cytosine DNA methyltransferase; DHFR, dihydrofolate reductase;
DHF, 7,8-dihydrofolate; THF, 5,6,7,8-tetrahydrofolate; TS, thymidylate
synthase; MTX, methotrexate; TMP, trimethoprim; TMECG, 3-O-(3,4,5-
trimethoxybenzoyl)-(-)-epicatechin; DMAP, 4-(dimethylamino)pyridine;
Trolox, 6-hydroxy-2.5,7,8-tetramethylchroman-2-carboxylic acid; TEAC,
Trolox equivalent antioxidant activity; SOD, superoxide dismutase; rHDH-
FR, recombinant human DHFR; TQD, (R)-6-{[methyl-(3,4,5-trimethoxy-
phenyl)amino]methyl}-5,6,7,8-tetrahydroquinazoline-2,4-diamine; MTHFR,
methyltetrahydrofolate reductase; ATCC, American Type Tissue Culture
Collection; FCS, fetal calf serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; HeM, human epidermal melanocytes; HRP,
horseradish peroxidase; ABTS, 2,2′-azinobis-(3-ethylbenzothiazoline-6-
sulfonic acid).
10.1021/jm701346h CCC: $40.75
2008 American Chemical Society
Published on Web 03/07/2008

Synthesis and ActiVity of a Tea Catechin Analogue
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2019
Chart 2
Chart 3
biological properties have scarcely been studied and its molec-
ular structure has not been characterized. The purpose of the
present study was to present an inexpensive alternative for
TMECG synthesis, accompanied by a high degree of recovery,
and to study the biological activity of this compound in different
cancer cell systems. In addition, any change in the antioxidant/
prooxidant profile of TMECG with respect to naturally occurring
tea catechins, but where the 3-gallyl moiety is maintained, might
contribute to understanding the mechanism(s) of action of this
biologically active compound.
Results and Discussion
Syntheses. Although TMECG was previously synthesized,20
experiments performed in our laboratory to synthesize this ECG
analogue following the described methodology rendered very
low recovery. A major problem was associated with unspecific
blockage of the 3-hydroxyl group of the epicatechin after the
benzylation reaction with benzyl bromide and K2CO3. It is well-
known that the benzylation of epicatechin provides a product
of low purity even after recrystallization.21 Thus, TMECG was
successfully synthesized following the five-step reaction se-
quence shown in Scheme 1, starting from the commercially
available catechin, which is considerably cheaper than epicat-
echin. In the first step, the phenolic hydroxyl groups, but not
of the alcoholic hydroxyl group, were protected by treating the
catechin with benzyl bromide in the presence of K2CO3, thus
yielding the corresponding tetra-O-benzylcatechin 1 in a 45%
yield.22 Next, the tetra-O-benzylepicatechin 3 was obtained from
1 following a synthetic methodology that involved an inversion
of the stereochemistry at C-3 through an oxidation–reduction
process. Oxidation of 1, using 1,1,1-triacetoxy-1,1-dihydro-1,2-
benziodoxol-3(1H)-one (Dess-Martin periodinane)23 in moist
CH2Cl2, gave the ketone derivative 2, which was subsequently
reduced to 3 with lithium tri-sec-butylborohydride (L-Selectride)
in THF at –78 °C. At this point, 3,4,5-trimethoxybenzoyl
chloride, previously prepared by reaction of the corresponding
carboxylic acid with PCl5 in toluene under reflux temperature,
was esterified with 3, in a CH2Cl2 solution and in the presence
of 4-(dimethylamino)pyridine (DMAP), giving 5 in 71% yield.
In the final step of this synthesis, the benzyl-protecting groups
were removed, giving rise to 6 (62% yield), using a strategy
based on a hydrogenolysis reaction that was carried out by
treating 5 over 20% Pd(OH)2/C in THF/MeOH. Compounds 5
and 6 were characterized by 1H and 13C NMR, elemental
analysis, and FAB mass spectrometry.
tetrahydrofolate, NADP+ oxidoreductase, EC 1.5.1.3) catalyzes
the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydro-
folate (THF) in the presence of coenzyme NADPH as follows:
DHF + NADPH + H+ f THF + NADP+. This enzyme is
necessary for maintaining intracellular pools of THF and its
derivatives, which are essential cofactors in one-carbon me-
tabolism. Coupled with thymidylate synthase (TS),12 it is directly
involved in thymidylate (dTMP) production through a de novo
pathway. DHFR is therefore pivotal in providing purines and
pyrimidine precursors for the biosynthesis of DNA, RNA, and
amino acids. In addition, DHFR is the target enzyme for
antifolate drugs, such as the antineoplastic drug methotrexate
(MTX) and the antibacterial drug trimethoprim (TMP).13 The
antifolate character of EGCG and ECG could explain their
anticarcinogenic and chemopreventive action, their anti-inflam-
matory properties, and even their claimed ability to promote
weight loss.11 Moreover, this mechanism may also explain the
high antimicrobial and antimycotic action of these compounds,
which protect against dental caries and infections produced by
pathogens such as Helicobacter pylori or Candida albi-
cans.14–17
However, despite these excellent properties, tea catechins have
at least one limitation, their low bioavailability.18 Factors
influencing this low bioavailability could be related to their low
stability in neutral or slightly alkaline solutions and their inability
to cross cellular membranes.19 In an attempt to solve such
bioavailability problems, we synthesized a 3,4,5-trimethoxy-
benzoyl analogue of ECG [3-O-(3,4,5-trimethoxybenzoyl)-(-)-
epicatechin, TMECG] (Chart 3), which showed significant
antiproliferative activity against different cancer cell lines.
