Oxidised derivatives of silybin and their antiradical and antioxidant activity

Article abstract of DOI:10.1016/j.bmc.2004.07.064

Carboxylic acids derived from silybin (1) and 2,3-dehydrosilybin (2) with improved water solubility were prepared by selective oxidation of parent compounds and a new inexpensive method for preparation of 2,3-dehydrosilybin from silybin was developed and

Full text of DOI:10.1016/j.bmc.2004.07.064

Bioorganic& Medicinal Chemistry 12 (2004) 5677–5687
Oxidised derivatives of silybin and their antiradical and
antioxidant activity
a,b
Radek Gazˇak, Alena Svobodova, Jitka Psotova, Petr Sedmera, Vera Prikrylova,
b
b
a
a
´
´
Daniela Walterova and Vladimır Kren
´
ˇ
ˇ
´
b
a,
*
´
´
ˇ
a
´ ˇ ´
Institute of Microbiology, Academy of Sciences of the Czech Republic, Laboratory of Biotransformation, Vıdenska 1083,
CZ-142 20 Prague, Czech Republic
Institute of Medical Chemistry and Biochemistry, Faculty of Medicine, Palacky University, Hnevotınska 3,
CZ-775 15 Olomouc, Czech Republic
b
´
ˇ
´
´
Received 13 April 2004; accepted 27 July 2004
Available online 3 September 2004
Abstract—Carboxylicacids derived from silybin ( 1) and 2,3-dehydrosilybin (2) with improved water solubility were prepared by
selective oxidation of parent compounds and a new inexpensive method for preparation of 2,3-dehydrosilybin from silybin was
developed and optimised. The antioxidative properties of the above-mentioned compounds and of side product 3a from oxidation
of compound 1 were determined by cyclic voltammetry, free radical scavenging (DPPH, superoxide) assays, and by inhibition of in
vitro generated liver microsomal lipid peroxidation. Dehydrogenation at C(2)–C(3) in flavonolignans (silybin vs 2,3-dehydrosilybin;
silybinic acid vs 2,3-dehydrosilybinic acid) strongly improved antioxidative properties (analogously as in flavonoids taxifolin vs
quercetin). Thus, in antioxidative properties, dehydrosilybin was superior to silybin by one order, but its water solubility is too
low for application in aqueous milieu. On the other hand, 2,3-dehydrosilybinic acid is a fairly soluble derivative with antilipoper-
oxidation and antiradical activities better than that of silybin.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
mechanisms operating at various cell levels. Silybin acts
as a radical scavenger3 and chain breaking antilipoper-
oxidant.4 Other important antioxidative effects of 1 are
due to its influence on the superoxide dismutase5 and
the enzyme system associated with glutathione.4 Re-
cently, low density lipoprotein (LDL) antioxidant activ-
ity of 1 has been reported,6 demonstrating thus its
antiatherosclerotic effects.7 Cell regenerating activity is
associated with its ability to activate the proteosynthesis
by DNA-dependent RNA-polymerase I stimulation.8
Flavonolignan silybin (1), isolated from seeds of the
milk thistle (Silybum marianum), is an active component
in a number of phytopreparations, for example, Silyma-
rin-Forteꢁ (Natur-Produkt, CZ), Legalonꢁ (Madaus,
D), widely used in human therapy for liver function
improvement and as a protectant against a number of
hepatotoxins (CCl4, galactosamine, tert-butylhydroper-
oxide, phalloidine, a-amanitine).1 Natural 1 consists of
two diastereomers, silybin A (3-(R),5,7-trihydroxy-2-
(R)-[3-(R)-(4-hydroxy-3-methoxyphenyl)-2-(R)-(hydroxy-
methyl)-2,3-dihydro-1,4-benzodioxin-6-yl]chroman-4-
one) and silybin B (3-(R)-5,7-trihydroxy-2-(R)-[3-(S)-(4-
hydroxy-3-methoxyphenyl)-2-(S)-(hydroxymethyl)-2,3-
dihydro-1,4-benzodioxin-6-yl]-chroman-4-one)2––in the
ratio ca. 45:55. Cytoprotectivity of 1 consists in several
The bioavailability and therapeutic efficiency of silybin
is rather limited by its very low water solubility
(430mg/L). The solubility was improved by the prepara-
tion of silybin 3,23-O-bis-hemisuccinate9 that enabled
intravenous application of silybin (Legalon-SIL) for
the treatment of acute liver intoxication by mycotoxins.
Glycosylation was demonstrated to be another way of
improving silybin solubility.2,10
Keywords: Silymarin; Flavonoid; Flavonolignan; Antioxidant; Radical
scavenger.
All commercial preparations of silybin suffer from spon-
taneous oxidation. The raw material for silybin prepara-
tion, for example, complex extract from seeds of
*
Corresponding author. Tel.: +420 296 442 510; fax: +420 296 442
509; e-mail: kren@biomed.cas.cz
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.07.064

´
R. Gazˇak et al. / Bioorg. Med. Chem. 12 (2004) 5677–5687
5678
S. marianum denoted as silymarin, contains some oxida-
tion products, which were practically neglected in the
studies of the silybin (silymarin) biological activity.11
2,3-Dehydrosilybin (2), (3,5,7-trihydroxy-2-[3-(4-hydr-
oxy-3-methoxyphenyl)-2-(hydroxymethyl)-2,3-dihydro-
1,4-benzodioxin-6-yl]-4H-chromen-4-one), was found as
a common component in the extracts from seeds of
S. marianum subsp. anatolicum12 but according to our
(unpublished) experience it is present in almost all sily-
marin and silybin preparations. This compound (and
probably other derivatives) causes the characteristic yel-
low colour of silybin preparations, whereas pure silybin
is practically colourless. 2,3-Dehydrosilybin (2) may be
also one of the products of in vivo silybin oxidation dur-
ing its antioxidative action.
R1 R2 R3 R4 R5
16
B
5
1
4
5
6
H
H
H
H
H
H
H
H
CH2OH
R
O
D
10
11
CH2OTBDMS
19
20
3
17
OCH3
7
2
O
C
R O
O
14
Ac Ac Ac Ac CH2OTBDMS
Ac Ac Ac Ac CH2OH
E
A
4
OR
1
3
OR
5
10 Ac Ac Ac Ac COOH
2
O
OR
11
H
H
H
H
COOH
23
16
1
O
10
11
CH2OH
O
D
HO
7
OH
3
B
A
C
19
17
OCH3
OH
O
14
2
E
4
5
O
HO
20
3a
R1 R2 R3 R4 R5
5
R
O
O
2
7
8
9
H
H
H
H
H
H
H
H
H
H
H
CH2OH
CH2OTBDMS
H
The preparation of 2 from silybin was accomplished by
H2O2 oxidation in NaHCO3 solution13 or alternatively
by iodine oxidation in glacial acetic acid.14 Its activity
against the toxins of Amanita phalloides is considerably
lower compared to silybin13 and this was probably the
reason why this compound was somehow ignored in fur-
ther biological studies. However, in our preliminary
experiments 2 was better antioxidant than silybin. This
compound has positive effect on some skin diseases as,
for example, psoriasis and atopical dermatitis (our
unpublished results). C-Isoprenylated or geranylated
derivates of 2 also constitute potentially effective modu-
lators of P-glycoprotein.15
3
OCH3
O
R O
Ac
Ac
Ac Ac CH2OTBDMS
Ac Ac CH2OH
4
OR
1
OR
2
O
12 Ac
Ac Ac COOH
OR
13
H
H
H
COOH
Chart 1.
2. Results and discussion
2.1. Chemistry
2.1.1. Preparation of 3,5,7-trihydroxy-2-[3-(4-hydroxy-3-
methoxyphenyl)-2-(hydroxymethyl)-2,3-dihydro-1,4-ben-
zodioxin-6-yl]-4H-chromen-4-one (2,3-dehydrosilybin, 2).
