Synthesis of (-)-epicatechin 3-(3-O-methylgallate) and (+)-catechin 3-(3-O-methylgallate), and their anti-inflammatory activity

Article abstract of DOI:10.1002/cbdv.200800224

A concise synthesis of (-)-epicatechin 3-(3-O-methylgallate) (1; ECG3′′Me), which is a minor constituent of tea, and (+)-catechin 3-(3-O-methylgallate) (2; CG3′′Me) via condensation of equimolar amount of catechin and gallate derivatives has been achieved

Full text of DOI:10.1002/cbdv.200800224

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Synthesis of (ꢀ)-Epicatechin 3-(3-O-Methylgallate) and (þ)-Catechin 3-(3-O-
Methylgallate), and Their Anti-Inflammatory Activity
by Takashi Iijimaa), Yoshihiro Mohria), Yasunao Hattorib)c), Atsushi Kashimad), Tsunashi Kamod),
Mitsuru Hirotad), Hiromasa Kiyotae), and Hidefumi Makabe*a)
a) Sciences of Functional Foods, Graduate School of Agriculture, Shinshu University,
8304 Minami-minowa, Kami-ina, Nagano 399-4598, Japan
(phone: þ81-265-77-1630, fax: þ81-265-77-1700, e-mail: makabeh@shinshu-u.ac.jp)
b) Interdisciplinary Graduate School of Science and Technology, Shinshu University,
8304 Minami-minowa, Kami-ina, Nagano 399-4598, Japan
c) Satellite Venture Business Laboratory, Shinshu University, 3-15-1, Tokida, Ueda, Nagano 386-8567,
Japan
d) Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University, 8304
Minami-minowa, Kami-ina, Nagano 399-4598, Japan
e) Department of Applied Bioorganic Chemistry, Division of Bioscience & Biotechnology for Future
Bioindustry, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiya,
Aoba-ku, Sendai 980-8578, Japan
A concise synthesis of (ꢀ)-epicatechin 3-(3-O-methylgallate) (1; ECG3’’Me), which is a minor
constituent of tea, and (þ)-catechin 3-(3-O-methylgallate) (2; CG3’’Me) via condensation of equimolar
amount of catechin and gallate derivatives has been achieved. The anti-inflammatory effect of the
synthetic compounds on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation of mouse
ears was examined. Compounds 1 and 2 suppressed the TPA-induced inflammation of mouse ears by 50
and 43%, respectively, at a dose of 200 mg. Their activities are stronger than those of indomethacin and
glycyrrhetinic acid, the normally used anti-inflammatory agents.
Introduction. – It is well-known that tea (Camellia sinensis) and its constituents
have a variety of pharmacological properties [1] such as anti-allergic [2][3], anti-
oxidative [4], anticarcinogenic [5], anti-atherosclerotic [6], and antibacterial activities
[7]. Especially (ꢀ)-epigallocatechin 3-(3-O-methylgallate) (EGCG3’’Me) attracted
most attention because of its strong anti-allergic activity [1]. On the other hand, little
has been known about the biological activity of (ꢀ)-epicatechin 3-(3-O-methylgallate)
(ECG3’’Me; 1), which is a minor constituent of tea isolated by Saijo [8]. We thus
decided to synthesize ECG3’’Me (1) and its diastereoisomer, CG3’’Me (2), to
investigate their biological activities. To the best of our knowledge, the synthesis of 1
was reported by Chan and co-workers; however, the key condensation step needed an
excess amount of gallate (¼ 3,4,5-trihydroxybenzoate) derivative [9]. We have
developed a more concise and efficient synthesis of 1 and 2 using equimolar amounts
of catechin and gallate derivatives. Here, we describe the synthesis of 1 and 2, and their
anti-inflammatory activities (Fig.).
Results and Discussion. – 1. Synthesis. We have chosen tetrabenzylated epicatechin
(3) [10] and/or tetrabenzylated catechin (4) [11], an alcohol unit, prepared according to
ꢀ 2009 Verlag Helvetica Chimica Acta AG, Zꢁrich

