Regioselective synthesis of methylated epigallocatechin gallate via nitrobenzenesulfonyl (Ns) protecting group

Article abstract of DOI:10.1016/j.bmcl.2009.05.111

Regioselective synthesis of methylated epigallocatechin gallate from epigallocatechin was accomplished using a 2-nitrobenzenesulfonyl (Ns) group as a protecting group for phenols. This methodology provided several methylated catechins, which are naturally

Full text of DOI:10.1016/j.bmcl.2009.05.111

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4171–4174
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Regioselective synthesis of methylated epigallocatechin gallate via
nitrobenzenesulfonyl (Ns) protecting group
Yoshiyuki Aihara a, Atsusi Yoshida a, Takumi Furuta a, , Toshiyuki Wakimoto a, Toshifumi Akizawa b,
Motomi Konishi b, Toshiyuki Kan a,
*
a School of Pharmaceutical Sciences, University of Shizuoka and Global COE Program, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
b Faculty of Pharmaceutical Sciences, Setsunan University, Hirakata, Osaka 573-0101, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Regioselective synthesis of methylated epigallocatechin gallate from epigallocatechin was accomplished
using a 2-nitrobenzenesulfonyl (Ns) group as a protecting group for phenols. This methodology provided
several methylated catechins, which are naturally scarce catechin derivatives.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 8 May 2009
Revised 27 May 2009
Accepted 28 May 2009
Available online 2 June 2009
Keywords:
Epigallocatechin gallate
Nitrobenzenesulfonyl (Ns) group
Methylated catechin
Matrix metalloproteinases
Epigallocatechin gallate (EGCG: 1), which exhibits various bio-
logical activities, including cancer prevention, antiviral, or antimi-
crobial activities, is a major component of catechin derivatives
derived from tea.1 Recently, methylated derivatives, such as
(–)-300-Me-epigallocatechin gallate (300-Me-EGCG: 2), have been
identified as scarce catechins in natural tea leaves and mammal
metabolites of tea catechins.2a Because 2 and its regioisomer (400-
Me-EGCG: 3) exhibit potent inhibitory activities against type I
allergic reactions in mice2a,b and matrix metalloproteinases,2c–g
other methylated catechin derivatives are also expected to possess
therapeutic potential.
However, due to the availability of 2 and 3 from natural and
synthetic sources,3 investigations of the structure and activity rela-
tionship have been limited to these compounds. Considering natu-
rally available catechins (EGC, GC, EC, and C), the ready supply of
all possible regio- and stereoisomers of methylated catechins
(Fig. 1), should be significant in systematic biological evaluation
of these molecules. Hence, we are pursuing the concise synthesis
of methylated catechins (2–11). Although many synthetic investi-
gations of the catechin skeleton have been reported,1a including
our group,4 conversion of methylated catechins from natural cate-
chins, which can be represented epigallocatechin (EGC) (12)5 and
other derivatives (GC, EC, and C), should be suitable due to an inex-
pensive and readily available natural source.
OH
4'
OH
B
R1
R2
OH
R5
B
HO
O
C
HO
O
C
3'
A
A
O
O
R3
R4
OH
R6
R7
3"
OH
O
O
D
D
A
B
4"
OH
OH
R1
R2
R3
R4
A
(–)-EGCG (1) :
OH OH OH OH
OH OH OMe OH
OH OH OH OMe
OH OMe OH OH
OMe OH OH OH
(–)-3"-Me-EGCG (2) :
(–)-4"-Me-EGCG (3) :
(–)-3'-Me-EGCG (4) :
(–)-4'-Me-EGCG (5) :
(–)-3"-Me-ECG (6) :
(–)-4"-Me-ECG (7) :
OH
OH
H
H
OMe OH
OH OMe
R5
R6
R7
B
(–)-3"-Me-GCG (8) : OH OMe OH
(–)-4"-Me-GCG (9) : OH OH OMe
R5
H
H
R6
R7
ent-B
(+)-3"-Me-CG (10) :
(+)-4"-Me-CG (11) :
* Corresponding author.
OMe OH
OH OMe
E-mail address: kant@u-shizuoka-ken.ac.jp (T. Kan).
Present address: Institute for Chemical Research, Kyoto University, Uji, Kyoto
611-0011, Japan.
Figure 1. Structure of (ꢀ)-EGCG and methylated derivatives.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.111