Although TMECG has been synthesized previously,20 its

2020 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Sánchez-del-Campo et al.
Scheme 1. Synthesis of 3-O-(3,4,5-Trimethoxybenzoyl)-(-)epicatechin 6a
a Reagents and conditions: (i) benzyl bromide, K2CO3, DMF, -15 °C to room temp; (ii) Dess-Martin periodinane, moist CH2Cl2, room temp; (iii)
L-Selectride, n-Bu4NCl, THF, -78 °C; (iv) 3,4,5-trimethoxybenzoyl chloride, 4, CH2Cl2, DMAP, -15 °C to room temp; (v) H2, 20% Pd(OH)2/C, THF/
MeOH, room temp.
Stability and Cellular Uptake of TMECG. The biological
effects of tea catechins have been extensively investigated in
cell lines, but the stability and cellular uptake of these
compounds are poorly understood. The most active tea polyphe-
nols are unstable in neutral or alkaline medium. Zhu and co-
workers24 demonstrated that ester cleavage of gallate catechins
is unlikely, and therefore, oxidation at different molecular levels
(especially in the phenolic hydroxyl groups of rings B and D)
is the most plausible mechanism for explaining their instability.
Thus, at high pH values, the basic environment can easily
deprotonate the phenolic groups, generating phenoxide anions.
The anion form is much more reactive toward electrophilic
agents, such as free radicals, and also forms the semiquinone
radical, which can undergo further dimerization or other
reactions. Because the pH value of the intestine and body fluid
is neutral or slightly alkaline, these tea catechins will be very
unstableinsidethehumanbody,leadingtoreducedbioavailability.24,25
The stability of EGCG has been studied in detail and seen to
vary with the cell culture conditions.19 EGCG is not stable in
cell culture systems, and its oxidation leads to the formation of
dimers and H2O2. Under many conditions, the half-life of EGCG
is <2 h in the presence of HT-29 human colon adenocarcinoma
cells and even shorter in the absence of cells.19 Methoxy
protection of the reactive hydroxyls groups in the D-ring of ECG
yielded a stable derivative in cell culture conditions. TMECG
was considerably more stable than ECG in a culture medium
in the absence of cells. Although the first time point was not
statistically different for either compound, the successive time
points showed statistically significant differences. The data
showed that more than 75% of the initial concentration of
TMECG remained in the culture medium after an incubation
period of 2 days, while only 30% of the initial concentration of
ECG remained at this time (Figure 1A).
character of ECG made its cellular uptake slower (Figure 1B).
The data indicated that TMECG is more efficiently transported
through the Caco-2 cell membranes than its natural counterpart
tea catechin.
Biological Activity of TMECG on Cancer Cells. Tea
polyphenols have demonstrated their antiproliferative activity
against human cells lines from breast,28 lung,29 colon,30 and
melanoma,31 among others. To observe the possible wide
spectrum of the antiproliferative activity of TMECG, we
investigated its effects against different human cancer lines
obtained from breast (MCF7), lung (H1264), colon (Caco-2),
and melanoma (SkMel-28) using the 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The ef-
fectiveness of TMEG on these cells followed the order SkMel-
28 > H1264 > Caco-2 > MCF7, with a half-maximal inhibitory
concentration (IC50) at 6 days of 2.9 ( 0.5, 18 ( 2.0, 33 ( 3.1,
and 80 ( 5.5 µM, respectively. The calculated IC50 values were
statistically different between cell lines but in the same
concentration range as those reported for the activity of TMECG
on other cellular systems.20 The factors that modulate this
different cell response to TMECG are currently being investi-
gated in our laboratory. Next, we investigated the cancer cell
inhibitory capacity of TMECG in comparison with naturally
occurring tea polyphenols. After 6 days of exposure of Caco-2
cells to 25 µM catechins, EGCG, ECG, and TMECG showed
remarkable growth inhibition activity compared with an un-
treated control (Figure 2A). However, catechins lacking the
3-gallyl moiety, such as EGC and EC, showed much lower
growth inhibitory activity (Figure 2A). SkMel-28 melanoma
cells were highly susceptible to TMECG treatment, and after 6
days of exposure to 20 µM TMECG, the cells showed
microscopic signs of apoptosis, including cell shrinkage, loss
of cell-cell contact, and the fragmentation of plasmatic and
nuclear membranes (Figure 2B).