Refluxing of silychristin (a flavonolignan from silymarin
group containing the same benzopyran-4-on moiety as
silybin) in dry pyridine (116ꢂC) with access to air leads
to 2,3-dehydrosilychristin almost quantitatively.16 How-
ever, silybin polymerises under the same conditions and
forms hemiacetal 3a. The decrease in the reaction tem-
perature to 80–90ꢂC significantly reduced formation of
these by-products (see Scheme 1). Use of pure oxygen
did not give better results than the use of air.
An important side product arising from oxidation of
silybin13 is a ꢀdiketoneꢁ 3-[3-(4-hydroxy-3-methoxy-phen-
yl)-2-hydroxymethyl-2,3-dihydro-benzo[1,4]dioxin-6-yl]-
1-(2,4,6-trihydroxy-phenyl)-propane-1,2-dione (3), which
is also a plausible product of silybin oxidation in vivo
and a contaminant of silybin preparations (Fig. 1). This
compound can form under specific conditions an intra-
molecular hemiacetal (3a) (2,4,6-trihydroxy-2-[3-(4-hyd-
roxy-3-methoxyphenyl)-2-(hydroxymethyl)-2,3-dihydro-
1,4-benzodioxin-6-yl]-1-benzofuran-3(2H)-one), which
was isolated and characterised in this study for the first
time.
The reaction of silybin with aqueous solution of N-
methyl-D-glucamine in an inert atmosphere yields dike-
tone 3, however, under access of air also 2 is formed.13
Weak aprotic base such as pyridine probably catalyses
the formation of thermodynamically stabilised hemiac-
Another, so far unknown, oxidation products of both 1
and 2, e.g., silybinic( 11) and 2,3-dehydrosilybinic( 13)
acids are presented in this paper. It is expected that these
compounds bearing easily ionisable carboxy functional-
ity will be more hydrophilicwhile maintaining beneficial
biological effects of their precursors (1, 2). All the new
compounds prepared in this study will also help to
understand molecular mechanisms of antiradical and
antioxidative activity of silybin and further optimise its
applications (for the structures see Chart 1).
1
etal 3a. Comparison of H NMR spectrum of the com-
pound 3a with that of silybin (1) showed that the AB
system of vicinal protons H-2 and H-3 was replaced
by another one (in fact two, presumably stemming from
silybin A and B, ratio ca. 45:55) representing geminal
protons (2J = 13.7 or 14.2Hz) coupled to the same car-
bon resonating at 40.49ppm. This methylene group is
located at the benzylic position to the ring B, as evident
from long-range interproton couplings to H-13 and H-
15, as well as from heteronuclear couplings of these pro-
tons to C-13, C-14 and C-15. The H-2 protons are also
coupled to carbons resonating at 192.7 (C-4) and
105.4ppm (assigned to C-3). Therefore, the C-ring is
five-membered and contains a hemiacetal group (3a).
OH
O
O
O
CH2OH
HO
O
OCH3
OH
OH
2.1.2. Preparation of the carboxy derivatives of 1 and 2.
The silylation of 1 or 2 by tert-butyldimethylsilyl chlo-
Figure 1. ꢀa-Diketoneꢁ 3––degradation product of silybin in alkaline
aqueous solutions.

´
R. Gazˇak et al. / Bioorg. Med. Chem. 12 (2004) 5677–5687
5679
O
CH2OH
OCH3
OH
HO
O
O
23
16
B
O
10
11
O
D
CH2OH
19
17
OCH3
OH
OH
HO
7
2
3
14
O
O
a
OH
E
A
C
2 (51 %)
20
5
OH
+
OH
O
1
23
10
16
1
O
CH2OH
HO
7
O
OH
3
17
19
OCH3
4
14
11
O
5
2
OH
O
20
OH
3a (5- 10 %)
Scheme 1. Reagents and conditions: (a) pyridine, air, 95ꢂC, 100h.
CH2OR
CH2OH
O
O
O
O
OCH3
OH
OCH3
OH
HO
HO
O
O
O
a
OH
OH
4
R = TBDMS
OH
OH
O
O
1
CH2OR
b
OCH3
AcO
O
O
OAc
OAc
c
OAc O
5 R =T BDMS
6 R =H
Scheme 2. Reagents and conditions: (a) TBDMSCl, pyridine, AgNO3 (cat.), 40ꢂC, 2h; (b) Ac2O, pyridine, rt, 24h; (c) BF3ÆEt2O, CHCl3, rt, 12h.
17
ride in pyridine catalysed by AgNO3 yielded 23-O-
TBDMS ethers 4 or 7 (67% and 60%, respectively).
The analogous alkylation of 1 by triphenylmethyl chlo-
ride (or bromide) afforded only traces of the correspond-
ing trityl ether. The ethers 4 and 7 were acetylated by a
standard Ac2O/pyridine procedure yielding correspond-
ing acetates 5 or 8. The TBDMS group was removed
with boron trifluoride etherate in CHCl3 giving respec-
tive alcohols 6 or 9 (Scheme 2).18 The use of Bu4NF in
THF failed in this case.
tions.2,20 We have chosen DMSO-d6 as a solvent in or-
der to use the OH resonances for the experimental
signal assignment (Fig. 2). The chemical shift differences
were small in the carbon dimension so that both diaster-
eomers mostly gave common crosspeaks. However, for
some atoms different signals were observed (Tables 1
and 2) but were not assigned to the individual silybins.
On the contrary, only one set of NMR signals was ob-
served in the 2,3-dehydrosilybin series (Tables 3 and 4).
The structures of compounds 4 and 7 were confirmed by
NMR. The position of TBDMS group was determined
indirectly, proving the presence of hydroxyls by HMBC.
Similarly, with acetyl derivatives 5, 6, 8 and 9, the acet-
ylated positions were inferred from gHMQC (relying
The oxidation of the alcohols 6 and 9 to the correspond-
ing acids 10 and 12 was carried out using solution of
H5IO6/CrO3 in wet CH3CN (0.75% v/v water) (Scheme
3).19
4
upon the J couplings21,22 between the acetyl protons
The NMR data of individual silybin A and B were
already published in CD3OD and CD3COCD3 solu-
and carbons at the site of acetylation in the case of enol
acetates). Because of signal doubling in the H NMR
spectra of 6 and 10, the actual number of acetyl groups
1
COOR
O
O
OCH3
OR
a
RO
O
O
6
CH2OH
O
O
HO
OCH3
OH
O
O
OR
OR
b
10 R = Ac
11 R = H
OH
OH
Scheme 3. Reagents and conditions: (a) H5IO6/CrO3, CH3CN, 0ꢂC,
1.5h; (b) K2CO3, MeOH, H2O, rt, 24h.
Figure 2. Diagnosticheteronulcear ocuplings in
HMBC (DMSO-d6).
