Metal coordination protocol for the synthesis of-2,3-dehydrosilybin and 19-O-demethyl-2,3-dehydrosilybin from silybin and their antitumor activities

Article abstract of DOI:10.1016/j.tetlet.2018.03.052

An efficient and practical method to access bioactive 2,3-dehydrosilybin and 19-O-demethyl-2,3-dehydrosilybin using naturally abundant flavonolignan silybin in the presence of metal salt as a chelating agent is described. The procedure presented here has

Full text of DOI:10.1016/j.tetlet.2018.03.052

Accepted Manuscript
Metal Coordination Protocol for the Synthesis 2,3-Dehydrosilybin and 19-O-
Demethyl-2,3-dehydrosilybin from Silybin and Their Antitumor Activities
Yong-ju Wen, Zong-yuan Zhou, Guo-lin Zhang, Xiao-xia Lu
PII:
S0040-4039(18)30366-6
https://doi.org/10.1016/j.tetlet.2018.03.052
TETL 49821
DOI:
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 February 2018
14 March 2018
19 March 2018
Please cite this article as: Wen, Y-j., Zhou, Z-y., Zhang, G-l., Lu, X-x., Metal Coordination Protocol for the Synthesis
2,3-Dehydrosilybin and 19-O-Demethyl-2,3-dehydrosilybin from Silybin and Their Antitumor Activities,
Tetrahedron Letters (2018), doi: https://doi.org/10.1016/j.tetlet.2018.03.052
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Tetrahedron Letters
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Metal Coordination Protocol for the Synthesis 2,3-Dehydrosilybin and 19-O-
Demethyl-2,3-dehydrosilybin from Silybin and Their Antitumor Activities
Yong-ju Wen a,c,d, Zong-yuan Zhou a,c, Guo-lin Zhang a*, and Xiao-xia Lu a,b∗
a Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China
b Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, Hainan Normal University, Hainan, 571127, China
c University of Chinese Academy of Sciences, Beijing 100049, China
dChemistry and Biology Bioengneering College, Yichun University, Yichun, 336000, China
ARTICLE INFO
ABSTRACT
An efficient and practical method to access bioactive 2,3-dehydrosilybin and 19-O-demethyl-
2,3-dehydrosilybin using naturally abundant flavonolignan silybin in the presence of metal salt
as a chelating agent is described. The procedure presented here has several advantages including
one-pot, synthetic ease, and products in high yields with no side reactions, and large-scale
feasibility. The dehydrogenation and demethylation proceed smoothly via a one-pot process
using the AlCl3/Pyridine system and I2 as the additive. Furthermore, 2,3-dehydrosilybin and 19-
O-demethyl-2,3-dehydrosilybin can inhibit the expression of intracellular mature miRNA-21
with IC50 values of 4.46 µM and 8.25 µM, respectively, and show moderate anticancer activities
against HeLa cell lines.
Article history:
Received
Received in revised form
Accepted
Available online
Keywords:
Silybin
2,3-dehydrosilybin
19-O-demethyl-2,3-dehydrosilybin
Synthesis
2009 Elsevier Ltd. All rights reserved.
Metal Chelating
Antitumor
Introduction
The first synthesis of 2,3-dehydrosilybin (DHS) was via the
oxidation of silybin with iodine in K-acetate buffer.15,
16
Silymarin applied in the treatment of cirrhosis, hepatitis, and
alcohol-induced liver disease is a crude extract of milk thistle
(Silybum marianum L. Gaertn., Asteraceae), which contains
more than 10 structurally closely related flavonolignans such as
silybin, silychristin, isosilybin, silydianin, taxifolin, quercetin,
and 2,3-dehydrosilybin etc.1-3 Among the flavonolignans,
silybin (SIL, Figure 1) is the most abundant flavonolignan and
the easiest to isolate from silymarin, but it exhibits quite poor
biological activities. The structure-activity relationship analysis
of silymarin flavonolignans reveals that 2,3 double bond or
phenolic hydroxyl group enhance biological activities. For
example, 2,3-dehydrosilybin (DHS, Figure 1) characterized by
the presence of the 2,3 double bond possesses higher biological
activities than silybin, such as anticancer,4-6 antioxidant,7-12 and
modulation of P-glycoprotein.13 Besides, 19-O-demethyl-2,3-
dehydrosilybin (DHDMS, Figure 1) replaced methoxy group by
the hydroxyl group at C-19 exhibits a much higher antioxidant
activity compared to SIL or DHS, and better inhibitory activity
against lipid peroxidation than quercetin.14 Unfortunately, DHS
and DHDMS are minor constituents in silymarin, therefore the
synthesis of the 2,3-dehydro derivative and minor silymarin
constituent DHS and DHDMS from the abundant silybin is of
high interest due to their biological activities.
However, these methods have certain drawbacks, including no
more than 50% yield, side products, the use of harmful solvents,
hydrolysis of the formation of acetates prior to final purification,
and low solubilities of silybin and iodine in acetic acid to scale-
up difficult. In another method, 2,3-dehydrosilybin have been
synthesized by the aerial or anaerobic oxidation of silybin with
base as catalyst, such as N-methylglucamine, pyridine and
alkaline milieu.17 The disadvantage of the base-catalyzed method
was the occurred side-reaction due to the unstable silybin in base
condition, including the free radical polymerization and
rearrangement reaction, to produce the insoluble substances and
decomposition products.7,18-20 19-O-demethyl-2,3-dehydrosilybin
(DHDMS) was prepared by the radical-coupling reaction of
quercetin with (E)-3-O-benzyl-3,4-dihydroxy-cinnamyl alcohol,21
only giving 9.6% yield which greatly limits it’s biological
applications. To the best of our knowledge, no reports existed to
date for the synthesis of DHDMS from SIL or DHS due to the
selective demethylation of 19-OMe was rather difficult.
———
∗ Corresponding author.
E-mail: luxx@cib.ac.cn (XX, Lu), zhanggl@cib.ac.cn (GL, Zhang)

2
Tetrahedron Letters
O
O
OH
Table 1. Survey of metal salts and solvents for the oxidation of SIL to
DHSa
Entry
HO
O
O
OCH3
OH
1) AlCl3-Pyridine
Mn+
H+
SIL
DHS
DHDMS
2) H+
O
Mn+
Pyridine/I2
Temp (°C)
110
Metal
MgCl2
CaCl2
ZnCl2
CuCl2
FeCl2
FeCl3
AlCl3
AlCl3
AlCl3
AlCl3
AlCl3
Solvent
Pyridine
Pyridine
Pyridine
Pyridine
Pyridine
Pyridine
Pyridine
MeOH
Yield (%)b
53
O
Mn+
1
2
1) AlCl3-Pyridine/I2
2) H+
110
37
Scheme 1.
Synthesis of DHS and DHDMS
3
110
55
4
110
43
Structurally, silybin is characterized by a flavonoid moiety
5
110
38
associated with a phenylpropanoid unit. The flavonoid part
consists of carbonyl and hydroxyl groups, they can coordinate
metal ions and form complexes. In fact, a large number of
experimental and theoretical studies demonstrate that metal ions
such as Mg2+, Zn2+, Ca2+, Cu2+, Fe2+, Fe3+ and Al3+ ions can
chelate with silybin at 3-OH, 5-OH, and 4-carbonyl group.22-24
Besides, a survey of literature show that methoxy group of ortho-
hydroxyphenyl alkyl ethers can be cleaved to provide catechol in
a direct route with aluminum trichloride-pyridine.25 Considering
the unique ability of flavonoids to coordinate metal ions, it is
envisaged that introduction of the metal ions to coordinate with
the carbonyl and hydroxyl groups to stabilize the structure of
silybin in base condition to avoid being oxidized or rearranged.