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Y. Aihara et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4171–4174
OMe
The Ns group should be an ideal protecting group for labile poly-
MeI
Li2CO3
R1O
OR1
phenols because it can be easily deprotected under mild condi-
tions.6,7 Furthermore, the electron-withdrawing nature of the Ns
group should enhance the stability of polyphenol under various
conditions. Herein, we report the efficient synthesis of methylated
catechins (2–11) by exploiting the Ns protecting group for phenols.
As shown in Scheme 1, we initially investigated the selective
incorporation of the methyl group to 3- and 4-OH of allyl gallate
(14). Gallic acid (13) was converted to allyl ester 14 by treating
with allyl alcohol and EDCI. Upon treating 14 with Li2CO3 and
methyl iodide, selective deprotonation of the most acidic 4-OH
and alkylation proceeded smoothly to provide 15. On the other
hand, 3-OH selective alkylation was performed utilizing a bridged
boronic ester intermediate8 between the ortho phenolic hydroxyl
groups. After forming the boronic ester by treating borax and 14
in the presence of NaOH, methylation with Me2SO4 and subse-
quent acidic hydrolysis of the boronic ester provided 18 exclu-
sively. The Ns group into the resultant phenols of 15 and 18 was
incorporated by treating with NsCl and Et3N to give 16 and 19,
respectively. Upon treating 16 and 19 with catalytic amounts of
Pd(PPh3)4 and p-tolSO2Na,9 deprotection of the allyl group pro-
ceeded smoothly to provide desired 17 and 20, respectively.
Next, we focused on incorporating gallate derivatives of 17 and
20 into protected epigallocatechin derivatives and deprotection of
the Ns group (Scheme 2). Protection of EGC (12) with the Ns group
was carried out by treating with NsCl and Et3N to provide 21. Upon
treating 21 with 20 and EDCI, condensation reaction proceeded
smoothly to provide desired 22 in high yield. A similar condensa-
tion of 21 and 17 provided desired methylated EGCG derivatives
23. Deprotection of the Ns groups of 22 and 23 was accomplished
by treating with thiophenol and cesium carbonate to provide 300-
methylated EGCG (2) and 400-methylated EGCG (3), respectively.
During this transformation, epimerizations at the 2-position of
the benzopyran ring and decomposition of gallate ester were not
observed. A similar protocol, which employed natural catechin
DMF
68%
CO2R2
15 : R1 = H
R2 = Allyl
NsCl, Et3N
DMAP
OH
16 : R1 = Ns
Pd(PPh3)4
p-tolSO2Na
H2O / THF
R2 = Allyl
HO
OH
17 : R1 = Ns
82% (2 steps)
CO2R
Allyl alcohol
R2 = H
13 : R = H
EDCI, DMAP
87%
OR1
14 : R = Allyl
borax
aq NaOH;
MeO
OR1
Me2SO4;
conc H2SO4
85%
CO2R2
18 : R1 = H
R2 = Allyl
NsCl, Et3N
DMAP
19 : R1 = Ns
Pd(PPh3)4
p-tolSO2Na
H2O / THF
R2 = Allyl
20 : R1 = Ns
95% (2 steps)
R2 = H
Scheme 1. Synthesis of selective methylated gallic acids.
A significant task in the catechin synthesis is selecting a suitable
protecting group for phenol. Although benzyl ether has been em-
ployed for catechin synthesis due to its readily deprotectable fea-
ture under hydrogenolysis conditions, ether formation of phenol
is often troublesome, for example, epimerization of the 2-position
occurs under basic conditions. Recently, the 2-nitrobenzenesulfo-
nyl (Ns) group has been employed as a novel protecting and acti-
vating group for amines.6 Moreover, the Ns group has been used
in the synthesis of nitrogen containing complex natural products.
OH
5'
NsCl
OH
H3BO3
NaOH
7
NsO
O
OR1
3'
ONs
(–)-EGC (12)
toluene
H2O
40%
OR1
OR1
OR1
OH
5
OR1
OR1
R1O
O
ONs
24
R1O
O
OTBDPS
OR
O
OR1
OR2
OR3
OH
O
TBDPSCl
Et3N
OR1
NsO
O
ONs
C
D
OR1
OH
CH3CN
-20 °C
83%
ONs
(–)-EGC (12)
(C : R1 = H)
20, EDCI
DMAP
NsCl, Et3N
CH3CN
-20 °C
25 : R = H
22
CH2N2, CH3CN
0 °C, 91%
(D: R1 = Ns, R2 = Me
CH3CN
92%
94%
3 = Ns)
26 : R = Me
R
21
OH
1) 27, EDCI, DMAP
CH3CN, 86%
2) TBAF, AcOH
THF
(C : R1 = Ns)
OMe
OH
17, EDCI
DMAP
HO
O
23
(D: R1 = Ns, R2 = Ns
CH3CN
97%
3) PhSH, Cs2CO3
CH3CN, 0 °C
R3 = Me)
O
77% (2 steps)
OH
OH
OH
PhSH
PhSH
O
ONs
Cs2CO3
Cs2CO3
3"-Me-EGCG (2)
22
23
4"-Me-EGCG (3)
HO2C
ONs
CH3CN
0 °C
81%
CH3CN
0 °C
81%
OH
4'-Me-EGCG (5)
Scheme 3. Synthesis of 40-Me-EGCG (5).
ONs
27
Scheme 2. Synthesis of 300-Me-EGCG (2) and 400-Me-EGCG (3).