2 observable by

´
R. Gazˇak et al. / Bioorg. Med. Chem. 12 (2004) 5677–5687
5680
Table 1. 1H NMR data (399.89MHz, 30ꢂC) of silybin derivatives
Proton
2
4a
6b
10b
11a
5.081 d (11.3)
5.359 d (12.1)
5.368 d (12.1)
5.682 d (12.1)
5.706 d (12.1)
6.593 d (2.2)
5.342 d (12.2)
5.349 d (12.2)
5.676 d (12.2)
5.679 d (12.2)
6.594 d (2.2)
6.596 d (2.2)
6.785 d (2.2)
6.791 d (2.2)
5.308 d (5.3)
5.315 d (5.3)
4.836 d (5.3)
4.841 d (5.3)
7.098 d (2.0)
7.142 d (2.0)
6.988 dd (8.2, 2.0)
7.000 dd (8.0, 2.0)
7.022 d (8.2)
5.066 d (11.2)
5.068 d (11.2)
4.597 d (11.2)
3
4.592 dd (11.3, 6.2)
4.603 dd (11.3, 6.2)
5.199 d (2.1)
6
5.919 d (2.1)
5.887 d (2.1)
6.596 d (2.2)
6.784 d (2.2)
6.792 d (2.2)
8
5.871 d (2.1)
5.876 d (2.1)
10
4.230 ddd (8.0, 3.7, 2.4)
4.240 ddd (8.0, 3.7, 2.4)
4.881 d (8.0)
4.037 ddd (8.2, 3.6, 2.4)
4.053 ddd (8.2, 3.6, 2.4)
5.061 d (8.2)
4.975 d (4.7)
4.986 d (4.7)
5.285 d (4.7)
5.289 d (4.7)
7.089 d (2.2)
11
5.065 d (8.2)
7.126 d (2.0)
13
7.082 d (2.0)
7.093 d (2.0)
15
7.016 dd (8.2, 2.0)
7.025 dd (8.2, 2.0)
6.957 d (8.2)
6.980 dd (8.2, 2.0)
6.894 dd (8.3, 2.0)
7.016 d (8.2)
6.999 dd (8.3, 2.2)
7.005 dd (8.3, 2.2)
6.938 d (8.3)
16
6.962 d (8.2)
7.009 d (1.9)
7.020 d (8.2)
7.061 d (1.7)
6.941 d (8.3)
7.007 d (2.0)
18
6.987 d (2.0)
6.993 d (2.0)
7.038 d (8.1)
7.043 d (8.1)
6.953 dd (8.1, 2.0)
6.969 dd (8.1, 2.0)
––
7.075 d (1.7)
7.100 d (8.0)
21
6.814 d (8.2)
6.735 d (8.1)
6.738 d (8.1)
6.848 dd (8.1, 2.0)
22
6.865 dd (8.2, 1.9)
3.521 dd (12.0, 3.7)
7.047 dd (8.0, 1.7)
7.052 dd (8.0, 1.7)
3.870 dd (12.6, 2.4)
23dc
23uc
OMe
––
––
––
––
3.775 dd (12.0, 2.4)
3.777 dd (12.0, 2.4)
3.776 s
3.599 dd (12.6, 3.6)
––
––
––
3.799 s
3.803 s
3.871 s
3.879 s
3.726 s
3.729 s
3.779 s
Additional signals––4: 0.009 (3H, s, Si–Me), 0.018 (3H, s, Si–Me), 0.857 (9H, s, Me3C), 5.786 (1H, d, J = 6.2, 3-OH), 9.138 (1H, s, 20-OH), 10.832
(1H, s, 7-OH), 11.877 (1H, s, 5-OH); 6: 2.051 s, 2.055 s, 2.299 s, 2.331 s, 2.377 s, 2.379 s, (4 · Ac); 10: 1.992 s, 1.998 s (3-Ac), 2.296 s (7-Ac), 2.304 s
(20-Ac), 2.372 s, 2.375 s (5-Ac), 5.921 (1H, br s, COOH); 11: 5.796 (1H, d, J = 4.4, 3-OH), 9.094 (1H, s, 20-OH), 11.138 (1H, s, 7-OH), 11.855, 11.889
(1H, s, 5-OH).
a In DMSO-d6.
b In CDCl3.
c d––Downfield, u––upfield.
was deduced from the number of crosspeaks between
acetyls and carbonyls in HMBC. The formation of acids
10–13 was confirmed by the transformation of the par-
tial structure –CH(O-)CH(O-)CH2OH to the –CH(O-)
CH(O-)COOH moiety, also corroborated by heteronu-
clear couplings (Fig. 3).
in a comparison with the parent compounds 1, 2 and
structurally related flavonoids taxifolin (14) and querce-
tin (15) (Fig. 5, Table 5).
The reducing potency of 1, 2, 3a, 11 and 13 has been
evaluated using cyclic voltammetry (CV) and character-
ised as the anodicpeak potential ( Epa) values deter-
mined from cyclic voltammograms (Table 5). The Epa
values, although obtained at the specific in vitro condi-
tions, are considered to be able to predict radical scav-
enging properties and antioxidant behaviour in
biological systems.26
However, whereas both in silybin and dehydrosilybin
series the value of J10,11 is 7.8–8.2Hz, it amounts 5.3,
4.7, 4.3 and 3.8Hz in the acids 10–13. Neolignans caf-
feicins A–D having a cis-configuration at the dioxane
ring, used as models, exhibit J = 3 and 4Hz.23 Since
the inversion of configuration at C-11 is unlikely for
our reaction sequence, we explain the observed values
by an equilibrium between two conformations (Fig. 4)
shifted towards the right. Similar conformational equi-
librium is well known with C-ring of flavans24 or
dihydroisoflavanols.25
In voltammograms of the parent compound 1 and of its
derivatives 3a, 11 and 13 one anodicwave was detected
at peak potentials ranging from 524 to 564mV. The Epa
values observed qualify these substances as rather poor
antioxidants as compared, for example, with antioxidant
vitamins (ascorbic acid, Epa = 370mV),26 phenolicacids
(caffeic acid, Epa = 100mV)27 or flavonoids 14, 15 (Table
5), and do not suggest significant change of antioxidant
potency in derivatives 3a, 11 and 13 in comparison with
1. Only compound 2 showed two anodicwaves ( Fig. 6)
that indicates the presence of two sites with different
2.2. Antioxidant activity
The new compounds 3a, 11 and 13 were tested for their
electron-donating potency and for their free radical
scavenging ability and inhibition of lipid peroxidation

´
R. Gazˇak et al. / Bioorg. Med. Chem. 12 (2004) 5677–5687
5681
Table 2. 13C NMR data (100.55MHz, 30ꢂC) of silybin derivatives
The wave at lower potential characterises stronger
reducing properties of 2 associated most likely with
the unsaturated hydroxy-chromanone moiety (in the
ring C) of flavonolignan molecule. This observation is
consistent with conclusions of structure–antioxidant
activity relationship studies of flavonoids, which have
demonstrated that the 3-OH group attached to the
2,3-double bond in conjugation with the 4-oxo function
in the C ring is very important for effective radical scav-
enging.29 This is also clearly shown by the Epa values of
14 (containing a structural motif of 1) and 15 (contain-
ing a structural motif of 2) demonstrating stronger anti-
oxidant activity of 2. However, substantially stronger
antioxidant capacity of quercetin (15) and taxifolin
(14) compared to silybin derivatives are mostly due to
the activity of catechol portion of their molecules be-
cause the intramolecular hydrogen bond in the semiqui-
none radical produced after the H-atom transfer to the
abstracting radical (e.g., alkyl peroxyls) is much stronger
than that in the parent phenol.30 Surprisingly, the wave
at lower potential was not detected in the voltammo-
gram of acid 13 (Fig. 6), which may be explained by
the presence of carboxylic moiety––see hereunder.
Carbon
2
4a
82.53
6b
81.03
10b
80.85
11a
82.53
82.57
71.44
71.51
81.10
73.23
82.55
71.41
3
73.21
73.26
4
197.68
185.24
185.25
185.27
110.55
110.57
151.33
151.39
110.99
197.69
100.44
163.31
96.14
4a
5
100.50
163.33
96.10
110.63
110.66
151.48
151.60
111.19
111.21
156.39
6
7
166.85
95.05
156.34
156.35
108.98
109.00
162.45
162.47
75.76
167.00
95.12
8
108.92
8a
10
11
12a
13
14
15
16
16a
17
18
19
20
21
22
23
19-OMe
162.48
77.72
162.52
162.56
78.32
162.47
75.77
78.36
75.98
76.03
75.85
75.27
75.99
75.09
75.32
142.72
143.24
143.27
116.55
116.71
130.11
130.15
121.26
121.47
116.21
116.28
143.64
143.67
127.13
143.72
143.81
116.44
142.23
142.25
116.76
116.86
130.45
130.47
121.39
121.47
116.29
116.31
142.72
In fact, the poor superoxide radical scavenging capacity
of silybin (1) was previously reported.3 Our results have
confirmed weaker ability of 1 to scavenge both chemi-
cally generated superoxide and a stable 1,1-diphenyl-2-
picrylhydrazyl (DPPH) radical (Table 5). Oxidation of
C-23 of silybin (1) to carboxy derivative 11 further re-
duced radical scavenging properties. However, signifi-
cant improvement of these properties was observed in
both 2,3-dehydroderivatives 2 and 13. Consistently, oxi-
dation at C-23 led again to the scavenging activity sup-
pression. This phenomenon was recently explained by
Foti et al.31 who demonstrated by kineticmeasurements
that the reaction between phenols and DPPHÅ occurs in
alcohols by electron transfer from the phenoxide anions
to DPPHÅ. The presence of carboxylic acid groups in the
molecules or even of adventitious acids (in the solvents)
reduces the quantity of phenoxide anions and thus the
reactivity toward the radical. Hemiacetal 3a exhibited
better antioxidant properties than 1 itself.