On the other hand, due to the oxophilicity of aluminum ions, the
ether cleavage reaction of 19-methoxy in phenylpropanoid unit of
SIL possibly occurred during AlCl3-pyridine induced the
dehydrogenation of silybin. Hence, in the present work, we focus
on the synthesis of DHS and DHDMS from silybin by
dehydrogenation or(and) demethylation via metal coordination
6
110
44
7
110
56
8
Reflux
Reflux
110
Trace
Trace
Trace
Trace
9
EtOH
10
11
DMF
DMSO
110
a
Unless stated otherwise, reactions were performed on 0.2 mmol scale with
silybin /metal salts as 1:2 ratio in 4.0 mL of solvent in air for 24 h.
b
Isolated yield.
equiv, 2.5 equiv and 3.5 equiv AlCl3 in pyridine. It is noteworthy
that increasing the AlCl3 amount from 2.0 to 3.5 equiv caused a
substantial decrease in the yield of DHS and increase in the yield
of DHDMS. (Table 1, entries 15-18). In contrast, only trace
desired product of DHDMS were detected after 2 h using
synthesized DHS as substrate in AlCl3-pyridine at 110 oC with or
without I2 (Table 2, entries 20 and 21). Notably, the yield of
DHDMS increased to be 85% when the KI was added (Table 2,
entry 22). Like KI, similar effect was observed using AlI3 instead
of AlCl3 (Table 2, entry 23). Since the reaction conditions of
dehydrogenation and demethylation were quite similar, the in site
generated iodide ion during the dehydrogenation by I2 played a
key role in promoting the demethylation.
(Scheme.1)，along with the determination of their antitumor
activities.
Results and Discussion
Table 2. Optimization of the reaction conditionsa
According to the O2-induced oxidation protocols and the
condition reported by Křen and co-workers17, the reaction of
silybin in the presence of various metals in the air using various
solvents at reflux temperature for 24 h was initially conducted.
Unfortunately, the desired products were obtained in only low
isolated yields and most of original materials were observed
(Table 1, entries 1-7), when the metal salts such as MgCl2, CaCl2,
ZnCl2, CuCl2, FeCl2, FeCl3 and AlCl3 were used. The effect of the
solvents, including MeOH, EtOH, DMF, and DMSO, did not
show any affect in the transformation yields (Table 1, entries 8-
11). Encouragingly, excellent yields were achieved when the I2
was used as oxidizing agent with the addition of metal salts for 1
h (Table 2, entries 1-7). The reaction was found to give high
conversions for all metal salts. Among the common metal salts
studied for this reaction, MgCl2 was found to be the most
effective additive for this transformation since it resulted in the
highest conversion to the desired product (Table 2, entry 1).
Further optimization of the temperature revealed that without
improving effect on the transformation of DHS when the
temperature was decreased to 100°C (Table 2, entry 8), but
further decrease in temperature to 90 and 80 oC gave the DHS in
86% and 75% yield, respectively (Table 2, entries 9 and 10)。
We observed the yield was slight increased 92% when the
reaction time was increased from 1 h to 2 h (Table 2, entry 11).
Next, the amount of the MgCl2 additive was examined. No
substantial improvement in the yield of DHS was observed when
the amount of MgCl2 was increased from 2.0 to 2.2 eq (Table 2,
entry 14), while for the decreasing the amount of MgCl2 to 1.8
and 1.6 eq only gave DSH in 86% and 79% yield, respectively
(Table 2, entries 12 and 13). Interestingly, use of AlCl3, DHS and
DHDMS were isolated in 75% and 19% yields, respectively
(Table 2, entry 7), while no or trace DHDMS product was
detected for the other metal salts (Table 2, entries 1-6).
