Base-catalyzed oxidation of silybin and isosilybin into 2,3-dehydro derivatives

Article abstract of DOI:10.1016/j.tetlet.2012.11.049

The base-catalyzed oxidation of the flavonolignans, silybin, and isosilybin into the corresponding 2,3-dehydro derivatives has been studied and optimized. Various bases, solvents, and reaction conditions were tested to achieve the best yields. The influence of water on the course of the reaction was also observed. It was found that this oxidation reaction was probably based on the disproportionation of the (iso)silybin (or some intermediate) molecule, as the reaction also proceeds in the absence of oxygen. We report here for the first time the preparation of optically pure 2,3-dehydroisosilybins A and B.

Full text of DOI:10.1016/j.tetlet.2012.11.049

Tetrahedron Letters 54 (2013) 315–317
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Tetrahedron Letters
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Base-catalyzed oxidation of silybin and isosilybin into 2,3-dehydro derivatives
a
a
a
Radek Gazák a,d, Patrick Trouillas b,c, David Biedermann , Katerina Fuksová , Petr Marhol ,
ˇ
ˇ
a
a,
⇑
ˇ
Marek Kuzma , Vladimír Kren
a
ˇ
Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenská 1083, CZ-142 20 Prague, Czech Republic
b Université de Limoges, LCSN-EA 1069, Faculté de Pharmacie, 2 rue du Docteur Marcland, 87025 Limoges, France
c Laboratoire de Chimie des Matériaux Nouveaux, Université de Mons Place du Parc 20, B-7000 Mons, Belgium
d Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 8, CZ-128 40 Praha 2, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
The base-catalyzed oxidation of the flavonolignans, silybin, and isosilybin into the corresponding 2,3-
dehydro derivatives has been studied and optimized. Various bases, solvents, and reaction conditions
were tested to achieve the best yields. The influence of water on the course of the reaction was also
observed. It was found that this oxidation reaction was probably based on the disproportionation of
the (iso)silybin (or some intermediate) molecule, as the reaction also proceeds in the absence of oxygen.
We report here for the first time the preparation of optically pure 2,3-dehydroisosilybins A and B.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 23 August 2012
Revised 26 October 2012
Accepted 8 November 2012
Available online 21 November 2012
Keywords:
Isosilybin
2,3-Dehydroisosilybin
2,3-Dehydrosilybin
Silybin
Oxidation
The flavonolignan silybin (1, Fig. 1; synonym ‘silibinin’) is a very
commonly reported component of the milk thistle (Silybum maria-
num). However, the crude extract from the seeds of this plant—
so-called silymarin—contains other constituents (isosilybin, sily-
christin, silydianin, 2,3-dehydrosilybin, taxifolin, and polymeric
polyphenolic compounds) whose biological activities have been
neglected. The situation is complicated by the fact that most
silymarin components occur as pairs of diastereoisomers (silybin,
isosilybin, silychristin) or enantiomers (2,3-dehydrosilybin).
At least two of these compounds—2,3-dehydrosilybin (2) and
isosilybin (3)—appear to be more potent than silybin in various
complex biological tests focusing on, for example, their antitumor
and antiproliferative activities.
mitochondria.13 Compounds 1 and 2 are inhibitors of glucose up-
take in adipocytes and Chinese hamster ovarian (CHO) cells.14
Natural isosilybin (3)—a silybin regioisomer—is, similar to sily-
bin, a mixture of the two diastereoisomers A (3a) and B (3b).
Recent studies have demonstrated that 3 is probably the most po-
tent anticancer agent present in silymarin. Compound 3 possesses
in vivo antiproliferative, anti-angiogenic, pro-apoptotic, and cell-
cycle modulatory properties.15 Moreover, isosilybin treatment
inhibits the growth of advanced human prostate cancer cells
in vivo without any toxic effects. Isosilybin B has more profound
effects upon most cell regulatory pathways than isosilybin A in
various prostate cancer cell lines.16,17
Although 4 has not yet been detected in silymarin, it should be
present in this natural mixture at least as a result of isosilybin
oxidation—as in compound 2. The oxidation of 1 into 2 improves
significantly its antioxidant and anticancer activities, therefore,
we expect that the analogous oxidation of 3 (being a more potent
anticancer agent than silybin) into 4 (which has not been described
as yet) should provide a more active anticancer compound than
both 2,3-dehydrosilybin and isosilybin.
