Concise synthesis of dideoxy-epigallocatechin gallate (DO-EGCG) and evaluation of its anti-influenza virus activity

Article abstract of DOI:10.1016/j.bmcl.2007.03.041

Dideoxy-epigallocatechin gallate (DO-EGCG) (2), a simplified analog of naturally occurring EGCG (1), was efficiently prepared by directly introducing a ketone group at C3 and successive reduction to the sec-alcohol with 2,3-cis stereochemistry. Compound 2 showed potent anti-influenza virus activity, indicating that the hydroxyl substituents on the A-ring are not crucial for anti-influenza virus activity.

Full text of DOI:10.1016/j.bmcl.2007.03.041

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3095–3098
Concise synthesis of dideoxy-epigallocatechin gallate
(DO-EGCG) and evaluation of its anti-influenza virus activity
Takumi Furuta,a,* Yasuo Hirooka,a Ayako Abe,a Yusuke Sugata,a Mitsuru Ueda,a
Kouki Murakami,b Takashi Suzuki,b Kiyoshi Tanakaa and Toshiyuki Kana,*
aDepartment of Synthetic Organic & Medicinal Chemistry, School of Pharmaceutical Sciences,
University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
bDepartment of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, CREST,
JST, and COE Program in the 21st Century, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan
Received 2 February 2007; revised 12 March 2007; accepted 13 March 2007
Available online 16 March 2007
This paper is dedicated to the memory of the late Professor Kiyoshi Tanaka, who passed away on December 8, 2004.
Abstract—Dideoxy-epigallocatechin gallate (DO-EGCG) (2), a simplified analog of naturally occurring EGCG (1), was efficiently
prepared by directly introducing a ketone group at C3 and successive reduction to the sec-alcohol with 2,3-cis stereochemistry. Com-
pound 2 showed potent anti-influenza virus activity, indicating that the hydroxyl substituents on the A-ring are not crucial for anti-
influenza virus activity.
Ó 2007 Elsevier Ltd. All rights reserved.
(À)-Epigallocatechin gallate (EGCG) (1),1 which is a
major constituent of green tea extract, has received spe-
cial attention for its cancer preventive,2 antiviral3, and
other important bioactivities.4 Although 1 is expected
to be a promising candidate for drug development, the
structure–activity relationship of 1 has not been suffi-
ciently investigated due to the limited availability of
EGCG derivatives, which are mainly provided from nat-
ural sources. Furthermore, selectively modifying the
densely substituted hydroxyl groups of 1 is difficult.
Because epigallocatechin derivative 3 should be easily in-
stalled by the reduction of the ketone functional group at
C3, compound 4 should be a key intermediate.7 Ketone 4
may be directly prepared from nitrochromene derivative
5, which possesses a tricyclic system that corresponds to
the A, B, and C-rings of the catechin framework. We envi-
sioned that nitrochromene 5 may be constructed by the
ring formation between two segments, salicylaldehyde (6)
and nitroolefin derivative 7. This divergent synthesis may
be applicable to EGCG related derivatives (Scheme 1).
Among the naturally occurring stereoisomeric catechins,
including (+)-catechin (2,3-trans), compound 1, which
possesses 2,3-cis stereochemistry, exhibits the most po-
tential bioactivities.
During the course of our investigation, we concisely syn-
thesized dideoxy-epigallocatechin gallate (DO-EGCG)
(2). Moreover, we found that 2 exhibits potent anti–
influenza virus activity, although this analog has simpli-
fied structure of 1 without the phenolic hydroxyl groups
on the A-ring. Herein, the detailed synthesis of 2 and the
preliminary investigation of its anti–influenza virus
activity are described (Fig. 1).
Therefore, an easy route to structurally diversified EGCG
analogs is needed.5 However, examples of a selective syn-
thesis of 1 and its related derivatives are limited due to the
difficulty of selectively constructing 2,3-cis stereogenic
centers on a C-ring where the chiral center at the C2 posi-
tion is easily epimerized under basic conditions.5,6
Initially a one-pot nitroaldol and dehydration reaction
of 3,4,5-tribenzyloxybenzaldehyde (8)5b with nitrometh-
ane under Knoevenagel conditions was performed to
afford desired 9 in good yield (Scheme 2).
Keywords: Epigallocatechin gallate; Nitrochromene derivative; Nef
reaction; Anti-influenza virus activity.
The successive ring construction between 9 and 6 in the
presence of a catalytic amount of DABCO8 was
*
Corresponding authors. E-mail: furuta@u-shizuoka-ken.ac.jp
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.03.041

