Synthesis and antiangiogenic activity of new silybin galloyl esters

Article abstract of DOI:10.1021/jm201034h

The synthesis of various silybin monogalloyl esters was developed, and their antiangiogenic activities were evaluated in a variety of in vitro tests with human umbilical vein endothelial cells (HUVECs). A structure-activity relationship (SAR) study found

Full text of DOI:10.1021/jm201034h

ARTICLE
pubs.acs.org/jmc
Synthesis and Antiangiogenic Activity of New Silybin Galloyl Esters
Radek Gaꢀzꢁak,† Kateꢀrina Valentovꢁa,‡ Kateꢀrina Fuksovꢁa,† Petr Marhol,† Marek Kuzma,†
§
||
‡
,†
ꢁ
Miguel Angel Medina, Ivana Obornꢁa, Jitka Ulrichovꢁa, and Vladimír Kꢀren*
†Centre of Biotransformation and Biocatalysis, Institute of Microbiology Academy of Sciences of the Czech Republic, Vídeꢀnskꢁa 1083,
Prague 4, CZ 142 20, Czech Republic
‡Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palackꢁy University, Hnꢀevotínskꢁa 3, CZ-775 15
Olomouc, Czech Republic
§Department of Molecular Biology and Biochemistry, University of Mꢁalaga, Campus de Teatinos, ES 29071 Mꢁalaga, Spain
Department of Obstetrics and Gynecology, Faculty of Medicine and Dentistry, Palackꢁy University and University Hospital in Olomouc,
I. P. Pavlova 6, 775 20 Olomouc, Czech Republic
S Supporting Information
b
ABSTRACT: The synthesis of various silybin monogalloyl
esters was developed, and their antiangiogenic activities were
evaluated in a variety of in vitro tests with human umbilical vein
endothelial cells (HUVECs). A structureꢀactivity relationship
(SAR) study found the regioselectivity of the silybin galloylation
to be highly significant. Silybin (as an equimolar mixture of two
diastereomers A and B) exhibited quite poor antiangiogenic
activities, whereas its B stereoisomer is more active than silybin
A. The galloylation of phenolic OH groups of natural silybin (a mixture of both isomers) leads to increases in their antiangiogenic
activities, which is more apparent with the 7-OH than the 20-OH. In contrast, gallates at aliphatic OH groups either had a
comparable activity to the parent compound or are even worse than silybin, which was observed in the case of 3-O-galloylsilybin. The
most effective compound from this series (7-O-galloylsilybin) has also been prepared from stereochemically pure silybins A and B to
evaluate the effect of stereochemistry on the activity. As with silybin itself, the B isomer of 7-O-galloylsilybin was more active than the
A isomer.
’ INTRODUCTION
respond to pegylated interferon plus ribavirin combination
therapy with high anti-HCV efficacy, a decrease in HIV replica-
tion, and increase in CD4+ cells.10
The flavonolignan silybin (1), a natural polyphenol contained
in the milk thistle (Silybum marianum) extract (so-called silymarin),
is a well-known flavonoid possessing multiple biological activities
at various cell levels.1 This compound is attracting increasing
attention, which is also reflected in a high number of papers
published recently1 (e.g., 204 papers in 2010; ISI, Web of
Science).
Natural silybin consists of an approximately equimolar mix-
ture of two diastereomers, silybin A (1a) and silybin B (1b)
(Figure 1), whose absolute configuration has been determined
recently.2,3 Their analytical separation is feasible using reverse-
phase HPLC,4,5 but preparative separation is extremely compli-
cated. It was satisfactorily achieved by ourselves quite recently
using lipases.6,7
Studies in approximately the past 20 years have shown that
silybin not only is an efficient hepatoprotectant and/or antiox-
idant but also possesses many other advantageous activities, such
as anticancer, chemopreventive, hypocholesterolemic, cardio-
protective, and neuroprotective activities.1,11 Silybin’s anticancer
effects were demonstrated in the treatment of some prostatic
diseases including adenocarcinoma,12 human non-small-cell lung
carcinoma H1299, H460, and H322 cells,13 and xenografts of
human colorectal cancer SW480.14 Silybin was found to be an
effective anticancer compound not only in the early stages of
many cancer types but also in the late phases, including
metastasis,12,15 which is difficult to treat using current commer-
cial anticancer agents.
Silybin is an active component in a plethora of phytoprepara-
tions used for the prevention and treatment of some liver diseases
and as an antidote against a number of hepatotoxins and
mycotoxins. The antioxidant and antiradical activities of silybin
have been studied, and respective molecular mechanisms were
described in detail.8,9 Recently, intravenous silybin was success-
fully used for the effective retreatment of HIVꢀHCV (HCV =
chronic hepatitis C virus) co-infected patients who did not
One of the mechanisms of silybin anticancer activity consists
of its antiangiogenic effects, involving several events connected
with this complex process, e.g., growth inhibition, cell cycle arrest,
apoptosis induction, inhibition of capillary tube organization,
reduced invasion, and migration of human endothelial cells.16
Received: August 2, 2011
Published: September 19, 2011
r
2011 American Chemical Society
7397
dx.doi.org/10.1021/jm201034h J. Med. Chem. 2011, 54, 7397–7407
|

Journal of Medicinal Chemistry
ARTICLE
Figure 1. Structures of silybin diastereomers.
The biological effects of optically pure silymarin components
have been mostly disregarded mainly because of their difficult
separation at a preparative scale. However, recent studies on the
pharmacological activity of pure silybins A and B clearly demon-
strated that the two silybin diastereomers 1a and 1b have
different biological activities; e.g., 1b has estrogenic activity,
whereas 1a exhibits no such activity.17 A study of the anticancer
efficacy of pure silymarin components on human prostate
carcinoma DU145 cells demonstrated that isosilybin B is the
most effective at suppressing the topoisomerase IIα gene pro-
moter and silybin B has the most significant effect on the G1 cell
cycle accumulation of these cancer cells.18 Another study of pure
silymarin components on advanced human prostate cancer PC3
cells showed that isosilybin B and silybin B are the most potent
compounds at inhibiting cell growth and colony formation.19
The potency of silybin B at inducing the cell death of PC3 cells
was lower than that of both isosilybins; however, it was still
significant and higher than that of silybin A.
Scheme 1. Synthesis of 7-O-Galloylsilybin (5)a
The presence of a galloyl moiety in the structure of flavonoids
(e.g., catechins) was found to be an important prerequisite for
their significant antiangiogenic properties (typically in epi-gallo-
catechin gallate, EGCG).20 Besides this, the introduction of a
galloyl moiety is expected to also improve the antioxidant and
chemoprotective activity of the resulting molecule. Therefore,
the synthesis of a series of silybin galloyl esters was developed in
this work with the aim of preparing new compound(s) with
significant antiangiogenic activity. The most active galloyl ester of
silybin was also prepared from stereochemically pure 1a and 1b
to assess the effect of stereochemistry on antiangiogenic activity.
a Reagents and conditions: (a) 3,4,5-tri-O-benzylgalloyl chloride (1.2
equiv), pyridine, DMAP (1 equiv), 0 °C, 1 h, 32%; (b) 3,4,5-tri-O-
benzylgalloyl chloride (1.2 equiv), Et3N (2 equiv), CH2Cl2/MeCN (1:1),
room temp, 1 h, 81%; (c) H2ꢀPd/C, EtOAc, room temp, 12 h, 91%.
(∼30%). As the 7-OH group is the most acidic group of the
silybin molecule (because of the -M effect of the carbonyl at C-4),
we tried to improve its regioselectivity in favor of the ester at the
7-OH by the selective generation of phenolate at this position
using a relatively weak base, Et3N. Optimizing the reaction con-
ditions with Et3N led to the development of a highly regioselective
method for the galloylation of the 7-OH (Scheme 1).