116.43
116.60
129.09
129.14
121.37
121.41
117.31
117.41
142.48
142.56
133.93
133.94
111.21
111.23
151.36
126.48
126.59
120.75
120.99
117.17
117.23
144.22
144.27
134.72
127.29
111.66
147.51
146.85
115.26
119.97
169.05
55.67
111.88
111.93
147.66
147.68
147.18
111.26
111.38
151.48
140.44
140.21
140.30
123.07
115.37
120.63
62.23
123.14
123.18
119.82
119.87
61.52
119.34
119.51
170.26
170.28
55.93
The results with radical scavenging capacity of various
mixtures of 1 and 2 (Fig. 7) show that possible contam-
ination of 1 by its oxidative product 2 strongly improve
radical scavenging properties of silybin preparations.
The mixtures containing P50% of 2 reached the IC50
values corresponding to those of pure 2. Already the
presence of 10% of 2 in 1 increases the antiradical–scav-
enging activity more than 3times compared to the pure
silybin. This clearly demonstrates synergistic effect of
both compounds. This finding is of a great importance
for the formulation and optimisation of mixed prepara-
tion based on both 1 and 2. Moreover, it helps to pro-
duce cheaper preparations of 2, where no complete
conversion is necessary and it also helps to avoid labori-
ous purification steps for removing of traces of unre-
acted 1 from the final preparations. These results also
demonstrate why some complex silybin-based prepara-
tions like silymarin (which is a mere mixture of flavo-
noids from S. marianum seeds with standardised
55.70
56.06
56.11
55.96
Additional signals––4: À5.45, À5.30 (2 · Si–Me), 17.98 (C–Si), 25.76
(Me3C); 6: 20.41, 20.63, 20.91, 21.13 (4 · Ac), 167.75, 168.76, 169.05
(2C), (4 · CO); 10: 20.27, 20.29 (3-Ac), 20.62 (20-Ac), 20.91 (5-Ac),
21.13 (7-Ac), 167.88 (7-CO), 169.03, 169.06 (20-CO), 169.21, 169.22 (5-
CO), 169.24, 169.27 (3-CO).
a In DMSO-d6.
b In CDCl3.
reducing power in the molecule. The wave at higher
peak potential, observed also in voltammograms of
other flavonolignan derivatives, most probably reflects
the electron donating ability of o-methoxy-phenolic
moiety at the E ring, inferred from the pulse radiolysis
as an exclusive target for one electron oxidation of 1.28

´
R. Gazˇak et al. / Bioorg. Med. Chem. 12 (2004) 5677–5687
5682
Table 3. 1H NMR data (399.89MHz, DMSO-d6, 30ꢂC) of dehydrosilybin derivatives
Proton
2
7
9
12
13
6
6.194 d (2.0)
6.459 d (2.0)
6.193 d (2.1)
6.454 d (2.1)
4.346 ddd (7.9, 3.8, 2.5)
4.930 d (7.9)
7.773 d (2.2)
7.752 dd (8.3, 2.2)
7.125 dd (8.6, 0.6)
7.039 d (1.9)
6.823 d (8.1)
6.884 dd (8.1, 1.9)
3.793 dd (12.0, 2.5)
3.543 dd (12.0, 3.8)
3.787 s
6.727 d (2.0)
7.135 d (2.0)
4.413 ddd (7.8, 4.4, 2.6)
5.154d (7.8)
7.406 d (2.0)
7.146 d (2.0)
5.536 d (3.8)
5.660 d (3.8)
7.619 d (2.1)
7.513 dd (8.7, 2.1)
7.170 d (8.7)
7.279 d (2.0)
7.086 d (8.2)
7.006 dd (8.2, 2.0)
––
6.265 d (2.1)
6.530 d (2.1)
5.306 d (4.3)
5.425 d (4.3)
7.490 d (2.2)
7.437 dd (8.7, 2.2)
7.137 d (8.7)
7.054 d (2.2)
6.748 d (8.2)
6.848 dd (8.2, 2.2)
––
8
10
11
13
15
16
18
21
22
23
4.275 ddd (7.9, 4.5, 2.5)
4.967 d (7.9)
7.669 d (2.2)
7.581 d (2.2)
7.532 dd (8.6, 2.2)
7.197 d (8.6)
7.284 d (1.8)
7.155 d (8.1)
7.090 dd (8.1, 1.8)
3.636 ddd (12.5, 4.8, 2.6)
3.407 ddd (12.5, 5.8, 4.4)
3.802 s
7.756 dd (9.0, 2.2)
7.120 d (9.0)
7.045 d (2.0)
6.853 d (8.1)
6.891 dd (8.1, 2.0)
3.573 ddd (12.3, 5.0, 2.6)
3.373 ddd (12.3, 5.1, 4.5)
3.792 s
––
3.758 s
––
3.740 s
19-OMe
3-OH
5-OH
9.532 s
12.404 s
9.537 s
12.402 s
––
12.130 s
––
12.111 s
not observed
12.116 s
11.080 s
7-OH
20-OH
23-OH
10.788 s
9.130 s
4.967 dd (5.1, 5.0)
10.800 s
9.159 s
––
––
––
––
––
9.142 s
––
5.095 dd (5.8, 4.8)
––
Additional signals––7: 0.009 (3H, s, Si–Me), 0.014 (3H, s, Si–Me), 0.851 (9H, s, Me3C); 9: 2.271 (3H, s, 20-Ac), 2.308 (3H, s, 7-Ac), 2.361 (3H, s,
3-Ac); 12: 2.239 (3H, s, 20-Ac), 2.312 (3H, s, 7-Ac), 2.358 (3H, s, 3-Ac).
Table 4. 13C NMR data (100.55MHz, DMSO-d6, 30ꢂC) of dehydrosilybin derivatives
Carbon
2
7
9
12
13
2
145.80
136.33
176.02
103.12
160.72
98.28
145.77
136.37
176.04
103.14
160.74
98.29
156.33
130.83
175.58
107.98
160.32
105.50
156.20
101.98
155.56
78.37
156.12
130.95
175.58
108.00
160.32
105.52
156.22
102.03
155.57
74.73
155.10
167.94
174.97
103.62
161.07
99.24
3
4
4a
5
6
7
164.07
93.58
164.09
93.59
164.86
94.42
8
8a
10
11
12a
13
14
15
16
16a
17
18
19
20
21
22
23
OMe
156.26
78.55
75.89
156.27
78.09
76.03
156.74
75.23
74.87
75.57
74.38
143.40
116.21
123.77
121.27
116.83
145.05
127.27
111.85
147.69
147.14
115.38
120.59
60.12
143.39
116.30
123.86
121.41
116.76
145.01
126.90
111.94
147.70
147.25
115.39
120.69
62.17
143.62
126.82
121.40
122.27
117.57
146.79
135.11
112.28
151.01
139.71
122.91
120.10
59.90
142.39
117.12
122.11
122.65
117.64
145.56
134.88
111.77
150.95
139.44
122.89
119.06
168.68
55.95
142.68
116.88
130.39
122.20
117.40
145.36
126.57
111.68
147.65
147.07
115.35
119.98
168.77
55.77
55.77
55.71
55.96
Additional signals––7: À5.46 (Si–Me), À5.36 (Si–Me), 17.97 (C–Si), 25.74 (CMe3); 9: 20.24 (3-Ac), 20.39 (20-Ac), 20.90 (7-Ac), 167.79 (3-CO), 168.34
(7-CO), 168.44 (20-CO); 12: 20.21 (3-Ac), 20.36 (20-Ac), 20.90 (7-Ac), 167.77 (3-CO), 168.34 (7-CO), 168.40 (20-CO).
O
O
COOH
H
C
C
H
OCH3
OH
HOOC
H
H
O
Ar
O
O
O
H
COOH
Figure 3. Diagnostic heteronuclear couplings in compounds 10–13.