Another factor reducing the bioavailability of tea catechins
is the low capacity of these compounds to cross cell membranes.
To study the cellular uptake of TMECG, we used Caco-2 cell
monolayers, which acted as a model of the human intestinal
epithelium.26,27 Figure 1B shows the concentrations of ECG and
TMECG in culture media in the presence of Caco-2 cells. As
can be seen, the TMECG concentration rapidly decreased in
the cell medium as a consequence of cellular uptake with a
maximal decrease after 4 h of exposure. The more hydrophilic
Antioxidant versus Prooxidant Activity of TMECG. Al-
though claims concerning the beneficial health effects of green
tea consumption are based on many epidemiological and
laboratory studies, the exact action mechanism of its polyphe-
nolic compounds is subject to continuous debate. Many
hypotheses and molecular targets for EGCG and other tea
catechins have been proposed in the past 10 years. Plant
polyphenols are natural antioxidants, and most of their phar-

Synthesis and ActiVity of a Tea Catechin Analogue
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2021
Figure 1. (A) Stability of ECG (b) and TMECG (0) in standard
culture medium in the absence of cells. Catechins at a concentration
of 50 µM were dissolved in 20 mL of cellular medium without cells
and incubated for 72 h in a CO2 incubator. (B) Uptake of ECG (b)
and TMECG (0) by Caco-2 cells. Caco-2 cells were grown until 90%
confluence was reached, and then 20 mL of cellular medium containing
50 µM catechins and 0.2 mM ascorbic acid was added. For both stability
and the cellular uptake experiments, 1 mL aliquots of culture medium
were taken at specified times and frozen in liquid N2 until catechin
concentration determination. The values presented are the mean
percentage determined from five independent experiments ( SD.
Figure 3. (A) Antioxidant activity of TMECG and tea catechins
expressed as TEAC and determined as detailed in the Experimental
Section. (B) Prooxidant activity of TMECG and tea catechins deter-
mined by their NADPH oxidation capacity in the absence or the
presence of SOD. The rate of the oxidation of NADPH (0.1 mM) was
calculated in the presence of 50 µM catechins in sodium phosphate
buffer at pH 7.4, 37 °C. The effect of SOD on catechin-dependent
NADPH oxidation was studied in the presence of 10 µg/mL of enzyme.
possess both antioxidant and prooxidant properties,34 and it has
been frequently suggested that the prooxidant action of polyphe-
nolics may be an important mechanism in their antiproliferative
and apoptosis-inducing properties.35 Whatever the case, the
mechanism whereby tea catechins change from antioxidant to
prooxidant activity, and vice versa, is uncertain. To test whether
the biological activity of TMECG was related to its antioxidant
and/or prooxidant properties, we determined the antioxidant and
prooxidant properties of this compound and compared them with
the properties of other natural occurring catechins in green
tea.The antioxidative activity of catechins was evaluated by
measuring the Trolox equivalent antioxidant capacity values
(TEAC).36 This method measures the relative ability of anti-
oxidant substances to scavenge the radical cation 2,2′-azinobis(3-
ethylbenzthiazoline-6-sulfonic acid) (ABTS•+) compared with
a standard amount of the synthetic antioxidant Trolox (6-
hydroxy-2.5,7,8-tetramethylchroman-2-carboxylic acid), an aque-
ous soluble vitamin E analogue. The TEAC value is defined as
the concentration of standard Trolox with the same antioxidant
capacity as 1 mM concentration of the antioxidant compound
under investigation. The results showed that the compounds with
the most hydroxyl groups apparently exert the greatest antioxi-
dant activity (Figure 3A), with ECG and EGCG at 4.9 and 4.8
mM, respectively, being more active antioxidants than vitamin
E (TEAC ) 1.0 mM). The enhanced values for the catechin-
gallate esters compared with nongallated catechins (EC or EGC)
reflect the additional contribution from the trihydroxybenzoate,
gallic acid, in ring D. Substitution of the hydroxyl groups in
ring D by methoxy groups decreased the antioxidant activity
of TMECG (TEAC ) 1.9 ( 0.1 mM) with respect to the other
catechin-gallate esters but had no appreciable effect on the
growth inhibition of Caco-2 cells (Figure 2A). It therefore seems
Figure 2. (A) Caco-2 viability after treatments with TMECG and tea
catechins and with respect to a control experiment (100% viability)
with no compound added. Cells were treated for 6 days with 25 µM of
each compound. The values presented are the mean percentage
determined from three independent experiments ( SD. (B) Morpho-
logical aspect (magnification, 800×) of untreated SkMel-28 cells
(control) compared with those subject to 6-days of treatments with 20
µM TMECG.
macological properties are considered to be due to their
antioxidant action.32 This is generally considered to reflect their
ability to scavenge endogenously generated oxygen radicals or
those radicals formed by various xenobiotics, radiation, etc.