’ CHEMISTRY
The galloylation of the primary 23-OH of 1 using reported
chemical methods failed (Mitsunobu reaction and Lewis acid
catalysis), as well as the use of lipase (Novozym 435), which is
generally feasible with aliphatic acids but with aromatic acids
does not proceed at all, as we found in all related previous
studies25ꢀ30 and tried ourselves without success. Therefore, to
achieve monoesterification of the silybin 23-OH group, a suitable
protected derivative had to be prepared before the intended
esterification. The higher acidity of phenolic OH groups allows
their selective alkylation in the presence of alcoholic OH groups,
e.g., benzylation, which leads to an appropriate partially pro-
tected silybin derivative, 5,7,20-tri-O-benzylsilybin (10).8 This
compound still contains two free OH groups (at C-3 and C-23),
but the use of benzyl- or MOM-protected gallic acid with DCC/
DMAP at 0 °C leads to a selective esterification of the 23-OH
(Scheme 2). On the basis of these experiments, we ascertained
that the use of the MOM group for gallic acid protection
(compound 4) instead of the benzyl group considerably simpli-
fied purification of the product from the reaction mixture. The
use of this orthogonal protection enabled catalytic hydrogeno-
lysis of the crude reaction mixture after esterification and made
The silybin molecule contains five hydroxyl groups with
various properties, which makes selective silybin functionaliza-
tion a difficult task. Recently, we reported a relatively complete
toolbox for the selective protection/deprotection of silybin,
including regioselective esterification.21 Selective acylation of
the phenolic OH group at C-7 can be achieved using the
corresponding acyl chloride or anhydride in pyridine.22 The
primary alcoholic group at C-23 is another OH group that can be
regioselectively esterified without the prior protection of other
OH groups, using the following methods: (i) the Mitsunobu
reaction (carboxylic acid, Ph3P, and DEAD);23 (ii) Lewis acid
catalysis (acyl chloride/anhydride, BF3 Et2O);22 (iii) biocatalytic
3
methods using lipases (Novozym 435, carboxylic acid or
vinyl ester).7,24
Existing methods of regioselective monoesterification of sily-
bin were utilized for the synthesis of gallates. Only the use of acyl
chloride in pyridine led to the intended monogalloylation of the
silybin molecule, specifically at the 7-OH (Scheme 1). However,
the regioselectivity of this reaction was not very high, which
resulted in a rather low yield of the corresponding product 9
7398
dx.doi.org/10.1021/jm201034h |J. Med. Chem. 2011, 54, 7397–7407

Journal of Medicinal Chemistry
ARTICLE
Scheme 2. Synthesis of 3-O-Galloylsilybin (7) and 23-O-Galloylsilybin (6)a
a Reagents and conditions: (a) BnBr (5 equiv), NaH (3 equiv), DMF, 0 °C for 1 h, then room temp 1 h;8 (b) 4(1.2 equiv), DCC (2 equiv), DMAP (0.5 equiv),
CH2Cl2, 2 h at 0 °C, 2 h at room temp, 44% (after step c); (c) H2ꢀPd/C, AcOEt; (d) HCl (cat.), MeOH, 3 h, 88% from 11, 83% from 13; (e) TBDMSCl
(1.2 equiv), Py, AgNO3 (cat.), 45 °C, 2 h, 62%; (f) 4 (2 equiv), DCC (2 equiv), DMAP (1 equiv), CH2Cl2, 48 h at room temp, 62% (after step c).
purification of the 23-O-[30,40,50-tri-O-(methoxymethyl)galloyl]-
silybin (11) easier. Final deprotection of 11 was accomplished by
acidic hydrolysis using MeOH/HCl, yielding 23-O-gallate 6
(Scheme 2).
Scheme 3. Alternative Synthesis of 3-O-Galloylsilybin (7)a
The synthesis of 3-O-galloylsilybin (7) started again from
benzylated derivative 10, which was subsequently protected at
the 23-OH with a TBDMS group,31 yielding compound 12
(Scheme 2). Galloylation of 12 with MOM-protected gallic acid
4 required a substantially longer reaction time than galloylation
of the 23-OH (48 h) to achieve sufficient conversion. Two
deprotection steps of fully protected intermediate which began
with hydrogenolysis of the benzyl groups and was followed by
acidic hydrolysis yielded the final 3-O-galloylsilybin (7). It is
worth mentioning that during the attempts to synthesize 20-O-
galloyl ester starting from 5,7-di-O-tritylsilybin (14),32 an ex-
clusive formation of galloyl ester at 3-OH was observed
(Scheme 3). Moreover, esterification of the 3-OH proceeds
substantially more quickly using 14 than when starting from 12.
Synthesis of the 20-O-gallate 8 was based on the procedure
developed for galloylation of the 7-OH, using Et3N as a base
producing phenolate at an appropriate position and thus directed
the regioselectivity of esterification. The more reactive 7-OH had
to be previously protected with a benzyl group, yielding com-
pound 16 (Scheme 4). Esterification of 16 using acyl chloride 3
and Et3N led to benzyl-protected ester 17, which after hydro-
genolysis gave 20-O-galloylsilybin (8) (Scheme 4).
a Reagents and conditions: (a) TrCl (2 equiv), Et3N (3 equiv), CH2Cl2,
3 h at room temp;32 (b) 3,4,5-tri-O-benzylgallic acid, DCC/DMAP,
CH2Cl2; (c) HCl (cat.), MeOH, 3 h, 52% (after two steps); (d)
H2ꢀPd/C, AcOEt, 12 h, 71%.
Since the starting natural silybin was a nearly equimolar mix-
ture of the A and B isomers, some NMR signals were doubled.
7399
dx.doi.org/10.1021/jm201034h |J. Med. Chem. 2011, 54, 7397–7407

Journal of Medicinal Chemistry
ARTICLE
The correct proton signal multiplicity was therefore obtained
from HOM2DJ and COSY. Fortunately, the chemical shift
differences were in the ppb range so that common cross-peaks
for both isomers were usually observed in the heterocorrelated
experiments. The unambiguous NMR signal assignment is
essential for the structure elucidation. Therefore, the measure-
ments were performed in DMSO-d6, in which the observation of
OH signals is possible. The assignment of all hydroxyl groups
represents an indirect way of determining the site of substitution
(missing signal of OH proton indicates substitution), providing
the information on sites that are not affected. Some substituents
(OCH3, OCH2Ph) offer direct means of their localization: NOE
to their neighboring protons (OCH3), H,H long-range couplings
(5, 5a, 5b, and 9) can be inferred from chemical shift changes of
C-6, C-7, C-8, and C-4a. An acylation at 20-OH (compounds 8
and 17) was accompanied by corresponding changes of chemical
shift of C-19, C-20, and C-21 observable in 13C NMR.
’ BIOLOGICAL RESULTS
Cytotoxicity. All the compounds studied (Figure 2) were first
tested for cytotoxicity on HUVEC, measured as the ability to
reduce MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetra-
zolium bromide). Silybin was the least toxic, with 50% viability
at 72.58 ( 4.18 μM, and 7-O-galloylsilybin exhibited the lowest
IC50 (7.85 ( 0.52 μM for the B stereoisomer; see Table 1).
However, unusual behavior with this model was noted for 7-O-
galloylsilybins, which exhibited approximately 50% viability at all
concentrations above their IC50, but even up to 100 μM it did not
drop below 40% of the control. At the same time, silybin and its
derivatives have limited solubility and concentrations higher than
30 μM are hard to achieve, even in the presence of DMSO. No
consistent effect was noted on LDH leakage (data not shown).
Cell Growth Assay. Angiogenesis involves the local prolifera-
tion of endothelial cells. We investigated the ability of silybin and
its gallates to inhibit the growth of endothelial cells. Table 2 and
(OCH3, OCH2Ph), and 3JC,H
.