Ar
H
silybin content) give often controversial, unreproducible
results,11 which may be caused by varying content of the
oxidation products of original flavonoids, namely 2.
Figure 4. The equilibrium between diaxial and diequatorial substitu-
ents of the D-ring.

´
R. Gazˇak et al. / Bioorg. Med. Chem. 12 (2004) 5677–5687
5683
OH
OH
OH
I (µA)
0.4
1
2
13
HO
O
HO
O
O
OH
OH
OH
0.0
OH
O
OH
15
14
-0.4
-0.8
-1.2
-1.6
Figure 5. Flavonoids taxifolin (14) and quercetin (15).
Reaction mixtures of 1 and 2 (and its mixtures) with
DPPHÅ were analysed by HPLC and MALDI-MS, to
disclose the reaction products of the both radical scav-
engers. Silybin upon reaction with DPPHÅ gives very
complex reaction mixture composed mostly of insoluble
brownish precipitates, which are obviously polymers
(radical-initiated polymerisation). Dehydrosilybin reacts
with DPPHÅ faster than 1 yielding dimer (according to
MS analysis) of unknown structure and some other non-
identified products. All substances tested were found to
be efficient inhibitors of ADP/Fe3+/NADPH-induced
lipid peroxidation of rat liver microsomal membranes,
2 being the most effective. Again, the presence of a carb-
oxyl at C-10 of 11 and 13 reduces partly their inhibitory
efficiency. The differences in the antioxidant activity of
the flavonolignans 1, 2, 3a, 11 and 13 and structurally
related flavonoids 14, 15 at the hydrophilic–hydropho-
bic interphase of microsomal membranes are much less
pronounced than in the scavenging activities evaluated
in the hydrophilicenvironment.
0
200
400
600
800
1000
1200
E (mV)
Figure 6. Cyclic voltammograms of silybin (1), 2,3-dehydrosilybin (2)
and 2,3-dehydrosilybinicacid ( 13).
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
To assess the contribution of lipophilicity to the inhibi-
tory effect on lipid peroxidation the partition co-effi-
cients of 1, 2, 3a, 11 and 13 were determined in the
system 1-octanol/water at pH7.4 of the aqueous
phase (Table 6). The observed order of lipophilicity
2 > 1 > 3a > 13 > 11 is not fully related to the antilipop-
eroxidation effect order 2 > 13 > 1 > 3a > 11. This result
indicates also that at hydrophilic–hydrophobic interpha-
ses typical for biological systems structural determinants
are important for antioxidant activity.
0
10
20
30
40
50
2 (%)
60
70
80
90
100
Figure 7. Radical scavenging activity of the mixtures 1 and 2. Data are
expressed as means from three measurements, SD < 0.02.
acids derived from 1 and 2 were prepared for the first
time. 2,3-Dehydrosilybin is more lipophilicand less
water-soluble than silybin, which predetermines it for
an application in lipophilic milieu (cell membrane anti-
oxidant, ointments). However, respective carboxylic
derivative combines its considerably better antioxidative
properties together with improved hydrophilicity.
2.3. Conclusion
New, cheaper and faster method for synthesis of 2,3-
dehydrosilybin (2) was developed and new carboxylic
Table 5. Cyclic voltammetry peak potentials, radical scavenging data and antilipoperoxidant activity of silybin, its derivatives and structural
analogues
CVa Epa (mV)
DPPH IC50 (lM)
O2 IC50 (lM)
LPx IC50 (lM)
ÅÀ
Compound
Silybin (1)
2,3-Dehydrosilybin (2)
524
1745 65
73
55.2 2.8
4.2 0.2
8.2 0.4
52.0 2.6
15.5 0.8
2.4 0.1
0.5 0.03
33.6 1.2
3.6 2.7
47.3 6.7
61.5 2.0
30.8 3.1
16.2 0.2
4.7 1.4
573; 397
544
557
3
ꢀHemiacetalꢁ 3a
646 42
2645 10
934 10
Silybinicacid ( 11)
2,3-Dehydrosilybinicacid ( 13)
Taxifolin (14)
564
258
21
11
6
1
Quercetin (15)
174
a CV––Cyclic voltammetry; Epa (mV)––the anodic peak potential; DPPH––1,1-diphenyl-2-picrylhydrazyl radical scavenging; IC50 (lM)––the con-
centration of the tested compound required to reduce the absorbance of DPPH by 50%; O2ÅÀ––superoxide scavenging––the antiradical activity of
the tested compound is expressed as the concentration required to reduce the absorbance by 50% (IC50); LPx––inhibition of microsomal lipid
peroxidation––the activity was calculated as the concentration of the tested compound inhibiting the colour reaction with thiobarbiturate by 50%
(IC50).

´
R. Gazˇak et al. / Bioorg. Med. Chem. 12 (2004) 5677–5687
5684
Table 6. Octanol/water partition coefficients (logP7.4) of silybin and its
derivatives
lary held at 3.3kV into Finnigan ESI source via a linear
syringe pump at a flow rate of 40lL/min. For high-res-
olution experiments the instrument was tuned to a reso-
lution of about 8000 (10% of valley definition) and the
measurements were carried out by the peak-matching
method using a mixture of polypropylene glycols (aver-
age Mr = 725) as an internal standard.
Compound
LogP7.4
Silybin (1)
2.2726
3.9165
2,3-Dehydrosilybin (2)
ꢀHemiacetalꢁ 3a
Silybinicacid ( 11)
0.51490
À0.88430
0.28510
2,3-Dehydrosilybinicacid ( 13)
Analytical HPLC was carried out on a Spectra Physics
analytical system (San Jose, USA) comprised of an SP
8800 ternary gradient pump, an SP 8880 autosampler
and a Spectra Focus scanning UV/vis detector. A Nucleo-
sil 100–5, C 18 AB column 250 · 4mm (Macherey–
The presence of a 2,3-double bond in the C-ring of mol-
ecule is connected with an increase in scavenging/
antioxidative potency of the compounds. 2,3-Dehydro-
silybin is obviously a superior silybin derivative both
from the point of view of radical scavenging and antilipo-
peroxidant activity. Compound 2 is a 25times better
radical scavenger (DPPHÅ) and a 10times better inhibi-
tor of lipid peroxidation than 1 but it has ca. 100times
higher hydrophobicity. 2,3-Dehydrosilybinic acid (13)
has a 100times higher hydrophilicity compared to 2
but 10times lower antioxidative activity than 2, still
better than 1. Silybinicacid ( 11) has an approximately
10times higher water solubility than 1 but only half of
its antioxidative activity. Due to the absence of catechol
ring flavonolignans 1, 2, 11, 13 are significantly less
potent radical scavengers compared to flavonoids 14,
15. On the other hand, antilipoperoxidant activities of
1 versus 14 and 2 versus 15 are comparable. Obviously,
an interaction with the lipidic phase, which might be
similar in both heterocyclic systems, plays here an
important role.
Nagel) with
a
mobile phase MeOH/H2O/CH3-
COOH = 42:58:0.01, flow rate 0.6mL/min was used at
60ꢂC, UV detection at 275nm.
3.2. Chemistry
3.2.1. 3,5,7-Trihydroxy-2-[3-(4-hydroxy-3-methoxyphen-
yl)-2-(hydroxymethyl)-2,3-dihydro-1,4-benzodioxin-6-yl]-
4H-chromen-4-one (2). Silybin (1, 6g, 12.44mmol) was
dissolved in dry pyridine (400mL) to give 0.031mol/
mL and the mixture was heated to 95ꢂC for 100 h under
stirring with access of air under CaCl2 drying tube
(TLC; chloroform/acetone/formic acid = 9:2:1). Reac-
tion mixture was evaporated in vacuo and the residual
pyridine was removed by co-evaporation with toluene.
Remaining solid was dissolved in ethyl acetate
(500mL), filtered through a silica gel pad (30g), which
was washed with hot acetone (400mL). The collected fil-
trates were evaporated to dryness and dissolved in hot
EtOH. The cooled solution afforded after filtration the
product 2 (3.05g, 51%) as a yellow microcrystalline solid.