However, some data in the literature suggest that the antioxidant
properties of polyphenolic compounds may not fully account
for their chemopreventive effects.33 Most plant polyphenols

2022 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Sánchez-del-Campo et al.
that the antioxidant potential of this synthetic catechin was not
responsible for the described biological effects.
In addition, phenolics can exert prooxidant effects by au-
toxidation, in which the initial step leads to semiquinone and
superoxide radical formation.37 These radicals are very reactive
and may damage major macromolecules such as proteins or
DNA. Moreover, both radicals will accelerate autoxidation,
generating H2O2 in the process. It has been suggested that the
prooxidant activity of catechins may be due to the flavonoid
unit of catechin (ring B). An additional group in this unit, as
occurs in EGC and EGCG, enhances the prooxidant activity of
the catechins.32 Figure 3B, which shows the extent of NADPH
oxidation in the presence of several catechins, seems to confirm
this hypothesis. EGC and EGCG enhanced NADPH oxidation,
which was inhibited by the presence of superoxide dismutase
(SOD). However, the oxidation of NADPH in the presence of
ECG, EC, or TMECG was negligible throughout the experi-
ments. If the biological activity of catechins is related to their
prooxidant activity, we would expect a stronger activity in the
presence of either EGC or EGCG; however, EGC was inactive
and EGCG showed similar activity to ECG and TMECG (Figure
2A). As observed in Figure 2A, the effect of catechins on Caco-2
cells was more related to the presence of a gallate ester moiety,
leading us to check other hypotheses to explain the biological
activity of TMECG.
Figure 4. (A) Relative human DHFR inhibition by TMECG and tea
catechins (all compounds at 100 µM) with respect to a control
experiment with no compound added. Each bar represents the mean of
five separate experiments. (B) Titration fluorescence experiments for
the binding of TMECG and EGC to human DHFR. The points are
experimental (after correction for enzyme dilution), and the lines are
best-fit theoretical curves. The enzyme concentration was 0.1 µM. (C)
View of TMECG modeled into the folate-binding site of human DHFR.
Atoms of the TMECG ligand are colored green. Four different ligands
from human and chicken DHFR crystal structures were used to define
a binding envelope. These were placed in a common orientation by
superimposing backbone atoms from a common set of protein residues
located around the ligands. Ligands from the following PDB structure
files were used: 1DR1 (biopterin), 1S3V (TQD), 1S3W, and 1DLR.
Binding of TMECG to Human DHFR. Green tea extracts
containing significant amounts of tea catechins strongly inhibited
the activity of recombinant human DHFR (rHDHFR).11 In order
to detect which components of these extracts were responsible
for such inhibition, we assayed DHFR activity in the presence
of EC, EGC, ECG, and EGCG. The results showed that both
ECG and EGCG were potent inhibitors of the human enzyme,
while polyphenols lacking the ester bound gallate moiety (EGC
and EC) did not inhibit rHDHFR (Figure 4A). These results
indicate that the ester-bound gallate moiety is essential for the
inhibition of this enzyme, as has been determined in the case
of bovine DHFR.9,10 TMECG with an ester-bound gallate unit
was also a good inhibitor of rHDHFR (Figure 4A). The effective
binding of TMECG to free rHDHFR was determined by
following the decrease in enzyme fluorescence that occurs on
formation of the enzyme-inhibitor complex (Figure 4B). When
rHDHFR fluorescence was excited at 290 nm, its emission
spectrum showed a maximum at 340–350 nm. The binding of
TMECG quenched this fluorescence, and the data showed that
the dissociation constant of free rHDHFR for TMECG (2.1 (
0.2 µM) was of the same order as that for EGCG (0.92 µM)
and ECG (1.78 µM). As expected, the addition of EGC to
rHDHFR did not modify enzyme fluorescence (Figure 4B).
Structural modeling of TMECG into human DHFR indicated
that the compound is orientated in a way similar to that described
for EGCG,9 with specific hydrogen bonding interactions, most
notably involving Glu-30 (O· · ·O distance 2.6 Å) (Figure 4C).
Comparison with a range of other DHFR structures containing
folate or various inhibitors showed that most of the TMECG
lies within the consensual substrate/inhibitor envelope, with the
exception of the non-ester trihydroxybenzoyl moiety (ring B).
The ester-bound gallate moiety (ring D) is accommodated in
an amphiphilic region of DHFR involving residues Ile7, Gln35,
Asn64, and Leu67.38 Although folate, TQD, MTX, and tea
catechins differ significantly in terms of their structural formulas,
they have a similar 3D shape,9 which appears to be an important
determinant in their binding to DHFR.
TMECG Modulates Folic Acid Cycle Gene Expression.