The position of galloyl group in 6 and 7 was determined using
downfield acylation shift (H-3 or H-23). The heteronuclear
coupling between H-3 or H-23 and the carbonyl of respective
acyls was a direct proof of the acylation. An acylation at 7-OH
Scheme 4. Synthesis of 20-O-Galloylsilybin (8)a
Table 1. Effect of Silybin and Its Gallates on Viability of
HUVECa
IC50 (μM)
silybin (1)
72.58 ( 4.18a
62.33 ( 2.39b
60.47 ( 1.03b
64.95 ( 3.59a,b
9.21 ( 0.93c
17.14 ( 0.59
7.85 ( 0.52c
35.41 ( 2.10
55.02 ( 4.21
silybin A (1a)
silybin B (1b)
3-O-galloylsilybin (7)
7-O-galloylsilybin (5)
7-O-galloylsilybin A (5a)
7-O-galloylsilybin B (5b)
20-O-galloylsilybin (8)
23-O-galloylsilybin (6)
a The cells were grown in 96-well plates to confluency, then incubated
with the tested compounds for 16 h. MTT-reducing ability is expressed
as IC50 (mean ( SD from three independent experiments performed in
triplicate). Values marked by the same letter are not significantly
different (p < 0.05).
a Reagents and conditions: (a) BnBr (2 equiv), K2CO3 (7 equiv),
acetone, reflux, 3 h;21 (b) 3,4,5-tri-O-benzylgalloyl chloride (3, 1.2 equiv),
Et3N (2 equiv), CH2Cl2, room temp, 1 h, 39%; (c) H2ꢀPd/C, EtOAc,
room temp, 12 h, 84%.
Figure 2. Structures of the silybin gallates tested.
7400
dx.doi.org/10.1021/jm201034h |J. Med. Chem. 2011, 54, 7397–7407

Journal of Medicinal Chemistry
ARTICLE
Table 2. Inhibitory Effects of the Studied Compounds on
structure could be observed were considered positive). These
MICs were as follows: for silybin, 75 μM; for 3-O-galloylsilybin,
75 μM; for 7-O-galloylsilybin, 25 μM; for 20-O-galloylsilybin,
60 μM; for 23-O-galloylsilybin, 75 μM.
HUVEC Proliferation (MTT Assay)a
IC50 (μM)
9.99 ( 0.11a
8.83 ( 0.44b
6.49 ( 0.33c
12.31 ( 0.27d
4.75 ( 0.13c
6.06 ( 0.15b
4.29 ( 0.30c
10.35 ( 0.35a,d
11.59 ( 0.71d
Quantification using the angiogenic score enabled us to
compare the tested compounds in more detail. In a typical
experiment, the angiogenic score in control wells was 3.57 (
0.64 and decreased to 0.73 ( 0.09 in wells treated with
2-methoxyestradiol (positive control). For 7-O-galloylsilybin,
significant inhibition of tube formation was already observed at
10 μM (79.9 ( 3.7% of control), and this effect was dose-
dependent. When its A and B isomers were evaluated, quite
similar results were obtained. In contrast, 3-O-galloylsilybin had
virtually no effect up to 75 μM and only a very slight (although
significant) effect was observed for 23-O-galloylsilybin (81.5 (
15.9% of control at 20 μM) and 20-O-galloylsilybin (94.5 ( 4.1%
of control at 20 μM and 49.5 ( 2.0% at 30 μM) (Figures 5 and
6). Taken together, these results clearly show that 7-O-galloylsily-
bin is the most potent inhibitor of this key step of angiogenesis.
silybin (1)
silybin A (1a)
silybin B (1b)
3-O-galloylsilybin (7)
7-O-galloylsilybin (5)
7-O-galloylsilybin A (5a)
7-O-galloylsilybin B (5b)
20-O-galloylsilybin (8)
23-O-galloylsilybin (6)
a HUVECs (4 ꢁ 103) in a total volume of 100 μL were incubated with
serial dilutions of the tested compounds for 3 days. MTT-reducing
ability is expressed as IC50 (mean ( SD from three independent
experiments performed in triplicate). Values marked by the same letter
are not significantly different (p < 0.05).
’ DISCUSSION
Silybin was recently reported to be a potent antiangiogenic
compound with significant effects on various phases of
angiogenesis.16 Moreover, the above-mentioned work only eval-
uated the antiangiogenic activity of natural silybin (mixture of
two diastereomers). The results of our study showed that silybin
has rather poor antiangiogenic effects, whereas silybin B is a
slightly better inhibitor of angiogenesis than silybin A.
On the basis of the results of our in vitro antiangiogenic assays
on HUVEC cells, it has been clearly demonstrated that galloyla-
tion of silybin is a suitable way to improve its antiangiogenic
effects. This SAR study had two important goals: (i) to determine
the most suitable position of the silybin molecule for galloylation;
(ii) to evaluate the influence of its stereochemistry on its
corresponding biological activity.
Figure 3. Effect of silybin gallates on HUVEC proliferation. Cell proli-
feration is presented as a percentage of control cell growth. Each point
represents the mean of three independent experiments performed in
triplicate. SD values were always below 10% and were omitted for clarity.
As for the position of silybin substitution, we identified two
appropriate sites of silybin molecule, i.e., its 7-OH and 20-OH
groups, finding the 7-OH to be more suitable. On the other hand,
galloylation of aliphatic OH groups was found to have a
deleterious effect on the antiangiogenic activity. Surprisingly,
3-O-galloylsilybin (7) was identified as the least active derivative,
even worse than the parent silybin (1), despite its highly similar
structure compared to epi-gallocatechin gallate (Figure 7), the
most active antiangiogenic component identified in green tea
extracts.20 Comparison of their structures raises several possible
explanations of their contradictory antiangiogenic efficacy: (i)
the presence of a coniferyl alcohol moiety in the structure of 7
can lead to the loss of its activity compared to EGCG; (ii) the
carbonyl group at the C-4 of 7 causes this ring to be more rigid
than in EGCG, which can again lead to lower activity; (iii) most
probably, the stereochemistry at C-3 of both compounds could
be a critical factor for their activities. It seems that both pyrogallol
moieties (B and G rings) present in EGCG have to be in one
plane, which is not the case with compound 7, which is in the
trans-configuration at C-2, C-3.
Figure 3 show 7-O-galloylsilybin and especially its B isomer
exerting the most potent inhibitory effect on HUVEC prolifera-
tion. Virtual increase in cell proliferation in the presence of very
high concentrations of 7-O-galloylsilybins as seen in Figure 3 was
due to their precipitation in the culture medium and interference
with formazan determination.
Endothelial Cell Migration Assay. The results obtained with
this assay (Figure 4), following the reoccupation of the “wound”
space after overnight (16ꢀ20 h) treatment, enabled us to deter-
mine the minimal inhibitory concentration (MIC) for each tested
compound. These MICs were as follows: for silybin, 50 mM
(virtually identical for both isomers); for 3-O-galloylsilybin,
50 mM; for 7-O-galloylsilybin, 10ꢀ20 mM (virtually identical
for both isomers); for 20-O-galloylsilybin, 20 mM; for 23-O-
galloylsilybin, 20 mM.
Endothelial Cell Differentiation Assay: Tube Formation on
Matrigel. After 6 h of treatment, we only detected a clear
inhibitory effect for 7-O-galloylsilybin at 50 μM. For the control
silybin, this kind of inhibitory effect could not be detected at such
a low concentration and it was only observed at 75 μM and
above. In extended incubations, after 24 h of treatment we
were able to determine the minimal inhibitory concentration
(MIC) for each tested compound (those assays where no tubular
On the basis of the correlation of the structures of galloylsi-
lybins with EGCG, the higher efficacy of both 7-O-galloylsilybin
(5) and 20-O-galloylsilybin (8) can also be explained. If the
coplanarity of the B and G rings serves as a structural motif for the
high antiangiogenic activity of EGCG, we can also find similar
motifs in the structures of both silybin gallates 5 and 8 (Figure 8).