Mother liquors afforded after partial evaporation to half
volume the second crop of less pure product (0.74g,
12%). The product was identified by HPLC comparing
with the authenticstandard and its structure was verified
by NMR (Tables 3 and 4) and MS-ESI (m/z): 481
[M+H]+.
3. Experimental
3.1. General methods
Reactions were monitored by TLC on Silica Gel F254
(Merck) and the spots were visualised by UV light and
by charring with 5% H2SO4 in ethanol.
Optical rotation was measured on Perkin–Elmer 141
polarimeter at 24ꢂC.
3.2.2. 2,4,6-Trihydroxy-2-[3-(4-hydroxy-3-methoxyphen-
yl)-2-(hydroxymethyl)-2,3-dihydro-1,4-benzodioxin-6-yl]-
1-benzofuran-3(2H)-one (3a). The mother liquor from
the second crystallisation of 2 was concentrated in vacuo
to the volume of 5mL and then added dropwise to the
excess (50mL) of saturated aqueous solution of NaHCO3.
The precipitate was filtered off and the filtrate was
extracted with ethyl acetate. The organic phase was
dried (Na2SO4). The dry extract (1g) was purified by
flash chromatography (chloroform/acetone/formic acid
5:1:0.1), which yielded 0.12g of title compound (3a) as
a white amorphous solid (12%).
NMR spectra were recorded on a Varian INOVA-400
1
spectrometer (399.89MHz for H, 100.55MHz for 13C)
in CDCl3 or DMSO-d6 (see text) at 30ꢂC. Chemical
shifts were referenced to the residual solvent signal (dH
7.265, dC 77.00; dH 2.50, dC 39.60). Digital resolution
used justified reporting the proton and carbon chemical
shifts to three and two decimal places, respectively. All
2D NMR experiments (HOM2DJ, gCOSY, TOCSY,
HMQC, HMBC) were performed using standard manu-
facturersꢁs software. The sequence for 1D-TOCSY
experiments32 was obtained through Varian User Li-
brary, the sequence gHMBC33,34 was obtained from
Varian Application Laboratory in Darmstadt (D).
1
[a]D +2.5 (c 0.16, acetone). H NMR (DMSO-d6): 2.877
and 2.933 (2H, AB, J = 13.7), 2.868 and 2.941 (2H, AB,
J = 14.2), 3.287 (1H, dd, J = 12.0, 2.1, H-23), 3.289 (1H,
dd, J = 12.0, 2.3, H-23), 3.474 (1H, dd, J = 12.0, 4.4, H-
23), 3.476 (1H, dd, J = 12.0, 4.6, H-23), 3.764 (6H, s,
2 · OMe), 4.055 (1H, ddd, J = 7.7, 4.6, 2.3, H-10),
4.061 (1H, ddd, J = 7.7, 4.4, 2.1, H-10), 4.796 (1H, d,
J = 7.7, H-11), 4.815 (1H, d, J = 7.7, H-11), 5.752 (1H,
d, J = 1.7, H-8), 5.756 (1H, d, J = 1.7, H-8), 5.802
Positive-ion electrospray ionisation (ESI) mass spectra
were recorded on a double-focusing instrument Finni-
gan MAT 95 (Finnigan MAT, Bremen, D) with BE
geometry. Samples dissolved in methanol–water (2:1,
v/v) were continuously infused through a stainless capil-

´
R. Gazˇak et al. / Bioorg. Med. Chem. 12 (2004) 5677–5687
5685
(2H, d, J = 1.7, 2 · H-6), 6.652 (2H, dd, J = 8.2, 2.0,
2 · H-15), 6.691 (2H, d, J = 2.0, 2 · H-13), 6.473 (2H,
d, J = 8.2, 2 · H-16), 6.779 (2H, d, J = 8.1, 2 · H-21),
6.808 (1H, dd, J = 8.1, 1.5, H-22), 6.812 (1H, dd,
J = 8.1, 1.7, H-22), 6.955 (1H, d, J = 1.5, H-18), 6.962
(1H, d, J = 1.7, H-18), 7.447 (2H, s, 2 · @CAOH),
9.107 (2H, s, 2 · @CAOH), 10.326 (1H, s, @CAOH),
11.864 (1H, s, @CAOH). 13C NMR (DMSO-d6): 40.49
(C-2); 55.67, 55.69 (OMe), 60.20 (C-23); 75.65, 75.76
(C-11); 77.98 (C-10), 89.87 (C-8); 95.89, 95.91 (C-6);
101.09 (C-4a); 105.32 (C-3); 111.70 (C-18); 115.36 (C-
21); 115.91 (C-16); 118.73, 118.75 (C-13); 120.47,
120.49 (C-22); 123.35 (C-15); 127.17 (C-14), 127.70 (C-
17); 141.96 (C-16a); 142.81 (C-12a); 146.98 (C-20);
147.65 (C-19); 158.23, 158.32 (C-5); 168.34 (C-7);
171.76 (C-8a); 192.79, 192.80, 192.81 (C-4). MS-ESI
(m/z): 483 [M+H]+.
1.9mmol/L in wet MeCN (0.75% v/v water) to make
114mL (complete dissolution typically required 1–2h).
The H5IO6/CrO3 solution (10mL) was added to a solu-
tion of the alcohol 6 (470mg, 0.722mmol) in wet aceto-
nitrile (5mL, 0.75 v/v% water) during 30min while
maintaining the reaction temperature at 0–5ꢂC. The
reaction was kept at 0ꢂC for 45min, and then it was
quenched by the addition of Na2HPO4 solution (0.5g
in 10mL H2O). The mixture was extracted with ethyl
acetate (2 · 15mL), organiclayer was washed with 1:1
brine/water mixture (2 · 25mL) then with an aqueous
solution of NaHSO3 (1g in 25mL H2O) and finally with
brine (25mL). The organicphase was dried (Na SO4).
2
After evaporation flash chromatography (chloroform/
acetone/formic acid 9:1:0.1) gave 10 (368mg, 77%) as a
white amorphous solid.
[a]D + 38.18 (c 0.22, acetone). MS-ESI (m/z): 665
3.2.3. 23-O-(tert-Butyldimethylsilyl)-silybin (2-(2-{[(tert-
butyldimethylsilyl)oxy]methyl}-3-(4-hydroxy-3-methoxy-
phenyl)-2,3-dihydro-1,4-benzodioxin-6-yl)chroman-4-one)
(4). Silybin (1, 1.50g, 3.11mmol) was dissolved at room
temperature in dry pyridine (20mL). tert-Bu(Me)2SiCl
(0.70g, 4.64mmol) and powdered AgNO3 (0.20g,
1.18mmol) were added and the stirred mixture was
heated under nitrogen at 40ꢂC. The reaction was
quenched after 2h with water (100mL), the mixture
was extracted with ethyl acetate (2 · 100mL) and the or-
[M+H]+. For 1H and 13C NMR data see Tables 1 and 2.
3.2.6. Silybinic acid (3-(4-hydroxy-3-methoxyphenyl)-6-
(3,5,7-trihydroxy-4-oxochroman-2-yl)-2,3-dihydro-1,4-
benzodioxine-2-carboxylic acid) (11). Compound 10
(0.29g, 0.436mmol) and K2CO3 (0.25g, 1.809mmol)
were dissolved in MeOH/water solution (16.5mL, 10:1
v/v). The reaction mixture was stirred 24h at room tem-
perature under argon. The reaction was quenched by an
addition of formicacid (1mL) and after 10min of stir-
ring it was evaporated to dryness. Dry solid afforded
after flash chromatography (chloroform/acetone/formic
acid 4:1:0.05) title compound (11) (150mg, 70%) as a
white amorphous solid.
ganicphase was dried over Na SO4. After evaporation,
2
flash chromatography (petroleum ether/AcOEt 2:1) gave
title compound (4) 1.24g (67%) as a white amorphous
solid.
[a]D À4.33 (c 0.30, CHCl3). MS-ESI (m/z): 597 [M+H]+.
[a]D + 7.5 (c 0.16, MeOH). MS-ESI (m/z): 497 [M+H]+.