Y. Aihara et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4171–4174
OMe
4173
of the amino group, the by-product, 2-(2-nitrophenylthio)aniline
(30), can be removed by washing the ethereal layer with a 1 M
HCl solution. Additionally, combining the selective methylation
strategy on the B- and D-rings of EGCG should yield several types
of double methylated EGCG derivatives. Furthermore, this regiose-
lective modification of EGCG should be applicable for alkylation as
well as acylation; thus, employing this synthetic strategy with the
other natural catechins (GC, EC, and C) suggests the possibility of
constructing a diverse catechin library.
With a variety of methylated catechin derivatives in hand, a
preliminary biological investigation tested the inhibition of matrix
metalloproteinases. As shown in Table 1, inhibitory activities were
examined in a recombinant matrix metalloproteinases (r-MMP-2
and r-MMP-7) and a recombinant membrane-type 1 metallopro-
tease (r-MT1-MMP)14,15 where catalytic domains were expressed
in Escherichia coli. Monomethylated catechins (2, 3, 6–10) exhib-
ited inhibitory activity against r-MMPs as shown in Table 1. The
IC50 values against r-MMPs varied according to the position of
methyl substitution. Among gallocatechin type derivatives, 300-Me
analogs (2, 8) displayed more potent inhibitory activity to r-MT1-
MMP and r-MMP-2 than 400-Me analogs (3, 9). In the case of r-
MMP-7, methylation decreased the inhibitory activity. The weaker
inhibitory activities of 6, 7, 10 and 11 against r-MMP-7 suggest
that the number of hydroxyl group is important for intensity and
specificity.
1) NsCl, Et3N
CH3CN, -20 °C
95%
ONs
NsO
O
25
ONs
2) TBAF, AcOH
THF, 0 °C, 86%
3) CH2N2, CH3CN
0 °C, 91%
OH
ONs
28
OMe
1) 20, EDCI
DMAP, CH 3CN
OH
OH
HO
O
2) o-H2NC6H4SH
Cs2CO3
CH3CN, 0 °C
63% (2 steps)
O
OH
OMe
OH
O
NO2
NH2
S
OH
3', 3"-diMe-EGCG (29)
30
Scheme 4. Synthesis of 30, 300-diMe-EGCG (29).
derivatives (GC, EC, and C), provided desired methylated gallate
catechin derivatives 4–11.10
Then we turned our attention to selectively incorporating a
methyl group at the B-ring (Scheme 3). Because to our knowledge,
(–)-EGCG derivatives methylated at the B-ring have yet to be re-
ported, a SAR study of these compounds should be significant.
However, there are few reports on selectively modifying the five
phenolic hydroxyl groups of EGC. We found that a bridged boronic
ester intermediate effectively distinguishes the hydroxyl groups at
the A-ring and B-ring. Treating 12 with NsCl and H3BO3 in the pres-
ence of NaOH11 causes the regioselective sulfonylation to proceed
to afford predominantly 30,5,7-Ns-EGCG (24). Next, selective incor-
poration of the TBDPS group at the less hindered hydroxyl group
(50-OH) was accomplished by treating with TBDPSCl and Et3N to
give 25. The 40-methylated EGCG derivatives were prepared after
methylation of 25 by diazomethane, condensation of 26 with Ns-
protected gallic acid 27,12 and stepwise deprotection of TBDPS
and Ns group to yield 40-Me-epigallocatechin gallate (40-Me-EGCG:
5).
In summary, we have developed an efficient synthetic method
to prepare methylated catechin derivatives utilizing the Ns pro-
tecting group for phenol. Furthermore, the present synthetic strat-
egy for regioselective alkylation at the B-ring and gallate group
should readily provide additional derivatives. Further exploitation,
including probing the biological activities, is under investigation in
our laboratory.
Acknowledgments
The authors thank Dr. Masayuki Suzuki (Mitsui Norin Co., Ltd)
for kindly providing (–)-ECGC, (–)-EGC, (–)-EC, and (–)-GC samples.
The authors also acknowledge Professor Tohru Fukuyama (Gradu-
ate School of Pharmaceutical Sciences, The University of Tokyo) for
his fruitful discussions. This work is financially supported by Tak-
eda Science Foundation, Naito Foundation, Nagase Science and
Technology Foundation, and a Grant-in-Aid for Scientific Research
on Priority Areas from the Ministry of Education, Culture, Sports,
Science and Technology (MEXT).
As shown in Scheme 4 and 30,300-diMe-EGCG (29) was also syn-
thesized from 25. Protection of 25 with a Ns group, deprotection of
the TBDPS group, and incorporation of a methyl group afforded 28.
Although condensation of 28 and 27, and deprotection of Ns group
readily provided 30-Me-EGCG (4), a modified preparation with dou-
ble methylated derivatives was demonstrated. After condensation
28 and 20, deprotection of the Ns group was accomplished using
2-aminothiophenol instead of thiophenol to afford 30,300-diMe-
EGCG (29).13 The advantage of this method is 2-aminothiophenol
is odorless compared to thiophenol. Furthermore, taking advantage
Supplementary data
Supplementary data (general experimental procedures and
characterization for all new compounds) associated with this arti-
cle can be found, in the online version, at doi:10.1016/j.bmcl.
2009.05.111.
References and notes
Table 1
Inhibitory effects on MMPs
1. For recent reviews on (–)-EGCG, see: (a) Nagle, D. G.; Ferreira, D.; Zhou, Y. D.
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Catechins
IC50 (lM)
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Sano, M. J. Agric. Food. Chem. 2000, 48, 5649; (g) Tachibana, H.; Sunada, Y.;
Miyase, T.; Sano, M.; Maeda-Yamamoto, M.; Yamada, K. Biosci., Biotechnol.,
Biochem. 2000, 64, 452.
r-MT1-MMP
r-MMP-7
r-MMP-2
(ꢀ)-EGCG (1)
6.8
1.7
12.5
15.6
3.7
3.1
10.5
52
21.5
20
72
>100
>100
20
32
>100
>100
9.6
8.7
18.7
88
34
5.4
19
(ꢀ)-300-Me-EGCG (2)
(ꢀ)-400-Me-EGCG (3)
(ꢀ)-300-Me-ECG (6)
(ꢀ)-400-Me-ECG (7)
(ꢀ)-300-Me-GCG (8)
(ꢀ)-400-Me-GCG (9)
(+)-300-Me-CG (10)
(+)-400-Me-CG (11)
28
22
>100