7401
dx.doi.org/10.1021/jm201034h |J. Med. Chem. 2011, 54, 7397–7407

Journal of Medicinal Chemistry
ARTICLE
Figure 4. Effect of 7-O-galloylsilybin on endothelial cell migration after overnight (16ꢀ20 h) incubation in 24-well plates: (A) T = 0; (B) control;
(C) 7-O-galloylsilybin, 10 μM; (D) 7-O-galloylsilybin, 20 μM.
As the structural similarity is higher for gallate 5, it should possess
an accordingly higher activity than the rather less similar 8.
To evaluate the effect of stereochemistry on the antiangio-
genic activity, the most active compound from the tested series,
7-O-galloylsilybin (5), was synthesized from stereochemically
pure silybins. Again, the effects of pure B-isomer 5b were signi-
ficantly higher than those of 5a.
HRMS measurements were performed using a commercial APEX-
Ultra FTMS instrument equipped with a 9.4 T superconducting magnet
and a dual II ESI/MALDI ion source (Bruker Daltonics, Billerica, MA,
U.S.). The analysis was performed using electrospray ionization (ESI),
and the spectra were acquired in negative ion mode. The interpretation
of mass spectra was done using DataAnalysis, version 4.0, software
package (Bruker Daltonics, Billerica, MA, U.S.).
Positive-ion electrospray ionization (ESI) mass spectra were re-
corded on a LCQDECA spectrometer (ThermoQuest, San Jose, CA,
U.S.). HPLC experiments were performed in a Shimadzu Prominence
UFLC system (Kyoto, Japan) with a PDA detector connected to a
Chromolith Performance RP-18e monolithic column (100 mm ꢁ 3 mm
i.d.) (Merck, Germany). Purity of the tested compounds was deter-
mined using HPLC, and in all cases the purity reached at least 95% (for
respective chromatograms see Supporting Information).
’ MATERIALS AND METHODS
Chemistry. General Methods. Silybin (mixture of A and B,
approximately 1:1) was kindly provided by Dr. L. Cvak (TAPI Galena,
IVAX Pharmaceuticals, Opava, CZ). 3,4,5-Tri-O-benzylgalloyl chloride
(3) was prepared by reacting 3,4,5-tri-O-benzylgallic acid (2) with an
excess of oxalyl chloride and used immediately after preparation without
further purification. All other chemicals were purchased from Sigma-
Aldrich and used as obtained. The reactions were monitored by TLC on
F254 silica gel (Merck), and the spots were visualized with UV light and
by charring with 5% H2SO4 in EtOH.
Stereochemically pure 1a and 1b were prepared using Novozym 435.7
5,7,20-Tri-O-benzylsilybin (10),8 7-O-benzylsilybin (16),21 3,4,5-tri-O-
benzylgallic acid (2),34 and 5,7-di-O-tritylsilybin (14)32 were prepared
according to published procedures, and their identity was verified by
appropriate structural analysis.
NMR spectra were recorded on a Bruker Avance III 400 MHz
1
spectrometer (400.13 MHz for H, 100.55 MHz for 13C at 30 °C)
3,4,5-Tri-O-methoxymethylgallic Acid (4). MOMCl (7.1 mL,
70.109 mmol, 75%) was added dropwise to a cooled solution of gallic
acid (3 g, 17.647 mmol) and NaH (3.2 g, 80 mmol, 60% dispersion in min
oil w/w) indry DMF (30 mL). The mixture was stirred for30 min at 0 °C,
then for 12 h at room temperature, diluted with ice-cold water, acidified
with an aqueous solution of tartaric acid (pH < 7), and extracted with
EtOAc (2 ꢁ 75 mL). The organic layers were combined, dried (Na2SO4),
and evaporated. The crude mixture was dissolved in EtOH (100 mL).
Aqueous KOH solution (1 M, 30 mL) was added, and the resulting
mixture was stirred for 12 h at room temperature. The reaction mixture
was concentrated in vacuo, then poured into ice-cold water and acidified
with tartaric acid (pH < 7). The aqueous portion was then extracted with
EtOAc (2 ꢁ 75 mL), and the organic portion was dried (Na2SO4) and
evaporated. Flash chromatography (CHCl3/toluene/acetone/HCO2H,
and a Bruker Avance III 600 MHz spectrometer (600.23 MHz for 1H,
150.93 MHz for 13C at 30 °C) in DMSO-d6 (99.9 atom % D, Sigma-
Aldrich, Steinheim, Germany). Residual signals of solvent were used as
an internal standard (δH 2.500 ppm, δC 39.60 ppm). All NMR
1
experiments, H NMR, 13C NMR, J-resolved, COSY, HSQC, HSQC-
TOCSY, HMBC, and 1D TOCSY were performed using the manufac-
1
turer’s software. H NMR and 13C NMR spectra were zero filled to
4-fold data points and multiplied by window function before Fourier
transformation. The two-parameter double-exponential LorentzꢀGauss
function was applied for 1H to improve resolution, and line broadening
(1 Hz) was applied to get better 13C signal-to-noise ratio. Chemical
shifts are given in δ scale with digital resolution, justifying the reported
values to three (δH) or two (δC) decimal places.
7402
dx.doi.org/10.1021/jm201034h |J. Med. Chem. 2011, 54, 7397–7407

Journal of Medicinal Chemistry
ARTICLE
Figure 5. Inhibition of HUVEC tube formation by silybin gallates. HUVECs were incubated overnight on Matrigel in the absence (control) or presence
of the tested compounds or 2-methoxyestradiol (2-ME, 10 μM, positive control), and the cultures were observed (40ꢁ magnification) and
photographed at 3ꢁ optical zoom with an Olympus CK40 inverted microscope. Three pictures in total covering the whole area were recorded per well
and transferred to the computer for image analysis. The extent of tube formation was quantified using an angiogenic score as proposed recently33 in a
representative optical field containing around 100 cells, and the results were expressed as a percentage of the nontreated control: /, p < 0.05 compared
with nontreated control.
Figure 6. Inhibition of HUVEC tube formation by silybin gallates. HUVECs were incubated overnight on Matrigel in the absence (control) or presence
of the tested compounds or 2-methoxyestradiol (2-ME, 10 μM, positive control), and cultures were observed (40ꢁ magnification) and photographed at
3ꢁ optical zoom with an Olympus inverted microscope CK40: (A) control; (B) 7-O-galloylsilybin, 10 μM; (C) 2-ME.
85:10:5:1) yielded title compound 4 (1.8 g, 34%) as a white amorphous
solid. 1H NMR (600 MHz) δ 3.413 (6H, s, OMe), 3.506 (3H, s, OMe),
5.127 (2H, s, p-OCH2), 5.236 (4H, s, m-OCH2), 7.395 (2H, s, H-ortho).
13C NMR (150 MHz) δ 55.88 (q, OMe), 56.56 (q, OMe), 94.82 (t, m-
OCH2), 97.80 (t, p-OCH2), 110.81 (d, C-ortho), 126.23 (s, C-ipso),
139.96 (s, C-para), 150.39 (s, C-meta), 166.64 (s, i-COOH). MS-ESI
(m/z): 325 (M+ + Na).
(1140 mg, 2.484 mmol), and DMAP (250 mg, 2.046 mmol) were
dissolved in dry pyridine (30 mL), and the mixture was stirred at 0 °C for
1 h. Reaction was stopped by diluting in ice-cold HCl (1 M, 150 mL).
The aqueous portion was extracted with EtOAc (2 ꢁ 50 mL), and the
organic layers were combined, dried (Na2SO4), and evaporated. The dry
solid left after flash chromatography (CHCl3/acetone/HCO2H, 95:
5: 1) was the title compound 9 (600 mg, 32%) as a white amorphous
solid. MS-ESI (m/z): 927 (M+ + Na). For 1H and 13C NMR data, see
Tables S1 and S2 in the Supporting Information.