HRMS (ESI) (m/z): 519.0921 (calcd for C25H20O11Na
[M+Na]+ 519.0903). For 1H and 13C NMR data see Ta-
bles 1 and 2.
1
For H and 13C NMR data see Tables 1 and 2.
3.2.4. 3,5,7,20-Tetra-O-acetylsilybin (3,5,7-triacetoxy-2-
[3-(4-acetoxy-3-methoxyphenyl)-2-(hydroxymethyl)-2,3-
dihydro-1,4-benzodioxin-6-yl]chroman-4-one) (6). Com-
pound 4 (1.00g, 1.675mmol) was dissolved in the mix-
ture of pyridine (15mL) and acetanhydride (15mL).
The reaction mixture was stirred 24h at room tempera-
ture. The mixture was then poured to saturated solution
of NaHCO3 (150mL), extracted with ethyl acetate
(2 · 100mL), the organicphase was washed with water,
dried over Na2SO4 and evaporated. The residue was dis-
solved in dry CHCl3 (30mL), BF3ÆEt2O (2.5mL, 50%
solution) was added and the mixture was stirred 12h
at room temperature. Then the mixture was poured into
the water (150mL), extracted with CH2Cl2 (2 · 100mL),
the organicphase was washed with water, dried
(Na2SO4) and evaporated. Dry solid afforded after flash
chromatography (petroleum ether/AcOEt 1:1) title com-
pound (6) (535mg, 49%) as a white amorphous solid.
3.2.7.
2-(2-{[(tert-Butyldimethylsilyl)oxy]methyl}-3-(4-
hydroxy-3-methoxyphenyl)-2,3-dihydro-1,4-benzodioxin-
6-yl)-3,5,7-trihydroxy-4H-chromen-4-one) (23-O-(tert-
butyldimethyl-silyl)-2,3-dehydrosilybin, 7). 2,3-Dehyd-
rosilybin (2, 0.980g, 2.040mmol) was dissolved in dry
pyridine (15mL). tert-Bu(Me)2SiCl (0.407, 2.698mmol)
and powdered AgNO3 (0.150gmmol) were added and
the stirred mixture was heated to 40ꢂC. The reaction
was quenched after 1.5h with water addition (100mL),
the mixture was extracted with ethyl acetate
(2 · 70mL) and the organicphase was dried (Na SO4).
After evaporation, flash chromatography (petroleum
ether/AcOEt 2:1) gave title compound (7) 0.730g
(60%) as a yellow amorphous solid.
2
[a]D 0 (c 0.34, acetone). MS-ESI (m/z): 595 [M+H]+.
1
For H and 13C NMR data see Tables 3 and 4.
[a]D +51.56 (c 0.32, CHCl3). MS-ESI (m/z): 651
[M+H]+. For 1H and 13C NMR data see Tables 1 and 2.
3.2.8. 3,7-Diacetoxy-2-[3-(4-acetoxy-3-methoxyphenyl)-
2-(hydroxymethyl)-2,3-dihydro-1,4-benzodioxin-6-yl]-
3.2.5. 3,5,7,20-Tetra-O-acetylsilybinic acid (3-(4-acetoxy-
3-methoxyphenyl)-6-[3,5,7-triacetoxy-4-oxochroman-2-yl]-
2,3-dihydro-1,4-benzodioxine-2-carboxylic acid) (10). A
stock solution of H5IO6/CrO3 was prepared by dissolv-
ing H5IO6 (11.4g, 50mmol) and CrO3 (0.023g,
5-hydroxy-4H-chromen-4-one
(3,7,20-tri-O-acetyl-2,3-
dehydrosilybin,9). Compound 7 (0.50g, 0.841mmol)
was dissolved in the mixture of pyridine (10mL) and
acetanhydride (10mL). The reaction and workup were
performed analogously as for 6. After evaporation, flash

´
R. Gazˇak et al. / Bioorg. Med. Chem. 12 (2004) 5677–5687
5686
chromatography (petroleum ether/AcOEt 1:1) gave the
title compound (9) 0.393g (77%) as a yellow amorphous
solid. [a]D +4.54 (c 0.44, acetone). MS (ESI) (m/z): 607
(M++H). For 1H and 13C NMR data see Tables 3 and 4.
MeOH) and 1.5mL of DPPH (20mgLÀ1, MeOH)
at 517nm for 10min.
(b) Superoxide scavenging: Superoxide was generated
from O2 in the presence of EDTA, MnCl2, NADH
and mercaptoethanol.35 Reaction mixture contain-
ing triethanolamine–diethanolamine buffer (0.8
mL, 100mM, pH7.4), NADH (40lL, 7.5mM)
EDTA–MnCl2 (25lL, 100/50mM), test sample
(0.1mL, 0.001–1mM) was incubated at 25ꢂC
for 10min. Reaction was started by the addition
of mercaptoethanol (0.1mL, 10mM) and the
decrease of absorbance at 340nm was monitored
for 20min.
3.2.9. 3-(4-Acetoxy-3-methoxyphenyl)-6-[3,7-diacetoxy-
5-hydroxy-4-oxo-4H-chromen-2-yl]-2,3-dihydro-1,4-ben-
zodioxine-2-carboxylic acid (3,7,20-tri-O-acetyl-2,3-
dehydrosilybinic acid,12). Stock solution of H5IO6/
CrO3 (6.4mL) was added to a solution of the compound
9 (0.30g, 0.495mmol) in wet acetonitrile (4mL, 0.75%
v/v water) during 30min while maintaining the reaction
temperature at 0–5ꢂC. The reaction and workup were
performed analogously as for 10. After evaporation,
flash chromatography (chloroform/acetone/formic acid
9:1:0.1) yielded the title compound (12) (262mg, 85%)
as a yellow amorphous solid.
3.5. Inhibition of microsomal lipid peroxidation
Microsomes were prepared from rat liver homogenate
and resuspended in 50mM Tris–HCl buffer with
100mM KCl and 0.1mM EDTA (pH7.4). Protein con-
centration in microsomal suspension was determined by
the Lowry method. The mixture of microsomal suspen-
sion (0.5mL, 0.5mg protein/mL), and test compounds
dissolved in DMSO (0.2mL, 0.05–1.0mM) were incu-
bated in the presence ADP (0.1mL, 20mM), FeCl3
(0.1mL, 1.2mM), NADPH (0.1mL, 1.25mM) in a shak-
ing water bath at 37ꢂC for 30min. The products of lipid
[a]D +8.33 (c 0.42, acetone). MS (ESI) (m/z): 621
(M++H). For 1H and 13C NMR data see Tables 3 and 4.
3.2.10. 3-(4-Hydroxy-3-methoxyphenyl)-6-(3,5,7-trihydr-
oxy-4-oxo-4H-chromen-2-yl)-2,3-dihydro-1,4-benzodioxine-
2-carboxylic acid (2,3-dehydrosilybinic acid, 13). Com-
pound 12 (0.2g, 0.322mmol) and K2CO3 (0.2g,
1.447mmol) were dissolved in MeOH/water solution
(11mL, 10:1 v/v). The reaction mixture was stirred 24h
at room temperature under argon. The reaction was
quenched by addition of formic acid (0.8mL) and after
10min of stirring evaporated to dryness. Dry solid
afforded after flash chromatography (chloroform/ace-
tone/formic acid 4:1:0.05) the title compound (13)
(104mg, 65%) as a yellow amorphous solid. [a]D +2.72
(c 0.22, MeOH). MS-ESI (m/z): 495 [M+H]+. HRMS-
ESI (m/z): 517.0752 (calcd for C25H18O11Na [M+Na]+:
517.0746). For 1H and 13C NMR data see Tables 3 and 4.
peroxidation were determined by a standard reaction
36
with thiobarbituricaicd.
The activity was calculated
as the concentration of the tested compound that inhib-
ited the colour reaction with thiobarbiturate by 50%
(IC50).