The folate cycle plays a central role in cell metabolism (Scheme
2). Among its important functions are the delivery of one-carbon
units to the methionine cycle for use in methylation reactions,
and the synthesis of pyrimidines and purines. Several enzymes,
including DHFR, TS, and methyltetrahydrofolate reductase
(MTHFR), participate in the activation and regeneration of folic
acid coenzymes. Several studies have shown that the protein
and mRNA levels of TS and DHFR are higher in tumor tissues
and cancer cells than in their normal counterparts.39,40 Tumors
with high levels of these enzymes are thought to undergo more
active cellular proliferation, which in turn is associated with
tumor invasiveness and metastasis. To investigate whether
TMECG could interfere with folate metabolism in cancer cells,
the levels of expression of these folate cycle genes were
analyzed in melanoma SkMel-28 cells using real time PCR. As
observed in other tumor tissues and cells, the genes involved
in the metabolism of folic acid were highly overexpressed in
melanoma cells compared with normal melanocytes (Figure 5).
The levels of DHFR, TS, and MTHFR mRNAs were calculated
to be about 400-, 22- and 4-fold higher, respectively, in
melanoma cells than in normal melanocytes. Treatment of
SkMel-28 with TMECG produced a substantial and rapid down-
regulation of these genes, involving significant changes in the
mRNA levels of the genes at 24 h of treatment. DHFR, TS,
and MTHFR mRNA levels were similar to that detected in

Synthesis and ActiVity of a Tea Catechin Analogue
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2023
Scheme 2. General Effects of Antifolates on the Folic Acid
Cyclea
a Abbreviations not included in the text: MS, methionine synthase; SAH,
S-adenosylhomocysteine; SAM, S-adenosylmethionine.
normal melanocyte cells after 5 days of TMECG treatment
(Figure 5). The data indicated that TMECG disturbs the folate
metabolism in melanoma cells and suggest that this might be
the mechanism by which TMECG induces cell growth inhibition
and death.
Conclusions
The synthesis of a 3,4,5-trimethoxybenzoyl ester analogue
of ECG was successfully carried out by using catechin and
inverting the stereochemistry at carbon-3. TMECG was more
stable and more effectively transported in colon cancer cell than
its natural counterpart ECG. In evaluating its antiproliferative
activity, we showed that TMECG efficiently inhibited the growth
of different human cells including cancer lines from breast, lung,
colon, and skin. The presence of methoxy groups in its ester-
bound gallyl moiety decreases its antioxidant and prooxidant
properties without affecting its cell-antiprofiferative effects. The
data indicated that the 3-gallyl moiety is essential for the
biological activity of TMECG. Recent investigations into
the action mechanism of tea catechins reveal that their 3-gallyl
moiety is required for the binding and inhibition of several
enzymatic targets including the proteasome,7 DNMT,8 or
DHFR.9–11 This moiety was also proposed as being necessary
to inhibit folic acid uptake in Caco-2 cells.28 We have shown
here that TMECG effectively binds to human DHFR and
controls the expression of the genes involved in folate metabo-
lism. We therefore conclude that TMECG, by inhibiting DHFR
and/or inhibiting folate transport and/or acting on another
proposed cellular target, acts as an antifolate metabolite, whose
action would result in disruption of the cell folate cycle (Scheme
2). Folate cycle disruption has an important effect on cells
because the depletion of folate coenzymes inhibits DNA
synthesis, decreases the cellular production of nucleotides, alters
DNA methylation patterns, and affects polyamine synthesis
(Scheme 2). These effects are more drastic in rapidly proliferat-
ing cancer cells because they have a high turnover of DNA and
therefore higher folate requirements. In conclusion, TMECG
could be an effective anticancer drug that shares an action
mechanism with other ester gallate catechins. Moreover, the
presence of a more lipophilic 3-gallyl moiety that enhances the
Figure 5. Time-dependent expression of DHFR, TS, and MTHFR in
SkMel-28 cells treated with 20 µM TMECG. Data were obtained by
real time PCR and compared with the relative expression of DHFR,
TS, and MTHFR in HeM cells (zero relative gene expression). Zero
time represents the level of DHFR in untreated SkMel-28 cells.
Differences between times were statistically significant (P < 0.025).
stability and cellular uptake could mitigate the main weakness
of tea catechins, their poor bioavailability.
Experimental Section
All reactions were carried out using solvents that were dried by
routine procedures. All melting points were determined on a Kofler
1
hotplate melting-point apparatus and are uncorrected. H and 13C
NMR spectra were recorded on a Bruker AC 400 MHz. The
following abbreviations are used to represent the multiplicity of
the signals: s (singlet), d (doublet), dd (double doublet), m
(multiplet), and q (quaternary carbon atom). Chemical shifts refer
to signals of tetramethylsilane in the case of 1H and 13C NMR
spectra. The FAB+ mass spectra were recorded on a Fisons
AUTOSPEC 5000 VG spectrometer, using 3-nitrobenzyl alcohol
as a matrix.