2,3-Dehydrosilybin is a minor component in silymarin1 with
significantly better anticancer2–4 and antioxidant5,6 activity than
silybin. Moreover, some C-prenylated7 and certain O-alkylated8
and 8-prenylated9 derivatives of 2,3-dehydrosilybin are effective
modulators of P-glycoprotein (Pgp). The 2,3-dehydro derivative 2
is an effective inhibitor of Plasmodium strains.10
2,3-Dehydrosilybin is a potent protectant against H2O2-induced
oxidative stress in human keratinocytes and mouse fibroblasts,11 it
suppresses UVA-caused oxidative stress in the skin12 and also
decreases reactive oxygen species (ROS) formation in rat heart
The major aim of this work was to develop a novel, robust, and
scaleable method for the oxidation of silybin and isosilybin to the
corresponding 2,3-dehydro derivatives; the method is based on
base-catalyzed disproportionation. The key part of this work was
the preparation and structural description of the optically pure
2,3-dehydrosilybins A and B 2, and 2,3-dehydroisosilybins A and
B 4.
⇑
Corresponding author. Tel.: +420 296 442 510; fax: +420 296 442 509.
ˇ
E-mail address: kren@biomed.cas.cz (V. Kren).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.11.049

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R. Gazák et al. / Tetrahedron Letters 54 (2013) 315–317
23
16
CH2OH
CH2OH
O
O
10
11
O
O
HO
O
O
OCH3
OH
2
3
17
19
HO
O
O
OCH3
OH
7
14
OH
OH
20
5
OH
OH
1a Silybin A (2R,3R,10R,11R)
1b Silybin B (2R,3R,10S,11S)
2a
2,3-Dehydrosilybin A (Δ2,3, 10R,11R)
2b
2,3-Dehydrosilybin B (Δ2,3, 10S,11S)
OH
OH
O
O
O
10
11
OCH3
OCH3
HO
O
HO
O
O
2
3
O
CH2OH
CH2OH
OH
OH
OH
O
OH
3b Isosilybin B (2R,3R,10S,11S)
4b
3a Isosilybin A (2R,3R,10R,11R)
4a
2,3-Dehydroisosilybin B (Δ2,3, 10S,11S)
2,3-Dehydroisosilybin A (Δ2,3, 10R,11R)
Figure 1. Silybin, isosilybin, and respective dehydro derivatives. Structures 1, 2, 3, and 4 occur as equimolar mixtures (natural) of both respective stereoisomers a and b.
The first method for the synthesis of 2,3-dehydrosilybin (2) in-
volved the oxidation of silybin with iodine in K-acetate buffer.18,19
This method gave a high yield of 2,3-dehydrosilybin (2, 70–80%),
which contained side products that were difficult to separate,
and an unreacted starting material. The reaction only gave a ca.
50% yield of pure 2 after repetitive crystallizations. Another draw-
back of this method was the formation of acetates that had to be
hydrolyzed (HCl) prior to final purification. Moreover, both silybin
and iodine have rather low solubilities in acetic acid, which made
scale-up difficult. Another approach leading to 2 is based on (sup-
posed) atmospheric oxidation of silybin in the presence of a base,
for example, treatment of silybin with N-methylglucamine where
2 was formed as one of the side products,20 or dehydrogenation
of silybin to 2 in the presence of air in boiling pyridine.5 Recently,
the oxidation of silybin to 2,3-dehydrosilybin was achieved in the
presence of air using potassium acetate in DMF at 50 °C.21 How-
ever, the yield strongly depended on the work-up (as observed
by ourselves) (Table 1).
presence of water in the solvent (up to 10%) did not affect the
course of the reaction.