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T. Furuta et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3095–3098
unstable under atmospheric conditions, the product
OR
OR
OR
OR
from the TiCl3 reaction was directly subjected without
purification to further reduction with L-Selectride to
give 12 as a single product with the desired stereochem-
istry.14 The relative configuration of 12 was confirmed
by the coupling constant for the protons at C2 and C3
OR
OR
selective
reduction
O
O
OH
O
3
4
3
1
on H NMR.6
OH
H
OR
B
OR
After condensation with 3,4,5-tribenzyloxybenzoic acid
(13),15 all of the benzyl groups were removed under cat-
alytic hydrogenation conditions with Pd(OH)2 to afford
2,16 which cannot be theoretically obtained through a
polyketide biosynthetic pathway.
OR
OR
OR
OR
O
6
O
C
A
ring
construction
NO2
O2N
7
5
Because EGCG is known to possess anti-influenza
virus activity,3a we then moved to a preliminary evalu-
ation of the activity of 2.17 As shown in Table 1, cate-
chin 2 showed a potent inhibitory effect of the infection
of the influenza virus (A/memphis/1/71, H3N2) to
MDCK cells with 11.92 lM of IC50, which is three
times higher than that of 1 (IC50: 41.25 lM). This re-
sult suggests that the hydroxyl groups on the A-ring
are unnecessary for the inhibition of the influenza virus
infection.
Scheme 1. Synthetic strategy.
OH
B
OH
OH
2
R
O
C
A
R
O
3
OH
OH
O
D
1: R = OH
2: R = H
OH
Furthermore, considering this potent bioactivity,
further chemical modifications such as introducing a
biotin tag and/or photoactivatable groups such as ben-
zophenone might be possible on the A-ring without
losing the bioactivities.18 This is advantageous for fur-
ther development of EGCG probe molecules.
Figure 1. EGCG (1) and dideoxy-EGCG (2).
achieved via a formal oxy-conjugate addition of the
phenolic hydroxyl group of 6 to 9, and subsequent
nitroaldol and condensation reactions gave desired
nitrochromene derivative 10 in moderate chemical
yield.9
Table 1. Inhibition of influenza A virus infection to MDCK cells
Next, we focused on the direct transformation of the
nitroolefin moiety to the ketone functional group.
Although several Nef-type reactions are available for
the transformation,10 only reductive conditions with
TiCl3 reliably afforded ketone derivative 11 without
the oxime intermediate.12,13 Ketone 11 was relatively
a
Compound
Complement inhibition IC50 (lM)
2
EGCG (1)
11.92 ( 4.50)
41.25 ( 15.45)
11
a Values are means of three experiments, standard deviation is given in
parentheses.
OBn
OH
OBn
OBn
OBn
OBn
OBn
OBn
OBn
O
6
CHO
b
a
OBn
OHC
NO2
O2N
10
8
9
OBn
OBn
OBn
OBn
OBn
OBn
OBn
OBn
OBn
HOOC
13
c
d
O
O
2
e, f
O
OH
11
12
Scheme 2. Preparation of dideoxy-EGCG (2). Reagents and conditions: (a) CH3NO2 (3.0 equiv), piperidine (3.0 equiv), AcOH (3.0 equiv), toluene,
80 °C, 63%; (b) salicylaldehyde (6) (1.5 equiv), DABCO (10 mol%), CH2Cl2, sealed tube, 60 °C, 54%; (c) TiCl3 (5.0 equiv), AcONH4 (2.0 equiv), 1,4-
dioxane–50% AcOH (5:1), rt; (d) L-Selectride (2.0 equiv), THF, rt, 38% (2 steps); (e) 3,4,5-tribenzyloxybenzoic acid (13) (1.0 equiv), EDCI
(1.5 equiv), DMAP (0.1 equiv), CH2Cl2, rt, 69%; (f) Pd(OH)2, H2, THF–MeOH (5:1), rt, 57%.