Compounds 1-3 were obtained using the experimental proce-
dures described elsewhere.21
3,4,5-Trimethoxybenzoyl Chloride, 4. A mixture of 3,4,5-
trimethoxybenzoic acid (3.0 g, 14.14 mmol) and phosphorus
pentachloride (3.53 g, 16.95 mmol) in dry toluene (50 mL) was
stirred at reflux temperature and under nitrogen for 3 h. The mixture
was allowed to reach room temperature, and the solvent was
removed under vacuum. The product was sufficiently pure to be
used in the following step without further purification.
5,7,3′,4′-Tetra-O-benzyl-3-(3′′, 4′′, 5′′-trimethoxybenzoyl)-
(-)-epicatechin, 5. To a solution of 3 (0.547 g, 0.84 mmol) and
DMAP (0.257 g, 2.10 mmol) in dry CH2Cl2 (50 mL) a solution of

2024 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7
Sánchez-del-Campo et al.
3,4,5-trimethoxybenzoyl chloride 4 (0.386 g, 1.68 mmol) in the
same solvent was added dropwise under nitrogen. The reaction
mixture was stirred at room temperature for 24 h, and then a solution
of saturated sodium bicarbonate (30 mL) and ethyl acetate (50 mL)
was added. Afterward, the mixture was extracted twice with water
(2 × 100 mL). The organic layers were dried with anhydrous
magnesium sulfate, and the solvent was removed under vacuum.
The resulting red oil was chromatographed on a silica gel column
using n-Hex/AcOEt (6:4, v/v) as solvent (Rf ) 0.64). The solvent
was removed under reduced pressure, and the solid was recrystal-
lized from Et2O/n-Hex to obtain a white solid (yield ) 71%). MS
antibiotics, under standard tissue culture conditions. Cell injury was
evaluated by a colorimetric assay for mitochondrial function using
the MTT cell proliferation assay.43 The assay is based on the ability
of a mitochondrial dehydrogenase enzyme from viable cells to
cleave the tetrazolium rings of the pale-yellow MTT and to form
dark-blue formazan crystals, which cannot pass through cell
membranes, and thus, they accumulated within healthy cells. The
number of surviving cells is directly proportional to the level of
the formazan product created, and the color can then be quantified
at 570 nm. For this assay, cells were plated in a 96-well plate at a
density of 1000 cells/well and grown until they reached 50–60%
confluence. Human epidermal melanocytes (HeM) were supplied
by Gentaur (Brussels, Belgium) and were cultured in HAM-F10
medium supplemented with 10% FCS, antibiotics and the human
melanocyte growth supplement (Gentaur).
Stability and Cellular Uptake of Catechins. Stability and
cellular uptake were determined by the difference between the initial
concentration of catechin and that calculated at specific times in
the cellular medium and expressed as a percentage of catechin at
zero time. Catechin concentrations were determined by HPLC
analysis. Briefly, a total of 50 µL of cellular medium was injected
into a HPLC system (Hitachi, LaChrom Elite) equipped with a 250
mm × 4.6 mm C18 column and a UV–vis detector. The mobile
phase was 0.1 M sodium dihydrogen phosphate buffer (pH 2.5)
containing 0.1 mM ethylenediaminetetraacetic acid disodium salt
(Na2EDTA)/acetonitrile (87/13 v/v), and the flow rate was 1 mL/
min. The column was maintained at 30 °C. Catechins were identified
by their characteristic elution times, and their concentrations were
calculated with respect to calibration curves for known concentra-
tions of catechins.