The reactions using various inorganic and organic bases were
monitored by HPLC (Scheme 1, Table 1). The choice of the base
was limited by its strength, because strong bases such as alkali me-
tal hydroxides cause rapid decomposition of silybin. The reaction
rate was relatively slow at room temperature, therefore all the
reactions were tested under reflux. Four equivalents of the base
versus 1 or 3 were found to be optimal for the product yield. Final-
ly, the reaction was tested both in the presence and absence of
atmospheric oxygen. Surprisingly, the reaction proceeded similarly
in both cases, which indicated that molecular oxygen was not re-
quired for this oxidation reaction. This observation can be corrob-
orated by the behavior of silybin during alkylation reactions, as it
always forms a small amount of 2,3-dehydrosilybin analogues, de-
spite working under strict anaerobic conditions. Moreover, a simi-
lar observation was reported recently in a study concerning the
The major disadvantage of the above base-catalyzed procedures
was the use of harmful solvents (pyridine or DMF), which are
incompatible with the large-scale preparation of 2,3-dehydrosily-
bin intended for pharmaceutical applications.
Table 1
Effect of the base on silybin oxidation
Base (equiv)
Solvent
Time (h)
Yield of 2 (%)
K2CO3 (4)
K2CO3 (4)
K2CO3 (4)
K2CO3 (4)
Na2CO3Á10H2O (4)
NaHCO3 (4)
Cs2CO3 (4)
Et3N (4)
EtOH/H2O (9:1)
EtOH
16
16
16
1.5
16
16
16
24 (72)
16 (48)
16
16 (94)
16 (42)
16 (48)
16 (48)
0.5a
45
All previous methods for 2,3-dehydrosilybin preparation, with
the exception of iodine oxidation, are similar in principle as they
proceed via oxidation of silybin by oxygen, in an alkaline milieu.
We assumed that this reaction was applicable to other 2,3-satu-
rated flavonolignans and its course depended only on the solvent,
base, and reaction conditions. The choice of suitable solvents was
rather limited by the relatively poor solubility of silybin in most or-
ganic solvents as well as in water. Silybin is virtually insoluble in
solvents such as CH2Cl2, toluene, hexane, and diethyl ether. It is,
however, relatively soluble in polar solvents. Moreover, its solubil-
ity increases in the presence of bases due to phenolate formation.
Accordingly, acetone and simple alcohols, such as MeOH and EtOH,
were tested as solvents for this reaction. Since most inorganic
bases, for example, carbonates and hydroxides, are rather insoluble
in organic solvents, solvent mixtures containing water were also
tested. Only negligible differences in the yields of 2 were observed
with the different solvents, keeping the conditions identical. The
40–45
40–45
48
37
51
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
DMFa
20
20 (40)
37 (49)
n.r.b
20 (50)
32 (50)
28 (40)
10 (27)
21a (78)c
KOAc (4)
Ba(OH)2 (4)
DMAP (4)
NH4OH (4)
Li2CO3 (4)
DBU (4)
KOAc (3)a
a
Experiment was performed according to the reported method21 with a slight
modification in the work-up that consisted of precipitation from an aqueous
solution of HCl.
b
No reaction.
c
Yield reported in the literature.21

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R. Gazák et al. / Tetrahedron Letters 54 (2013) 315–317
317
R1
R2
R1
O
O
O
10
11
10
base
HO
O
O
HO
O
O
11
O
R2
ROH, reflux
R = Me, Et
OH
OH
OH
OH
R
R
1 = CH2OH, R2 = 4-hydroxy-3-methoxyphenyl
2
4
1
3
R
R
1 = CH2OH, R2 = 4-hydroxy-3-methoxyphenyl
1 = 4-hydroxy-3-methoxyphenyl, R2 = CH2OH
1 = 4-hydroxy-3-methoxyphenyl, R2 = CH2OH
2a 10R,11R, 2b 10S,11S
1a 10R,11R, 1b 10S,11S
4a
4b
10S,11S
10R,11R,
3a
3b
10S,11S
10R,11R,
Scheme 1. Base-catalyzed oxidation of silybin and isosilybin into 2,3-dehydro derivatives.