Entry
Metal
Temp
(°C )
110
110
110
110
110
110
110
100
90
Time
(h)
1
Yield (%)b
DHDMS
Salt (equiv)
DHS
91
1
2
MgCl2 (2.0 eq)
ZnCl2 (2.0 eq)
CaCl2 (2.0 eq)
CuCl2 (2.0 eq)
FeCl2 (2.0 eq)
FeCl3 (2.0 eq)
AlCl3 (2.0 eq)
MgCl2 (2.0 eq)
MgCl2 (2.0 eq)
MgCl2 (2.0 eq)
MgCl2 (2.0 eq)
MgCl2 (1.8 eq)
MgCl2 (1.6 eq)
MgCl2 (2.2 eq)
AlCl3 (2.3 eq)
AlCl3 (2.5 eq)
AlCl3 (3.0 eq)
AlCl3 (3.5 eq)
AlCl3 (3.0 eq)
AlCl3 (3.0 eq)
AlCl3 (3.0 eq)
AlCl3 (3.2 eq)
AlI3 (3.2 eq)
Trace
Trace
N.r.c
N.r.c
Trace
Trace
19
1
89
3
1
84
4
1
60
5
1
87
6
1
85
7
1
75
8
1
91
N.r.c
N.r.c
N.r.c
N.r.c
N.r.c
N.r.c
N.r.c
32
9
1
86
10
11
12
13
14
15
16
17
18
19
20d
21e
22f
23h
80
1
75
100
100
100
100
110
110
110
110
110
110
110
110
110
2
92
1
86
1
79
1
91
2
62
2
50
43
2
Trace
Trace
Trace
92
2
91
2.5
2
90
Trace
Trace
85
2
2
2
92
a
Unless stated otherwise, reactions were performed on 0.2 mmol scale with
silybin in 1 equiv. I2 in 4.0 mL of pyridine in air.
b
Isolated yield.
Not reaction
c
In this regard, silybin was found to provide the 19-O-
demethyl-2,3-dehydrosilybin (DHDMS) in 19% yield using 2.0
equiv AlCl3, indicating a demethylation process. To explore if
such a side reaction could be developed into a useful new method
for the preparation of DHDMS, we directly treated SIL with 2.3
d
0.2 mmol of DHS instead of silybin as substrate without I2.
0.2 mmol of DHS instead of silybin as substrate in 1 equiv. I2.
e
f 0.2 mmol of DHS instead of silybin as substrate in 2 equiv. KI.
h 0.2 mmol of DHS instead of silybin as substrate without I2.

Based on the above experimental results and literature
reports,25-28 a plausible dehydrogenation and demethylation
mechanism using the AlCl3/Pyridine system and I2 as the additive
is depicted in Scheme 2. Reaction occurred in the flavonoid and
carbonyl and 5-hydroxyl group afford silybin aluminum
complexes A1, which subsequently undergoes keto-enol
tautomerization with the assistance of Al3+. Iodine then
encounters the nucleophilic attack of the C=C bond of enol A2 to
give intermediate A3, which subsequently undergoes elimination
of HI to furnish product 2,3-dehydrosilybin aluminum complexes
A4. On the other hand, according to the Lange’s mechanism for
AlCl3-pyridine-catalyzed cleavage of alkyl o-hydroxyphenyl
ethers,25 the deprotonation of the 18-hydroxyl group of
phenylpropanoid moiety by aluminum trichloride affords
aluminum phenolate A5. Coordination of the 19-methoxyl group
to the Lewis acidic center of aluminum phenolate A5 gives a five-
membered cyclic intermediate A6 after releasing methyl iodine.
Only a trace mount o product DHDMS was observed when DHS
was as substrate without iodine ion, whereas high yields of
DHDMS was afforded with the iodine ion. Therefore, it seems
possible that this demethylation reaction proceeds though
releasing methyl iodine. Finally, hydrolysis of aluminum
complexes A4 and A6 furnishes DHS and DHDMS, respectively.