1
(FAB+) m/z (%) 845 (M+ + 1, 10). H NMR (CDCl3, 400 MHz)
δ 7.30–7.43 (m, 20H, Ph), 7.17 (s, 2H, H2′′ and H6′′), 7.13 (d,
4
3
4
1H, J ) 1.9 Hz, H2′), 7.07 (dd, 1H, J ) 8.4 Hz, J ) 1.9 Hz,
3
4
H6′), 6.89 (d, 1H, J ) 8.4 Hz, H5′), 6.30 (d, 1H, J ) 2.2 Hz,
H6), 6.28 (d, 1H, 4J ) 2.2 Hz, H8), 5.11 (s, 4H, 2 × CH2O), 5.03
3
(s, 2H, CH2O), 5.02 (s, 2H, CH2O), 4.97 (d, 1H, J ) 11.7 Hz,
H2), 4.87 (d, 1H, 3J ) 11.7 Hz, H3), 3.83 (s, 3H, OCH3), 3.80 (s,
6H, OCH3), 3.10 (m, 2H, H4). 13C NMR (CDCl3, 100 MHz) δ
164.9 (q, -COO), 158.4 (q, Ar-O), 157.6 (q, Ar-O), 155.2 (q,
Ar-O), 152.5 (2 × q, Ar-O), 148.6 (q, Ar-O), 142.0 (q, Ar-O),
136.8 (q, PhCH2), 136.7 (q, PhCH2), 136.5 (q, PhCH2), 136.4 (q,
PhCH2), 130.8 (q, C1′), 128.3 (CH, PhCH2), 128.2 (CH, PhCH2),
128.1(CH, PhCH2), 128.0 (CH, PhCH2), 127.6 (CH, PhCH2), 127.5
(CH, PhCH2), 127.4 (2 × CH, PhCH2), 127.1 (CH, PhCH2), 127.0
(CH, PhCH2), 126.9 (CH, PhCH2), 126.8 (CH, PhCH2), 124.7 (q,
C1′′), 119.6 (CH, C6′), 114.4 (CH, C6), 113.6 (CH, C2′), 106.7
(CH, C2′′ y C6′′), 100.5 (q, C4a), 94.2 (CH, C6), 93.5 (CH, C8),
71.2 (CH2, CH2Ph), 70.9 (CH2, CH2Ph), 69.8 (CH2, CH2Ph), 69.6
(CH2, CH2Ph), 68.5 (CH, C3), 60.5 (CH3, OCH3), 55.8 (CH3,
OCH3), 25.5 (CH2, C4). Anal. Calcd for C53H48O10: C, 75.34; H,
5.73. Found: C, 75.57; H, 5.94.
Antioxidant Activity. The antioxidant capacity was measured
using a method based on the abilities of different substances to
scavenge the ABTS·+ radical cation compared with a standard
antioxidant (Trolox) in a dose–response curve.36 ABTS·+ radical
cation was prepared by oxidation of ABTS (150 µM) in the presence
of H2O2 (75 µM) and horseradish peroxidase (HRP) (0.25 µM) in
citrate-phosphate buffer, pH 4.5. This solution was then diluted
in 5 mM phosphate buffered saline (PBS), pH 7.4, to an absorbance
of 0.70 ((0.02) at 734 nm and preincubated at 30 °C prior to use.
Fresh ABTS·+ radical cation solution was prepared each day.
Trolox (2.5 mM) was prepared in PBS for use as stock standard.
Fresh working standards were prepared daily by diluting 2.5 mM
Trolox with PBS. All catechins were dissolved in PBS to a
concentration of 50 µM. After addition of 1 mL of ABTS•+ solution
to aliquots of Trolox or catechins (1–100 µL, depending on the
activity of the particular compound) the solutions were vortexed
for exactly 30 s and the absorbance at 734 nm was taken exactly
1 min after initiation of mixing in a Perkin-Elmer Lambda-35
spectrophotometer. PBS blanks were run in each assay. The
dose–response curve for Trolox consisted of plotting the absorbance
at 734 nm as a percentage of the absorbance of the uninhibited
radical cation and was based on triplicate determinations. The
activities of catechins were assayed at four different concentrations
previously determined to be within the range of the dose–response
curve. All the measurements were repeated on triplicate samples
at these four concentrations. By reference to the Trolox dose–re-
sponse curve, the mean Trolox equivalent antioxidant capacity
(TEAC) value was derived for each catechin compound.
3-O-(3, 4, 5-Trimethoxybenzoyl)-(-)-epicatechin, 6. Under
normal pressure, a solution of 5 (0.600 g, 0.71 mmol) and 10%
Pd/C (0.06 g of palladium, 0.56 mmol) in THF/MeOH (1:1, 40
mL) was treated with molecular hydrogen. The solution was stirred
for 18 h at room temperature and then filtered on a Celite pad,
which was then washed with methanol (300 mL). The solvent was
removed under vacuum, and the resulting solid was recrystallized
from Et2O (yield ) 62%). MS (FAB+) m/z (%) 485 (M+ + 1, 28).
1H NMR (acetone-d6, 400 MHz) δ 8.31 (bs, 1H, OH), 8.10 (bs,
1H, OH), 7.94 (bs, 1H, OH), 7.80 (bs, 1H, OH), 7.14 (s, 2H, H2′′
4
3
and H6′′), 7.11 (d, 1H, J ) 2.0 Hz, H2′), 6.90 (dd, 1H, J ) 8.2
Hz, 4J ) 2 Hz, H6′), 6.79 (d, 1H, 3J ) 8.2 Hz, H5′), 6.03 (d, 1H,
4
4J ) 2.2 Hz, H6), 6.02 (d, 1H, J ) 2.2 Hz, H8), 5.48 (m, 1H,
H3), 5.19 (bs, 1H, H2), 3.80 (s, 6H, OCH3), 3.73 (s, 3H, OCH3),
3.10 (m, 1H, Hgem, H4), 3.05 (m, 1H, Hgem, H4). 13C NMR
(acetone-d6, 100 MHz) δ 165.9 (q, -COO), 157.9 (q, Ar-O), 157.4
(q, Ar-O), 156.8 (q, Ar-O), 154.0 (q, Ar-O), 145.7 (q, Ar-O),
145.4 (q, Ar-O), 143.3 (q, Ar-O), 131.4 (q, C1′), 126.2 (q, C1′′),
118.7 (CH, C6′), 115.6 (CH, C5′), 114.6 (CH, C2′), 107.6 (CH,
C2′′ and C6′′), 98.5 (q, C4a), 96.4 (CH, C7), 95.5 (CH, C9), 77.7
(CH, C2), 70.7 (CH, C3), 60.6 (CH3, CH3O), 56.4 (CH3, CH3O),
26.0 (CH2, C4). Anal. Calcd for C25H24O10: C, 61.98; H, 4.99.