7-O-(30,40,50-Tri-O-benzylgalloyl)silybin (9): Method I. Sily-
bin (1, 1000 mg, 2.073 mmol), 3,4,5-tri-O-benzylgalloyl chloride (3)
7403
dx.doi.org/10.1021/jm201034h |J. Med. Chem. 2011, 54, 7397–7407

Journal of Medicinal Chemistry
ARTICLE
Figure 7. Comparison of structures of the least active silybin derivative and the most active component of green tea extract (congruous parts are
highlighted in red).
Figure 8. Correlation of the structures of the active galloylsilybins with EGCG (corresponding motifs are highlighted in the same color).
7-O-(30,40,50-Tri-O-benzylgalloyl)silybin (9): Method II. Et3N
(0.145 mL, 1.042 mmol) was added to a stirred solution of silybin (1,
250 mg, 0.518 mmol) in CH2Cl2/MeCN (20 mL, 1:3 v/v), and the
mixture was stirred at room temperature for 10 min. Then a solution of 3
(287 mg, 0.626 mmol) in CH2Cl2 (10 mL) was added dropwise and the
resulting mixture was stirred at room temperature for 1 h. Solvents were
evaporated to dryness by coevaporation with toluene. The dry solid left
after flash chromatography (CHCl3/acetone/HCO2H, 95:5:1) was the
title compound 9 (380 mg, 81%) as a white amorphous solid. MS-ESI
(m/z): 927 (M+ + Na). For 1H and 13C NMR data, see Tables S1 and S2
in the Supporting Information.
for 12 h. The Pd was then removed by filtration through Celite that was
washed with acetone, and the solvent was evaporated. The residue left
after flash chromatography (CHCl3/acetone/HCO2H, 5:1:0.1) was the
title compound 5 (320 mg, 91%) as a white amorphous solid. HRMS
calcd (M ꢀ H+) 633.1244, found 633.1243. For 1H and 13C NMR data,
see Tables S1 and S2 in the Supporting Information.
7-O-Galloylsilybin A (5a). Compound 5a was prepared via the
same procedure as for 5 (intermediate 9a was obtained using method II),
starting from pure 1a. HRMS calcd (M ꢀ H+) 633.1244, found 633.1243.
1
For H and 13C NMR data, see Tables S1 and S2 in the Support-
ing Information.
7-O-Galloylsilybin (5). Compound 9 (500 mg, 0.553 mmol) was
dissolved in EtOAc (30 mL), and then Pd/C (500 mg, 10% Pd) was
added. The reaction mixture was stirred under a hydrogen atmosphere
7-O-Galloylsilybin B (5b). Compound 5b was prepared via the
same procedure as for 5 (intermediate 9b was obtained using method
II), starting from pure 1b. HRMS calcd (M ꢀ H+) 633.1244, found
7404
dx.doi.org/10.1021/jm201034h |J. Med. Chem. 2011, 54, 7397–7407

Journal of Medicinal Chemistry
ARTICLE
1
633.1243. For H and 13C NMR data, see Tables S1 and S2 in the
3-O-Galloylsilybin (7): Method II. 5,7-Di-O-tritylsilybin (14,
470 mg, 0.487 mmol), 3,4,5-tri-O-benzylgallic acid (2, 257 mg, 0.584 mmol),
DCC (201 mg, 0.974 mmol), and DMAP (59 mg, 0.483 mmol) were
dissolved in dry CH2Cl2 (25 mL), and the mixture was stirred at room
temperature for 1 h. MeOH (25 mL) and HCl (1 mL, 36% v/v) were
then added, and the resulting mixture was stirred for another 2 h.
Reaction was stopped by the addition of saturated NaHCO3 solution
to neutral pH. The mixture was evaporated to dryness by coevapora-
tion with toluene, redissolved in CH2Cl2, and filtered. Flash chroma-
tography (CHCl3/toluene/acetone/HCO2H, 95:5:5:1) yielded 3-O-
[(30,40,50-tri-O-benzyl)galloyl]silybin (15, 230 mg, 52%) as a white
Supporting Information.
23-O-(30,40,50-Tri-O-methoxymethylgalloyl)silybin (11). 5,7,
20-Tri-O-benzylsilybin (10, 900 mg, 1.196 mmol), the acid 4 (434 mg,
1.436 mmol), DCC (494 mg, 2.394 mmol), and DMAP (73 mg,
0.598 mmol) were dissolved in dry CH2Cl2 (50 mL), and the mixture was
stirred at 0 °C for 2 h and then an additional 2 h at room temperature. The
mixture was then evaporated, redissolved in CH2Cl2, filtered to remove
DCU, and then evaporated. The crude reaction mixture was dissolved in
CH2Cl2 (6 mL) and then filtered through a pad of silica gel (3 cm). The
elution was performed using petroleum ether/EtOAc (1:1, 250 mL). The
resulting mixture (1.38 g) was subjected to hydrogenolysis using Pd/C
(720 mg, 10% of Pd) in EtOAc (40 mL) for 12 h. Solids were removed by
filtration through Celite, which was washed with acetone, and the
combined solution was evaporated. Flash chromatography (CHCl3/
toluene/acetone/HCO2H, 95:5:5:1) yielded title compound 11 (400 mg,
44%) as a white amorphous solid. MS-ESI (m/z): 789 (M+ + Na). For 1H
and 13C NMR data, see Tables S1 and S2 in the Supporting Information.
23-O-Galloylsilybin (6). HCl (0.2 mL, 36% v/v) was added to a
solution of 11 (220 mg, 0.287 mmol) in MeOH (30 mL), and the
resulting mixture was stirred at room temperature for 3 h. The reaction
was quenched by the addition of saturated NaHCO3 solution (to pH 7),
and the solvents were removed by coevaporation with pure EtOH. The
solid residue was dissolved in acetone, filtered to remove inorganic salts,
and evaporated. Flash chromatography (CHCl3/acetone/HCO2H,
5:1:0.1) yielded title compound 6 (160 mg, 88%) as a white amorphous
solid. HRMS calcd (M ꢀ H+) 633.1244, found 633.1243. For 1H and
13C NMR data, see Tables S1 and S2 in the Supporting Information.
5,7,20-Tri-O-benzyl-23-O-(tert-butyldimethylsilyl)silybin
(12). Powdered AgNO3 (359 mg, 2.113 mmol) was added to a solution
of 5,7,20-tri-O-benzylsilybin (10, 1590 mg, 2.112 mmol) and TBDMSCl
(382 mg, 2.534 mmol) in dry pyridine (15 mL), and the mixture was
stirred at 45 °C for 2 h. The mixture was then diluted with ice-cold water.
The precipitate was filtered off and dissolved with EtOAc. The organic
phase was dried over anhydrous Na2SO4 and evaporated to dryness.
Flash chromatography (petroleum ether/EtOAc, 3:1) yielded title
compound 12 (1130 mg, 62%) as a white amorphous solid. MS-ESI (m/z):
889 (M+ + Na). For 1H and 13C NMR data, see Tables S1 and S2 in the
Supporting Information.
1
amorphous solid. MS-ESI (m/z): 927 (M+ + Na). For H and 13C
NMR data, see Tables S1 and S2 of Supporting Information.
Hydrogenolysis of 15 (200 mg, 0.221 mmol) with Pd/C (100 mg,
10% w/w) in EtOAc (15 mL) for 12 h yielded crude title compound 7,
which was purified by flash chromatography (CHCl3/acetone/HCO2H,
5:1:0.1) to yield pure 7 (99 mg, 71%) as a white amorphous solid.
HRMS calcd (M ꢀ H+) 633.1244, found 633.1244. For 1H and 13
C
NMR data, see Tables S1 and S2 in the Supporting Information.
7-O-Benzyl-20-O-[(30,40,50-tri-O-benzyl)galloyl]silybin (17).