The dehydrogenation product DHS is major when the aluminum
trichloride amount is not more than 2.0 equiv, whereas increasing
the aluminum trichloride amount from 2.0 to 3.0 equiv caused the
DHDMS as the major product, which indicates that the stepwise
coordination of Al3+ to the chelation site and the demethylation
proceeds slower than the dehydrogenation.25
A
C
E
B
D
Figure 2. Effect of SIL, DHS and DHDMS on miR-21 production and
cell viability. (A) HeLa-luciferase-miR-21 cells were treated with SIL, DHS
and DHDMS at 10 μM for 24 h. (B) Cell viability and (E) cell proliferation
were measured by Alamar-Blue Regent. (C-D) Hela-luciferase-miR-21 cells
were treated with DHS and DHDMS at various concentrations for 24 h.
Luciferase was measured by coenzyme A sodium hydrate and D-luciferin
sodium salt and reduced firefly luciferase expression indicates the binding of
endogenous miRNAs to the cloned miR-21 target sequence. All the data
points represent means ± SD of triplicate times, *p < 0.05, **p < 0.01, ***p
< 0.001.
HeLa-luciferase-miR-21 cells were constructed by transfected
PCIneo-luciferase-miR-21 vector which quantitatively evaluate
microRNA-21 (miR-21) activity by the insertion of miR-21
target sites downstream and screening with G418 (300 µg/mL).
Luciferase is the primary reporter gene and reduced firefly
luciferase expression indicates the binding of endogenous
miRNAs to the cloned miR-21 target sequence. The cells were
seeded at a density of 105 cells/well in 96-well plates and
maintained at 37 °C under a humidified atmosphere of 5% CO2
and 95% air in RPMI-1640 medium supplemented with 10%
fetal bovine serum (FBS) and G418 (300 µg/mL). The cells were
treated with vehicle (0.1% DMSO) alone, 10 µM SIL, DHS,
DHDMS for 24 h, and then tested luminescence with D-luciferin
sodium salt. All of them showed some effect, DHS and DHDMS
inhibited production of miR-21 and proliferation with low IC50
values of 4.64 µM and 8.25 µM, respectively (Figure 2). Thus,
DHS and DHDMS can be an alternative drug targeting miR-21
which is relevant to the tumorigenesis and progression.
high yields. Metal coordination played a key role to avoid
oxidizing and rearranging of silybin in alkaline aqueous solutions,
which resulted in satisfactory yields of DHS and DHDMS, giving
91% and 92% yeild, respectively. Development of this method
will greatly facilitate the biological studies of DHS and DHDMS
that has demonstrated great potentials in pharmaceutical
applications.
Acknowledgments
This work is partly supported by Key Laboratory of Tropical
Medicinal Plant Chemistry of Ministry of Education Hainan
Normal University and Science and Technology Service Network
Initiative of Chinese Academy of Sciences (No. KFJ-SW-STS-
143-1) and National Science Foundation of China (No.
21272229).
In summary, we have developed an efficient method for the
preparation of biologically relevant flavonolignans DHS and
DHDMS via dehydrogenation or(and) demethylation of silybin in

4
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Synthesis of 2,3-dehydrosilybin and 19-O-demethyl-2,3-dehydrosilybin is
Metal Coordination Protocol for the
Synthesis 2,3-Dehydrosilybin and 19-O-
Demethyl-2,3-dehydrosilybin from Silybin
and Their Antitumor Activities
reported starting from naturally abundant flavonolignan silybin using
metal-pyridine/I2 with high yields of 91% and 92%, respectively.
Synthesized compounds are tested for their anticancer activities against
HeLa cell lines with IC50 values of 4.46 and 8.25 µM.
Yong-ju Wen a,c,d Zong-yuan Zhou a,c Guo-lin Zhang a,* and Xiao-xia Lu a,b, *

Metal Coordination Protocol for the Synthesis 2,3-Dehydrosilybin and 19-O-
Demethyl-2,3-dehydrosilybin from Silybin and Their Antitumor Activities
1. Metal coordination protocol provided direct access to silybin derivatives.
2. The dehydrogenation and demethylation proceed via AlCl3/pyridine/I2 system.
3. Silybin derivatives exhibited the better anticancer activities than the silybin.