Found: C, 61.69; H, 5.24.
Materials. Highly purified tea polyphenols, EGCG (>95%),
ECG (>98%), EGC (>98%) EC (>98%), and (-)-catechin (>98%)
were purchased from Sigma Chemical Co. (Madrid, Spain).
rHDHFR was purchased from Sigma and dialyzed exhaustively
against distilled water prior to use. The enzyme concentration was
determined by MTX titration of enzyme fluorescence.41 DHF (90%)
was obtained from Aldrich Chemical Co. (Madrid, Spain) and
NADPH from Sigma. The concentrations of NADPH and DHF
were determined enzymically at 340 nm using DHFR and a molar
absorbance change (∆ꢀ) of 11 800 M-1 cm-1 for the reaction at
this wavelength.42 SOD from bovine liver was obtained from Sigma
and used without further purification.
NADPH Oxidation by Catechins. NADPH oxidation in the
presence of catechins was determined by following the decrease
in absorbance of NADPH at 340 nm in a Perkin-Elmer Lambda-
35 spectrophotometer.
DHFR Assays. The activity of DHFR in the absence or presence
of catechins was determined at 25 °C by following the decrease in
the absorbance of NADPH and DHF at 340 nm. Experiments were
performed in a buffer containing 2-(N-morpholino)ethanesulfonic
acid (Mes, 0.025 M), sodium acetate (0.025 M), tris(hydroxy-
methyl)aminomethane (Tris 0.05 M), and NaCl (0.1 M) at pH 7.4.
To prevent the oxidation of catechins, the reaction mixture contained
Cell Cultures. Human cancer cells lines (SkMel-28, MCF7,
H1264, and Caco-2) were obtained from the American Type Tissue
Culture Collection (ATCC) and were maintained in appropriate
culture media supplemented with 10% fetal calf serum (FCS) and

Synthesis and ActiVity of a Tea Catechin Analogue
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 7 2025
1 mM ascorbic acid. Assays were started by adding enzyme. In
the absence of the enzyme, the rate of absorbance change was
negligible.
Supporting Information Available: 1H and 13C NMR spectra
for compounds 5 and 6. This material is available free of charge
via the Internet at http://pubs.acs.org.
Fluorescence Studies. The fluorescence of DHFR is reduced
upon binding of substrates and inhibitors, a property that can be
used as a convenient method for determining both the enzyme
concentration and the dissociation constants of enzyme-ligand
complexes. The dissociation constant for the binding of TMECG
to free rHDHFR was determined by fluorescence titration in an
automatic-scanning Perkin-Elmer LS50B spectrofluorimeter with
1.0 cm light path cells and equipped with a 150 W xenon (XBO)
light source. The formation of a binary complex between the
enzyme and the ligand was followed by measuring the quenching
of the tryptophan fluorescence of the enzyme upon addition of
microliter volumes of a concentrated stock solution of ligand.
Fluorescence emission spectra were recorded when rHDHFR
fluorescence was excited at 290 nm. Titrations were performed in
the same buffers as described for DHFR assays. Temperature was
controlled at 25 °C using a Haake D1G circulating bath with a
heater/cooler.
Molecular Modeling. Molecular modeling was carried out using
the Discover module of Insight II (Insight II, release 2000.1,
Accelrys Ltd., Cambridge, U.K.). Human DHFR X-ray crystal
structure 1S3V44 was retrieved from the Protein Data Bank,45 and
its TQD ligand was used as a template for positioning the TMECG
ligand. The composite protein/TMECG model was geometrically
optimized within Insight II using the consistent valence force field
and steepest descent algorithm to a derivative of 1.0. The refined
model was validated within InsightII using Prostat.
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Acknowledgment. This work was supported in part by
grants from PBL International and Ministerio de Educación y
Ciencia (Project SAF2006-07040-C02-01) to J.N.R.-L., from
Ministerio de Educación y Ciencia (Project SAF2006-07040-
C02-02) to J.C.-H., and from Fundación Séneca (CARM)
(Project 02970/PI/05) to A.T. L.S.-d.-C. has a fellowship from
the Fundación Séneca (Comunidad Autónoma de Murcia).

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