Et3N (0.243 mL, 1.748 mmol) was added to a stirred solution of 16 (500
mg, 0.874 mmol) in CH2Cl2 (50 mL), and the mixture was stirred
at room temperature for 10 min. A solution of 3 (441 mg, 0.962 mmol)
in CH2Cl2 (10 mL) was then added dropwise, and the resulting mixture
was stirred at room temperature for 1 h. Solvents were evaporated to
dryness by coevaporation with toluene. The dry solid left after flash
chromatography (CHCl3/toluene/acetone/HCO2H, 95:10:5:1) was the
title compound 17 (343 mg, 39%) as a white amorphous solid. MS-ESI
(m/z): 1017 (M+ + Na). For 1H and13C NMRdata, see TablesS1 andS2
in the Supporting Information.
20-O-Galloylsilybin (8). Hydrogenolysis of 17 (300 mg, 0.302 mmol)
with Pd/C (200 mg, 10% w/w) in EtOAc (30 mL) for 12 h yielded crude
title compound 8, which was purified by flash chromatography (CHCl3/
acetone/HCO2H, 5:1:0.1) to yield pure 8 (160 mg, 84%) as a white amor-
phous solid. HRMS calcd (M ꢀ H+) 633.1244, found 633.1242. For 1H and
13C NMR data, see Tables S1 and S2 in the Supporting Information.
Biological Testing. Reagents. Dimethylsulfoxide (DMSO) for
cell cultures, 2-ME (2-methoxyestradiol), MTT (3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide), and fetal calf serum
were purchased from Sigma-Aldrich Ltd., U.S. Collagenase was from Serva,
Germany. Endothelial cell basal medium with growth factors (endothelial
growth medium, EGM) was from Promocell, Germany. Other chemicals
and solvents were of analytical grade from Pliva-Lachema, Czech Republic.
The tested compounds were dissolved in DMSO at 10 mM, and these
stock solutions were kept at ꢀ20 °C.
23-O-tert-Butyldimethylsilyl-3-O-[(30,40,50-tri-O-methoxy-
methyl)galloyl]silybin (13). Compound 12 (500 mg, 0.577 mmol),
3,4,5-tri-O-methoxymethylgallic acid (4, 349 mg, 1.153 mmol), DCC
(238 mg, 1.153 mmol), and DMAP (70 mg, 0.577 mmol) were dissolved
in dry CH2Cl2 (30 mL), and the mixture was stirred at room tempera-
ture for 48 h. The mixture was then evaporated, redissolved in CH2Cl2
(10 mL), filtered to remove DCU through a pad of silica gel (3 cm),
eluted with petroleum ether/EtOAc (1:1, 250 mL), and evaporated. The
resulting mixture (920 mg) was subjected to hydrogenolysis (Pd/C,
500 mg, 10% ofPd) in EtOAc (30 mL) for12 h. The catalyst was removed
by filtration through Celite. The Celite pad was washed with acetone, and
the solvent was evaporated. Flash chromatography (CHCl3/toluene/
acetone/HCO2H, 95:10:5:1) yielded title compound 13 (315 mg, 62%)
as a white amorphous solid. MS-ESI (m/z): 903 (M+ + Na). For 1H and
13C NMR data, see Tables S1 and S2 in the Supporting Information.
3-O-Galloylsilybin (7): Method I. HCl (0.5 mL, 36% v/v) was
added to a solution of 13 (200 mg, 0.287 mmol) in MeOH (10 mL), and
the reaction mixture was stirred at room temperature for 3 h. After the
reaction was quenched with saturated NaHCO3 solution (to neutral pH),
the solvents were removed by coevaporation with absolute EtOH. The
solid residue was dissolved in acetone, filtered, and evaporated. Flash
chromatography (CHCl3/acetone/HCO2H, 5:1:0.1) yielded title com-
pound 7(120 mg, 83%) as a white amorphous solid. HRMS calcd (M ꢀ H+)
633.1244, found 633.1244. For 1H and 13C NMR data, see Tables S1 and
S2 in the Supporting Information.
Human Umbilical Vein Endothelial Cells (HUVECs). HU-
VECs were obtained from human umbilical cords of healthy nonsmoker
women (18ꢀ35 years) at the Department of Obstetrics and Gynecol-
ogy, University Hospital in Olomouc, Czech Republic. All volunteers
had to fulfill the requirements for study submission and sign a personal
awareness declaration. The choice of volunteers was in accordance with
the principles of the International Ethics Committee for Biomedical
Research (CIOMS, Geneva, 1993), and the study was approved by the
Ethic Commission FNO and LF UP. After delivery, part of the umbilical
cord (minimum 100 mm) was cut off and placed in sterile Earle’s balanced
salt supplemented with antibiotics at 4 °C. Endothelial cells were isolated
by collagenase digestion up to 20 h after removal. The umbilical cord was
washed with EBS and infused with a solution of collagenase in EBS. After
incubation (30 min, 37 °C) the cord was washed with Hanks buffer
without Ca2+ or Mg2+. The cells were cultivated in EGM and used in
passages 2ꢀ6.
Cytotoxicity. HUVECs were plated on gelatin-coated 96-well cell
culture plates in EGM and grown to confluency. The tested compounds
were placed in fresh EGM in serial dilutions (100 μL/well) and
7405
dx.doi.org/10.1021/jm201034h |J. Med. Chem. 2011, 54, 7397–7407

Journal of Medicinal Chemistry
ARTICLE
incubated overnight (16ꢀ20 h). After incubation, 50 μL of medium was
removed for determining LDH leakage35 and 50 μL of fresh medium
containing 1 mg/mL of MTT was added.36 The plates were incubated
for a further 3 h (37 °C). The resulting formazan was dissolved in 50 μL
of 1% NH3 in DMSO, and absorbance was read at 540 nm.
these two stereoisomers. Evaluation of their antiangiogenic
effects clearly showed that the gallate prepared from the B-isomer
(5b) is significantly more effective than the corresponding
A-isomer 5a. To the best of our knowledge, this is the first study
of the antiangiogenic activities of silybin gallates, including their
synthesis and the synthesis of stereochemically pure 7-O-galloyl-
silybins A and B. Moreover, this study is one of relatively few
studies of the biological activities of stereochemically pure
silybins and even fewer studies of the effects of stereochemically
pure silybin derivatives.
Cell Growth Assay. 4 ꢁ103 HUVECs in a total volume of 100 μL
were incubated with serial dilutions of the tested compounds for 3
days. After incubation, an amount of 10 μL of MTT (5 mg/mL) was
added and the remainder of the method was the same as for the
cytotoxicity assay.
Endothelial Cell Migration Assay. The migratory activity of
HUVECs was assessed using a wound migratory activity.37 Confluent
monolayers in 24-well cell culture plates were wounded with pipet tips
(10ꢀ200 μL), giving rise to one acellular 1-mm-wide line per well. After
the cells were washed twice with PBS, the cells were supplied with
500 μL of fresh EGM in the absence (controls) or presence of tested
compounds. Wounded areas were observed (40ꢁ magnification) and
photographed at 3ꢁ optical zoom with an Olympus inverted micro-
scope CK40 at time zero and after overnight incubation at 37 °C.
Endothelial Cell Differentiation Assay: Tube Formation on
Matrigel. An amount of 10 μL of Matrigel (9 mg/mL, BD Biosciences)
was applied to the inner well of a μ-Slide Angiogenesis (ibidi) at 4 °C and
allowed to polymerize at 37 °C for a minimum of 30 min. Approximately
1 ꢁ 104 HUVECs were added to 50 μL of EGM in the absence (control)
or presence of the tested compounds or 2-methoxyestradiol (10 μM,
positive control). The mixture was incubated overnight, and cultures
were observed (40ꢁ magnification) and photographed at 3ꢁ optical
zoom with an Olympus CK40 inverted microscope. Three pictures, in
total covering the whole area, were recorded per well and transferred to
the computer for image analysis. The extent of tube formation was
quantified using an angiogenic score as proposed recently33 in a
representative optical field containing approximately 100 cells, and the
results were expressed as a percentage of the nontreated control:
’ ASSOCIATED CONTENT
S
Supporting Information. HPLC chromatograms and
b
HRMS spectra of studied compounds (5ꢀ8, including 5a and
5b) and 1H and 13C NMR data and spectra of compounds 4ꢀ17.