T. Furuta et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3095–3098
RO
3097
In conclusion, the synthesis of EGCG analog 2 has been
OH
performed via a concise synthetic route with the selective
construction of the 2,3-cis stereochemistry. The method-
ology presented here should be easily applicable to fur-
ther functionalized analogs. Thus, this methodology
should promote a detailed structure–activity relation-
ship study. Further preparation of EGCG derivatives,
investigation of the anti-influenza virus activity mecha-
nism, and a survey of other bioactivities such as anti-
Alzheimer’s disease effect4 are currently underway.
CHO
14: R = Bn
15: R = Ms
OR
10. For a review of Nef-type reaction, see: Ballini, R.; Petrini,
M. Tetrahedron 2004, 60, 1017.
11. For other examples of the direct conversion of
a
nitroalkene to a carbonyl group using TiCl3, see: (a)
Katoh, T.; Nishide, K.; Node, M.; Ogura, H. Heterocycles
1999, 50, 833; (b) Ballini, R.; Fiorini, D.; Palmieri, A.
Tetrahedron Lett. 2004, 45, 7027.
12. The reductive conditions with Zn/AcOH and Pb/AcOH
afforded only oxime derivative 16.
References and notes
OBn
OBn
1. For a review of chemistry and bioactivities of EGCG, see:
Nagle, D. G.; Ferreira, D.; Zhou, Y.-D. Phytochemistry
2006, 67, 1849.
O
OBn
2. For examples, see: (a) Tachibana, H.; Koga, K.; Fujimura,
Y.; Yamada, K. Nature Struct. Mol. Biol. 2004, 11, 380;
(b) Fujiki, H. The Chemical Record 2005, 5, 119.
3. For examples, see: (a) Nakayama, M.; Suzuki, K.; Toda,
M.; Okubo, S.; Hara, Y.; Shimamura, T. Antiviral Res.
1993, 21, 289; (b) Yamaguchi, K.; Honda, M.; Ikigai, H.;
Hara, Y.; Shimamura, T. Antiviral Res. 2002, 53, 19; (c)
Song, J.-M.; Lee, K.-H.; Seong, B.-L. Antiviral Res. 2005,
68, 66.
4. For the modulation of b-amyloid precursor protein
cleavage, see: Rezai-Zadeh, K.; Shytle, D.; Sun, N.; Mori,
T.; Hou, H.; Jeanniton, D.; Ehrhart, J.; Townsend, K.;
Zeng, J.; Morgan, D.; Hardy, J.; Town, T.; Tan, J.
J. Neurosci. 2005, 25, 8807.
5. For an example of asymmetric synthesis of EGCG, see: (a)
Li, L.; Chan, T. H. Org. Lett. 2001, 3, 739; For examples
of B- and D-ring modified analog of EGCG, see: (b)
Zaveri, N. T. Org. Lett. 2001, 3, 843; (c) Wan, S. B.;
Landis-Piwowar, K. R.; Kuhn, D. J.; Chen, D.; Dou, Q.
P.; Chan, T. H. Bioorg. Med. Chem. 2005, 13, 2177; (d)
Anderson, J. C.; Headley, C.; Stapleton, P. D.; Taylor, P.
W. Tetrahedron 2005, 61, 7703; For an example of 3-
amino derivative of EGCG, see: (e) Anderson, J. C.;
Headley, C.; Stapleton, P. D.; Taylor, P. W. Bioorg. Med.
Chem. Lett. 2005, 15, 2633.
6. Recently an efficient synthesis of EGCG, including the
direct formation of 2,3-cis stereochemistry by reductive
intramolecular etherification, was reported. See: Kitade,
M.; Ohno, Y.; Tanaka, H.; Takahashi, T. Synlett 2006,
2827.
7. For examples, see: Ref. 5a,b. In Ref. 5a, a satisfactory
yield of 2,3-cis derivative was obtained using this strategy,
but a small amount of 2,3-cis stereochemistry was directly
constructed via an intramolecular substitution reaction of
the phenolic hydroxyl group and the benzyl bromide
moiety.
8. To construct this ring, DABCO is the most effective. Other
basic conditions using Et3N, DBU, CsF, and NaH as well
as acidic conditions employing BF3 Æ Et2O, SnCl4, and
acetic acid were ineffective. These results suggest that the
reaction proceeds via Baylis-Hillman type catalytic cycles.
For other examples with similar ring formations, see: (a)
Yan, M.-C.; Jang, Y.-J.; Yao, C.-F. Tetrahedron Lett.
2001, 42, 2717; (b) Yao, C.-F.; Jang, Y.-J.; Yan, M.-C.
Tetrahedron Lett. 2003, 44, 3813.
N
16
OH
13. The one-pot transformation of the nitrochromene deriv-
ative to the ketone derivative, which includes two steps
(NaBH4 reduction and CrCl2 reduction in 10% aqueous
HCl), has been reported. However, further selective
reduction to the 2,3-cis catechin derivative has not been
investigated. See: Rao, T. S.; Trivedi, G. K. Indian J.
Chem. 1985, 24B, 1159.
14. Experimental procedure for 12: TiCl3 (872 lL,
1.35 mmol) was added to a solution of 10 (154 mg,
0.266 mmol) and AcONH4 (43 mg, 0.54 mmol) in
dioxane –50% AcOH (5:1, 2.5 mL) under an Ar atmo-
sphere. The mixture was stirred for 17 h at room
temperature. Then H2O was added to the reaction
mixture, extracted with EtOAc, dried over anhydrous
MgSO4, and evaporated to afford a mixture (166 mg)
containing 11 as a yellow oil. The mixture (166 mg)
in THF (4 mL) was cooled at À78 °C and
L-Selectride (1.0 M solution in THF, 540 lL, 0.54 mmol)
was added at À78 °C under an Ar atmosphere. The
mixture was allowed to warm to room temperature and
stirred for 24 h. Then satd. NaHCO3 aq was added to
the reaction mixture, extracted with EtOAc, washed with
brine, dried over anhydrous MgSO4, and evaporated.
The residue was purified by chromatography on a silica
gel column (n-hexane–EtOAc, 3:1) to afford 12 (55 mg,
38%, 2 steps) as a yellow oil. Spectral data of 12: 1H
NMR (500 MHz, CDCl3): d 2.95 (dd, J = 1.8, 16.4 Hz,
1H), 3.23 (dd, J = 4.3, 16.4 Hz, 1H), 4.22 (br s, 1H),
4.97 (s, 1H), 5.07 (s, 2H), 5.14 (s, 4H), 6.82 (s, 2H), 6.9–
7.5 (m, 19H); MS (FAB) m/z 545 (M+H)+; HRMS calcd
for C36H33O5 (M+H)+ 545.2328. found 545.2337.
15. Nakazono, M.; Ma, L.; Zaitsu, K. Tetrahedron Lett. 2002,
43, 8185.
16. Spectral data of 2: 1H NMR (270 MHz, acetone-d6): d 2.97
(d, J = 17.8 Hz, 1H), 3.41 (dd, J = 4.0, 17.8 Hz, 1H), 5.16
(s, 1H), 5.56 (br s, 1H), 6.63 (s, 2H), 6.98 (s, 2H), 6.8–7.2
(m, 4H); MS (FAB) m/z 427 (M+H)+; HRMS calcd for
C22H19O9 (M+H)+ 427.1029. found 427.1054.
17. A general procedure for the Inhibition of the influenza
virus infection by the analog of catechin. Each sample
(1 mg/ml) in a serum free medium (SFM) (hybridoma-
SFM complete DPM, Invitrogen Corp. NY, USA) was
serially diluted twofold with SFM. Seventy-five micro-
liters of each sample dilution was mixed with 75 ll of an
influenza virus A/Memphis/1/71 (H3N2) suspension
(100 FFU) in SFM and then incubated at 4 °C for
1 h. Confluent monolayers of Madine-Darby canine
9. Bn and Ms protected hydroxyl groups to, respectively,
construct rings with A-ring moieties 14 and 15, which both
possess electron donation and withdrawing groups, were
unsuccessful. In both cases, only the starting aldehydes
were recovered.