3.6. Hydrophobicity
The 1-octanol/water partition co-efficients (logP) were
determined by the shake-flask method using Britton–
Robinson I buffer (0.04M, pH7.4, ionicstrength
l = 0.15M) as an aqueous phase. The samples were dis-
solved in 1-octanol (5.10À5 M), the volumes of the parti-
tion phases were within 10–50mL, volume ratios within
1:5–5:1. The partitions were performed with a modified
rotary system (Unipan) at 25rpm and 22 2ꢂC for 3h.
The phases were separated by centrifugation at 3500rpm
for 30min and sample concentration was determined
spectrophotometrically at 289nm. The partition co-effi-
cients were determined at different volume ratios of
the partition phases and calculated according to equa-
tion P = kVw/Voct (k = coct/cw). The final logP were
obtained by extrapolation to the point of theoretical
equi-partition (k = 1).37
3.3. Cyclic voltammetry
Cyclic voltammetry measurements were performed
using the Potentiostat/Galvanostat Model 273 (EG&G
Princeton Applied Research, USA). A three-electrode
system consisting of a glassy carbon working electrode
MF2012 (Bioanalytical Systems, West Lafayette, IN,
USA), a platinum-wire auxiliary electrode and a Hg/
Hg2Cl2/saturated KCl reference electrode was used.
The potentials mentioned throughout this work are
referred to this electrode. The compounds (1.10À3 M,
DMSO) were diluted to concentrations of 1.10À4 M in
0.1M sodium-phosphate buffer pH7.0. All measure-
ments were performed at laboratory temperature and
200mVsÀ1 scan rate in the range 0–1200mV. The work-
ing electrode was polished with 0.05lm grade alumina
(Buehler, Lake Buffs, IL, USA) prior to each scan.
The anodic(oxidation) peak potential Epa was read
from the anodicwave of the voltammogram.
Acknowledgements
This work was supported by the Grant No 303/02/1097
from the Czech Science Foundation.
3.4. Antiradical activity
References and notes
(a) DPPH (1,1-diphenyl-2-picrylhydrazyl radical) scav-
enging: The absorbance change of DPPH was
measured in the reaction mixture containing
0.75mL of test compound solution (0.01–5.0mM,
1. (a) Morazzoni, P.; Bombardelli, E. Fitoterapia 1995, 66, 3;
(b) Flora, K.; Hahn, M.; Rosen, H.; Benner, K. Am. J.
Gastroenterol. 1998, 93, 139.

´
R. Gazˇak et al. / Bioorg. Med. Chem. 12 (2004) 5677–5687
5687
ˇ
2. Kren, V.; Kubisch, J.; Sedmera, P.; Halada, P.; Prikry-
lova, V.; Jegorov, A.; Cvak, L.; Gebhardt, R.; Ulrichova,
ˇ
17. Hakimelahi, C. H.; Proba, Z. A.; Ogilvie, K. K. Tetrahe-
dron Lett. 1981, 22, 4775.
18. Kelly, D. R.; Roberts, M. S.; Newton, R. F. Synth.
Commun. 1979, 9, 295.
´
ˇ
´
´
J.; Simanek, V. J. Chem. Soc., Perkin. Trans. 1 1997,
2467.
3. Mira, L.; Silva, M.; Manso, C. F. Biochem. Pharmacol.
1994, 48, 753.
4. (a) Valenzuela, A.; Silva, M.; Manso, C. F. Biol. Res.
1994, 27, 105; (b) Saller, R.; Meier, R.; Brignoli, R. Drugs
2001, 61, 2035; (c) Wellington, K.; Jarvis, B. Biodrugs
2001, 15, 465.
19. Zhao, M.; Li, J.; Song, Z.; Desmond, R.; Tschaen, D. M.;
Grabowski, E. J. J.; Reider, P. J. Tetrahedron Lett. 1998,
39, 5323.
20. Lee, D. Y. W.; Liu, Y. J. Nat. Prod. 2003, 66, 1171.
21. Araya-Maturana, R.; Delgado-Castro, T.; Cardona, W.;
´
Weiss-Lopez, B. E. Curr. Org. Chem. 2001, 5, 243.
5. Lang, I.; Deak, G.; Muzes, G.; Pronai, L.; Feher, J.
Biotechnol. Ther. 1993, 4, 263.
22. Martin, G. E. Ann. Rept. NMR Spectrosc. 2002, 46, 37.
23. Cilliers, J. J. L.; Singleton, V. L. J. Agric. Food Chem.
1991, 39, 1298.
24. Porter, L. J. In The Flavonoids Advances in Research since
1986; Hawthorne, J. D., Ed.; Chapman and Hill: London,
1994; pp 48–50.
ˇ
´
6. (a) Skottova, N.; Krecman, V.; Walterova, D.; Ulrichova,
J.; Simanek, V. Atherosclerosis 1997, 134, 134; (b) Skot-
ˇ
´
´
ˇ
ˇ
´
ˇ
´
tova, N.; Krecman, V.; Simanek, V. Phytother. Res. 1999,
13, 535.
ˇ
´
7. Steinberg, D.; Pathasarathy, S.; Carew, T. E.; Khoo, J. C.;
Witzum, J. L. New. Engl. J. Med. 1989, 320, 915.
8. Sonnenbichler, J.; Zetl, I. Prog. Clin. Biol. Res. 1986, 13,
319.
25. Pihlaja, K.; Ta¨htinen, P.; Klika, K. D.; Jokela, T.;
Salakka, A.; Wa¨ha¨la¨, K. J. Org. Chem. 2003, 68, 6864.
26. Kohen, R.; Gati, I. Toxicology 2000, 148, 149.
´
27. Psotova, J.; Lasovsky, J.; Vicar, J. Biomed. Papers 2003,
´
ˇ
147, 147.
9. Braatz, R.; Gurler, K.; Bergish, G.; Halbach, G.; Soicke,
H.; Schmidt, K. Czech. Patent 273,610, 1985; Chem. Abstr.
1985, 105, P127476b).
}
}
28. Gyorgy, I.; Antus, S.; Foldiak, G. Radiat. Phys. Chem.
´
1992, 39, 81.
ˇ
´
10. Kubisch, J.; Sedmera, P.; Halada, P.; Gazˇak, R.; Skot-
tova, N.; Simanek, V.; Kren, V. Heterocycles 2001, 54,
29. Pietta, P.-G. J. Nat. Prod. 2000, 63, 1035.
30. Foti, M. C.; Johnson, E. R.; Vinqvist, M. R.; Wright, J. S.;
Barclay, L. R. C.; Ingold, K. U. J. Org. Chem. 2002, 67,
5190.
31. Foti, M. C.; Daquino, C.; Geraci, C. J. Org. Chem. 2004,
69, 2309.
ˇ
´
´
ˇ
901.
ˇ
11. Simanek, V.; Kren, V.; Ulrichova, J.; Vicar, J.; Cvak, L.
´
Hepatology 2000, 32, 442.
ˇ
´
ˇ
12. (a) Bandopadhyay, M.; Pardeshi, N. P.; Seshadri, T. R.
Ind. J. Chem. 1972, 10, 808; (b) Meric¸li, A. H. Planta Med.
1988, 44.
13. Halbach, G.; Trost, W. Arzneim. Forsch. 1974, 24, 866.
14. Pelter, A.; Ha¨nsel, R. Chem. Ber. 1975, 108, 790.
15. Maitrejean, M.; Comte, G.; Barron, D.; El Kirat, K.;
Conseil, G.; Di Pietro, A. Bioorg. Med. Chem. Lett. 2000,
10, 157.
´
32. Uhrın, D.; Barlow, P. J. Magn. Reson. 1997, 126, 248.
33. Hurd, R. E.; John, B. K. J. Magn. Reson. 1991, 91, 648.
34. Wilker, W.; Leibfritz, D.; Kerssebaum, R.; Bermel, W.
Magn. Reson. Chem. 1993, 31, 287.
35. Paoletti, F.; Mocali, A. Methods Enzymol. 1990, 186, 209.
36. Buege, J. A.; Aust, S. D. Methods Enzymol. 1978, 52, 302.
37. Dearden, J. C.; Bressnen, G. M. Quant. Struct. Act. Relat.
1998, 7, 133.
16. Zanarotti, A. Heterocycles 1982, 19, 1585.