thermal rearrangement of taxifolin (flavanonol) into alphitonin, a
process during which quercetin (2,3-dehydroflavanonol) was
formed as a side product despite the work being performed under
a strictly inert atmosphere with a degassed solvent.22 Based on
these results, we conclude that an alternative oxidation mecha-
nism takes place in this situation. Since no oxidizing agent is pres-
ent and the oxidation reaction is always accompanied by
decomposition products, we infer that the formation of dehydrosi-
lybins from the respective silybin under alkaline conditions is a
disproportionation reaction. This hypothesis is also supported by
the fact that the maximum yield of 2,3-dehydrosilybin was very
close to 50%. During the reaction, the formation of polar com-
pounds (TLC) was observed, which however decomposed quickly
into polymers. These might be the reduced products of dispropor-
tionation, but without knowledge of their structure(s), we cannot
really speculate on the reaction mechanism.
The strength of the base influenced positively the kinetics of
2,3-dehydrosilybin formation. However, the best results were
more likely to be obtained with relatively weak bases such as NaH-
CO3, KOAc, NH4OH, or DMAP after a longer reaction time (16–94 h).
Probably, stronger bases (Cs2CO3 or DBU) cause simultaneous
decomposition of product 2, which results in the lower yield.
The optimized conditions23 were used for the first preparation
of both optically pure 2,3-dehydrosilybins A and B (2a and 2b)
starting from stereochemically pure 1a and 1b. Subsequently, the
synthesis of 2,3-dehydroisosilybins A and B starting from 3a and
3b was accomplished (the experimental procedure and analytical
data for these new compounds are described in the Supplementary
data associated with this work).
the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.
11.049. These data include MOL files and InChiKeys of the most
important compounds described in this article.
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23. Silybin (2.5 g, 5.183 mmol) and NaHCO3 (1.74 g, 20.798 mmol) were dissolved
in MeOH (100 mL) and the mixture was heated under reflux for 16 h. The
mixture was then left to cool to room temperature and poured into ice-cold
water containing HCl (400 mL, 5% v/v). The precipitate formed was filtered off,
In conclusion, we have described the base-catalyzed prepara-
tion of optically pure 2,3-dehydrosilybins A and B and 2,3-dehy-
droisosilybins A and B. This oxidation reaction was probably
based on the disproportionation of the silybin or isosilybin (or
some intermediate) molecule, as the reaction was found to proceed
in the absence of oxygen.
Acknowledgments
washed with H2O, dissolved in
a mixture of EtOAc/acetone (1:1), and
evaporated to give 2.17 g of dry residue. The solid was crystallized from
MeOH (1000 mg, 40% yield). The mother liquor was filtered through a silica gel
pad (CHCl3/acetone/HCOOH 90:10:1–70:30:1) to obtain, after concentration,
another portion of the product, which after recrystallization from MeOH
yielded pure 2 (270 mg, 11%). Thus, the total yield of 2 was 51%. Full spectral
characterization of compounds 2a, 2b, 4a, and 4b (1H and 13C NMR, MS, HPLC,
This work was supported by Grants from the Czech Science
Foundation P301/11/0662 (V.K.), P207/10/0288 (R.G.); ESF COST
Chemistry CM0804 (Grant MŠMT LD11051). The authors also
thank the ‘‘Conseil Régional du Limousin’’ for the financial support
and CALI (CAlcul en LImousin) for computing facilities.
[a]D, and CD) are given in the Supplementary data.
Supplementary data
Supplementary data (supplementary data (1H and 13C NMR, MS,
HPLC, [a]D, and CD)) associated with this article can be found, in