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T. Furuta et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3095–3098
kidney (MDCK) cells in 96-well microplates (Corning
caused 50% inhibition of FFU was determined by
plotting the percentage inhibition against the concentra-
tion of each sample. (a) Suzuki, T.; Takahashi, T.; Guo,
C.-T.; Hidari, IP. J. K.; Miyamoto, D.; Goto, H.;
Kawaoka, Y.; Suzuki, Y. J. Virol. 2005, 79, 11705; (b)
Aoki, C.; Hidari, IP. J. K.; Itonori, S.; Yamada, A.;
Takahashi, N.; Kasama, T.; Hasebe, F.; Islam, M. A.;
Hatano, K.; Matsuoka, K.; Taki, T.; Guo, C.-T.;
Takahashi, T.; Sakano, Y.; Suzuki, T.; Miyamoto, D.;
Sugita, M.; Terunuma, D.; Morita, K.; Suzuki, Y.
J. Biochem. 2006, 139, 607.
Costar Corporation, Cambridge, MA) were inoculated
with 100 ll of the mixture at room temperature. After
1 h at 34 °C, the inoculum was removed from each
plate, the monolayers were washed three times with
PBS, and incubated for 16 h at 34 °C in 100 ll SFM.
The monolayers in each well were washed three times
with PBS, fixed with 50 ll of methanol at room
temperature for 5 min, and washed three more times
with PBS. Infectious foci of cells were detected by focus-
forming assay as previously described (a,b) using an
Anti-NP monoclonal antibody and horseradish peroxi-
18. (a) Kan, T.; Tominari, Y.; Morohashi, Y.; Natsugari, H.;
Tomita, T.; Iwatsubo, T.; Fukuyama, T. Chem. Commun.
2003, 2244; (b) Morohashi, Y.; Kan, T.; Tominari, Y.;
Fuwa, H.; Okamura, Y.; Watanabe, N.; Sato, C.;
Natsugari, H.; Fukuyama, T.; Iwatsubo, T.; Tomita, T.
J. Biol. Chem. 2006, 281, 14670.
dase–conjugated goat anti-mouse immunoglobulin
M
G
plus (IgG + M) antibody. Infectious cells were
defined as the mean of three counts of blue-stained
cells within one well. The virus infection was determined
as focus-forming units (FFU). A concentration that