This material is available free of charge via the Internet at http://
pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: (+420) 296 442 510. Fax: (+420) 296 442 509. E-mail:
kren@biomed.cas.cz.
’ ACKNOWLEDGMENT
This work was supported by Grant P207/10/0288 (R.G.) and
Grant P301/11/0767 (V.K. and J.U.) from the Czech Science
Foundation, Grant MSM 6198959216 (K.V. and J.U.) and Grant
OC08049 (V.K.) from the Ministry of Education of the Czech
Republic, Research Concept of the Institute of Microbiology
Grant AV0Z50200510, Grant PS09/02216 (M.A.M.) from the
Spanish Ministry of Science and Innovation, Grant PIE P08-
CTS-3759 (M.A.M.) from the Andalusian Government, and ESF
COST CM0602 projects. Dr. P. Novꢁak (Institute of Microbiol-
ogy, Prague, Czech Republich) is thanked for ESI MS and FT MS
measurements. We also thank to Casimiro Cꢁardenas (University
of Mꢁalaga) for skillful technical assistance.
ꢀ
ꢁ
Nos ꢁ 1 þ Noc ꢁ 2 þ Nop ꢁ 3
angiogenic score ¼
( ½0; 1; or 2ꢂ
Not
where Nos is the number of sprouting cells, Noc is the number of
connected cells, Nop is the number of polygons, and Not is the total
number of cells. The presence of complex mesh (luminal structures
consisting of walls two to three cells thick) is given a score of 1 and is
added once per optical field. If the wall is four or more cells thick, a score
of 2 is awarded.33
Statistical Analysis. Data were analyzed with one-way ANOVA
using Statext, version 1.4.2b, statistical package. Differences were con-
sidered statistically significant when p < 0.05. IC50 values were obtained
using Microsoft Excel 2007.
’ ABBREVIATIONS USED
HUVEC, human umbilical vein endothelial cell; SAR, structureꢀ
activity relationship; EGCG, epi-gallocatechin gallate; HOM2DJ,
homo-J-related hydrogen-1 to hydrogen-1; MTT, 3-(4,5-dimethyl-
2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; LDH, lactate
dehydrogenase;2-ME, 2-methoxyestradiol; HSQC, heteronuclear
single quantum coherence; TOCSY, total correlation spectro-
scopy; HMBC, heteronuclear multiple bond correlation
’ CONCLUSIONS
This study of the antiangiogenic effects of regioisomeric
monogalloyl esters of the flavonolignan silybin provides clear
proof that this natural compound is a useful lead structure for
relatively simple semisynthetic modifications leading to potent
biologically active drugs. A SAR study of the corresponding
silybin gallates showed that the position of silybin substitution
plays a key role in the activity of the resulting galloyl ester.
Accordingly, galloylation of the phenolic OH groups at C-7 or
C-20 resulted in rather effective inhibitors of angiogenesis,
whereas esterification of the aliphatic OH groups at C-3 and
C-23 is connected with a very slight improvement in activity or
even loss of activity with the 3-O-gallate 7.
’ REFERENCES
(1) Gaꢀzꢁak, R.; Walterovꢁa, D.; Kꢀren, V. Silybin and silymarin: new and
emerging applications in medicine. Curr. Med. Chem. 2007, 14, 315–338.
(2) Lee, D. Y.-W.; Liu, Y. Molecular structure and stereochemistry of
silybin A, silybin B, isosilybin A, and isosilybin B, isolated from Silybum
marianum (milk thistle). J. Nat. Prod. 2003, 66, 1171–1174.
(3) Kim, N.-C.; Graf, T. N.; Sparacino, C. M.; Wani, M. C.; Wall,
M. E. Complete isolation and characterization of silybins and isosilybins
from milk thistle (Silybum marianum). Org. Biomol. Chem. 2003, 1,
1684–1689.
The most effective compound from the series, 7-O-gallate 5,
was also prepared from stereochemically pure silybins A and B
instead of natural silybin consisting of an equimolar mixture of
(4) Ding, T.-M.; Tian, S.-J.; Zhang, Z.-X.; Gu, D.-Z.; Chen, Y.-F.; Shi,
Y.-H.; Sun, Z.-P. Determination of active component in silymarin by
RP-LC and LC/MS. J. Pharm. Biomed. Anal. 2001, 26, 155–161.
7406
dx.doi.org/10.1021/jm201034h |J. Med. Chem. 2011, 54, 7397–7407

Journal of Medicinal Chemistry
ARTICLE
ꢀ
(5) Marhol, P.; Gaꢀzꢁak, R.; Bednꢁaꢀr, P.; Kꢀren, V. Narrow-bore coreꢀ
shell particles and monolithic columns in the analysis of silybin dia-
stereoisomers. J. Sep. Sci. 2011, 34, 2206–2213.
(6) Monti, D.; Gaꢀzꢁak, R.; Marhol, P.; Biedermann, D.; Purchartovꢁa,
K.; Fedrigo, M.; Riva, S.; Kꢀren, V. Enzymatic kinetic resolution of silybin
diastereoisomers. J. Nat. Prod. 2010, 73, 613–619.
(7) Gaꢀzꢁak, R.; Marhol, P.; Purchartovꢁa, K.; Monti, D.; Biedermann,
D.; Riva, S.; Cvak, L.; Kꢀren, V. Large-scale separation of silybin dia-
stereoisomers using lipases. Process Biochem. 2010, 45, 1657–1663.
(8) Trouillas, P.; Marsal, P.; Svobodovꢁa, A.; Vostꢁalovꢁa, J.; Gaꢀzꢁak, R.;
Hrbꢁaꢀc, J.; Sedmera, P.; Kꢀren, V.; Lazzaroni, R.; Duroux, J.-L.; Walterovꢁa,
D. Mechanism of the antioxidant action of silybin and 2,3-dehydrosily-
bin flavonolignans: a joint experimental and theoretical study. J. Phys.
Chem. A 2008, 112, 1054–1063.
(22) Gaꢀzꢁak, R.; Purchartovꢁa, K.; Marhol, P.; Zivnꢁa, L.; Sedmera, P.;
Valentovꢁa, K.; Kato, N.; Matsumura, H.; Kaihatsu, K.; Kꢀren, V. Anti-
oxidant and antiviral activities of silybin fatty acid conjugates. Eur. J. Med.
Chem. 2010, 45, 1059–1067.
(23) Wang, F.; Huang, K.; Yang, L.; Gong, J.; Tao, Q.; Li, H.; Zeng,
S.; Wu, X.; St€ockigt, J.; Li, X.; Qu, J.; Zhao, Y. Preparation of C-23
esterified silybin derivatives and evaluation of their lipid peroxidation
inhibitory and DNA protective properties. Bioorg. Med. Chem. 2009,
17, 6380–6389.
(24) Theodosiou, E.; Katsoura, M. H.; Loutrari, H.; Purchartovꢁa, K.;
Kꢀren, V.; Kolisis, F. N.; Stamatis, H. Enzymatic preparation of acylated
derivatives of silybin in organic and ionic liquid media and evaluation of
their antitumor proliferative activity. Biocatal. Biotransform. 2009,
27, 161–169.
(9) Gaꢀzꢁak, R.; Sedmera, P.; Vrbackꢁy, M.; Vostꢁalovꢁa, J.; Drahota, Z.;
Marhol, P.; Walterovꢁa, D.; Kꢀren, V. Molecular mechanisms of silybin and
2,3-dehydrosilybin antiradical activity: role of individual hydroxyl
groups. Free Radical Biol. Med. 2009, 46, 745–758.