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Y. Aihara et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4171–4174
3. For synthesis of 400-MeEGCG from EGCG, see: (a) Miyase, T.; Sano, M. Jpn. Kokai
Tokkyo Koho 2001, 253879; For solid phase synthesis of a benzyl protected
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Nitta, M.; Furuta, T.; Kan, T. Synlett 2008, 3234.
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2008, 108, 228.
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Fukuyama, T. Chem. Commun. 2004, 353.
7. During the course of this investigation, employment of Ns ester as a protecting
group for phenol has been independently reported, see: Koyama, Y.;
Yamaguchi, R.; Suzuki, K. Angew. Chem., Int. Ed. 2008, 47, 1084.
8. Chang, J.; Chen, R.; Guo, R.; Dong, C.; Zhao, K. Helv. Chem. Acta. 2003, 86,
2239.
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soft nucleophile is required, see: Honda, M.; Morita, H.; Nagakura, I. J. Org.
Chem. 1997, 62, 8932.
10. Detailed synthetic procedures are described in the Supplementary data.
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12. Synthesis of 27 was conducted by protection of 14 with the Ns group and
deprotection of the allyl ester. Detailed synthetic procedures are described in
the Supplementary data.
13. Similar deprotection of the Ns group using a similar odorless thiol 4-carboxy
phenylthiol has been reported, see: Matoba, M.; Kajimoto, T.; Node, M. Synth.
Commun. 2008, 38, 1194.
14. Purification and refolding of recombinant human proMMP-7 and expression in
Escherichia coli, and its characterization has been reported, see: Itoh, M.;
Masuda, K.; Ito, Y.; Akizawa, T.; Yoshioka, M.; Imai, K.; Okada, Y.; Sato, H.; Seiki,
M. J. Biochem. 1996, 119, 667.
15. Inhibitory effect of green tea polyphenols on membrane-type
1 matrix
metalloproteinase has been reported, see: Ref. 2b.