(25) Ardhaoui, M.; Falcimaigne, A.; Engasser, J. M.; Moussou, P.;
Pauly, G.; Ghoul, M. Enzymatic synthesis of new aromatic and aliphatic
esters of flavonoids using Candida antarctica lipase as biocatalyst.
Biocatal. Biotransform. 2004, 22, 253–259.
(10) Payer, B. A.; Reiberger, T.; Rutter, K.; Beinhardt, S.; Staettermayer,
A. F.; Peck-Radosavljevic, M.; Ferenci, P. Successful HCV eradication and
inhibition of HIV replication by intravenous silibinin in an HIVꢀHCV
coinfected patient. J. Clin. Virol. 2010, 49, 131–133.
(26) Guyot, B.; Bosquette, B.; Pina, M.; Graille, J. Esterification of
phenolic acids from green coffee with an immobilized lipase from
Candida antarctica in solvent-free medium. Biotechnol. Lett. 1997, 19,
529–532.
(11) Agarwal, R.; Agarwal, Ch.; Ichikawa, H.; Singh, R. P.; Aggarwal,
B. B. Anticancer potential of silymarin: from bench to bed side. Anti-
cancer Res. 2006, 26, 4457–4498.
(12) Singh, R. P.; Raina, K.; Sharma, G.; Agarwal, R. Silybin inhibits
established prostate tumor growth, progression, invasion, and metastasis
and suppresses tumor angiogenesis and epithelialꢀmesenchymal transi-
tion in transgenic adenocarcinoma of the mouse prostate model mice.
Clin. Cancer Res. 2008, 14, 7773–7780.
(27) Kodera, Y.; Takahashi, K.; Nishimura, H.; Matshushima, A.;
Saito, Y.; Inada, Y. Ester synthesis from substituted carboxylic acid
catalyzed by polyethylene glycol-modified lipase from Candida cylin-
dracea in benzene. Biotechnol. Lett. 1986, 8, 881–884.
(28) Otto, R. T.; Scheib, H.; Bornscheuer, U. T.; Pleiss, J.; Syldatk,
C.; Schmid, R. D. Substrate specificity of lipase B from Candida
antarctica in the synthesis of aryl-aliphatic glycolipids. J. Mol. Catal. B:
Enzym. 2000, 8, 201–211.
(13) Mateen, S.; Tyagi, A.; Agarwal, C.; Singh, R. P.; Agarwal, R.
Silibinin inhibits human nonsmall cell lung cancer cell growth through
cell-cycle arrest by modulating expression and function of key cell-cycle
regulators. Mol. Carcinog. 2010, 49, 247–258.
(14) Velmurugan, B.; Gangar, S. C.; Kaur, M.; Tyagi, A.; Deep, G.;
Agarwal, R. Silibinin exerts sustained growth suppressive effect against
human colon carcinoma SW480 xenograft by targeting multiple signal-
ing molecules. Pharm. Res. 2010, 27, 2085–2097.
(15) Deep, G.; Agarwal, R. Antimetastatic efficacy of silibinin:
molecular mechanisms and therapeutic potential against cancer. Cancer
Metastasis Rev. 2010, 29, 447–463.
(16) Singh, R. P.; Dhanalakshmi, S.; Agarwal, C.; Agarwal, R. Silibinin
strongly inhibits growth and survival of human endothelial cells via cell
cycle arrest and downregulation of survivin, Akt and NF-kappaB: implica-
tions for angioprevention and antiangiogenic therapy. Oncogene 2005,
24, 1188–1202.
(29) Shintre, M. S.; Ghadge, R. S.; Sawant, S. B. Lipolase catalysed
synthesis of benzyl esters of fatty acids. Biochem. Eng. J. 2002, 12,
131–141.
(30) Takahashi, K.; Yoshimoto, T.; Ajima, A.; Tamaura, Y.; Inada, Y.
Modified lipoprotein lipase catalyzes ester synthesis in benzene. Sub-
strate specificity. Enzyme 1984, 32, 235–240.
(31) Gaꢀzꢁak, R.; Svobodovꢁa, A.; Psotovꢁa, J.; Sedmera, P.; Pꢀrikrylovꢁa,
V.; Walterovꢁa, D.; Kꢀren, V. Oxidised derivatives of silybin and their
antiradical and antioxidant activity. Bioorg. Med. Chem. 2004, 12,
5677–5687.
(32) Lee, D. Y. W.; Zhang, X.; Ji, X. S. Preparation of tritium-labeled
silybin-A protectant for common liver diseases. J. Labelled Compd.
Radiopharm. 2006, 49, 1125–1130.
(33) Aranda, E.; Owen, G. I. A semi-quantitative assay to screen for
angiogenic compounds and compounds with angiogenic potential using
the EA.hy926 endothelial cell line. Biol. Res. 2009, 42, 377–389.
(34) Dehmlow, C.; Murawski, N.; de Groot, H. Scavenging of
reactive oxygen species and inhibition of arachidonic acid metabolism
by silibinin in human cells. Life Sci. 1996, 58, 1591–1600.
(35) Bergmeyer, H. U.; Bernt, E. Lactate Dehydrogenase: UV-Assay
with Pyruvate and NADH; Academic Press: New York and London,
1974.
(36) Sieuwerts, A. M.; Klijn, J. G. M.; Peters, H. A.; Foekens, J. A.
The MTT tetrazolium salt assay scrutinized. How to use this assay
reliably to measure metabolic-activity of cell-cultures in-vitro for the
assessment of growth-characteristics, IC50-values and cell-survival. Eur.
J. Clin. Chem. Clin. Biochem. 1995, 33, 813–823.
(17) Plíꢀskovꢁa, M.; Vondrꢁaꢀcek, J.; Kꢀren, V.; Gaꢀzꢁak, R.; Sedmera, P.;
ꢀ
Walterovꢁa, D.; Psotovꢁa, J.; Simꢁanek, V.; Machala, M. Effects of
silymarin flavonolignans and synthetic silybin derivatives on estrogen
and aryl hydrocarbon receptor activation. Toxicology 2005, 215,
80–89.
(18) Davis-Searles, P. R.; Nakanishi, Y.; Kim, N.-Ch.; Graf, T. N.;
Oberlies, N. H.; Wani, M. C.; Wall, M. E.; Agarwal, R.; Kroll, D. J. Milk
thistle and prostate cancer: differential effects of pure flavonolignans
from Silybum marianum on antiproliferative end points in human
prostate carcinoma cells. Cancer Res. 2005, 65, 4448–4457.
(19) Deep, G.; Oberlies, N. H.; Kroll, D. J.; Agarwal, R. Identifying
the differential effects of silymarin constituents on cell growth and cell
cycle regulatory molecules in human prostate cancer cells. Int. J. Cancer
2008, 123, 41–50.
(37) Martinez-Poveda, B.; Quesada, A. R.; Medina, M. A. Hypericin
in the dark inhibits key steps of angiogenesis in vitro. Eur. J. Pharmacol.
2005, 516, 97–103.
(20) Kondo, T.; Ohta, T.; Igura, K.; Hara, Y.; Kaji, K. Tea catechins
inhibit angiogenesis in vitro, measured by human endothelial cell
growth, migration and tube formation, through inhibition of VEGF
receptor binding. Cancer Lett. 2002, 180, 139–144.
(21) Dꢀzubꢁak, P.; Hajdꢁuch, M.; Gaꢀzꢁak, R.; Walterovꢁa, D.; Svobodovꢁa,
A.; Psotovꢁa, J.; Sedmera, P.; Kꢀren, V. New derivatives of silybin and 2,3-
dehydrosilybin and their cytotoxic and P-glycoprotein modulatory
activity. Bioorg. Med. Chem. 2006, 14, 3793–3810.
7407
dx.doi.org/10.1021/jm201034h |J. Med. Chem. 2011, 54, 7397–7407