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
521
Figure. The structures of ECG3’’Me (1), CG3’’Me (2), and EGCG3’’Me
the Kawamotoꢂs procedure [11], and carboxylic acid part 6, prepared from commer-
cially available 3,4-dihydroxy-5-methoxybenzaldehyde via protection of the phenolic
OH group as benzyl ether and subsequent oxidation with Ag2O (Scheme 1).
Condensation of equimolar amount of 3 and 6 was examined using various reagents
(Table 1), i.e., 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride
(EDCI), dicyclohexylcarbodiimide (DCC), and N,N’-diisopropylcarbodiimide
(DIC). We found that EDCI was the most effective condensing reagent. Using
2.0 equiv. of EDCI, 7 (cf. Scheme 3) was obtained in good yield. Although we tried the
combination of O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate (HATU) and 1-hydroxy-7-azabenzotriazole (HOAt) as condensing re-
agents, the condensed product was not observed at all. In the case of tetrabenzylated
catechin, 4, using 1.0 equiv. of DCC led to 8 in 77% yield, whereas EDCI afforded only
26% yield (Scheme 2).
Condensed products 7 and 8 were subjected to the debenzylation by Pd(OH)2
under the H2 atmosphere in THF/MeOH/H2O to give 1 and 2, respectively, in good
yields (Scheme 3).
2. Anti-Inflammatory Activity. The mouse-ear inflammation test was used to
evaluate the anti-inflammatory activity of each synthetic compound. The anti-
inflammatory activities of 1 and 2 are summarized in Table 2 [12]. Compounds 1 and
2 suppressed the TPA-induced edema up to IE of 50 and 43%, respectively, at 200 mg
when painted on the mouse ear. The different configuration at C(3) does not seem to
affect the activity. Indomethacin and glycyrrhetinic acid, the normally used anti-

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Scheme 1. Preparation of Alcohol Units 3 and 4, and Carboxylic Acid 6
Scheme 2. Equimolar Condensation Reaction between 4 and 6
inflammatory agents, only inhibited up to IE of 16 and 24%, respectively, at a 200 mg
application. Recently, Mizushina et al. reported the anti-inflammatory activity of (ꢀ)-
epicatechin gallate (ECG) and (þ)-catechin gallate (GC) [13]. According to their
report, ECG and GC suppressed the TPA-induced edema up to IE of 25.2 and 27.0%,
respectively, at 250 mg application. The MeO group at C(3’’) seems to play an important
role for the activity. Mizushina et al. also reported the inhibition of DNA polymerase a
(pol a) by catechin derivatives [13]. Their report showed that the inhibition of pol a by
catechin derivatives had a high correlation with the anti-inflammatory activity. Thus,

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
523
Table 1. Equimolar Condensation between 3 and 6a)
Reagentb)
Equiv.
Yield of 7 [%]
DCC
DCC
DIC
DIC
DIC
1.0
1.5
1.5
2.0
1.0
1.0
2.0
30c)
35
46
58
47
EDCI
EDCI
57
68
a) All reactions were carried out in CH2Cl2 at room temperature for 48 h. b) DCC¼N,N’-Dicyclohex-
ylcarbodiimide; DIC¼N,N’-diisopropylcarbodiimide; EDCI¼1-ethyl-3-[3-(dimethylamino)propyl]car-
bodiimide hydrochloride. c) The reaction time was 24 h.
Scheme 3. Synthesis of 1 and 2
tea catechin derivative 1 could be considered a possible candidate as an anticancer
agent (Table 2).

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Table 2. Anti-Inflammatory Activities of Compounds 1 and 2 in the Mouse-Ear Inflammatory Test
Test compound
Inhibitory effect (IE) [%]
1
2
50ꢂ1.44
43ꢂ1.43
16ꢂ0.77
24ꢂ1.18
Indomethacin
Glycyrrhetinic acid
Conclusions. – A concise synthesis of ECG3’’Me (1) and CG3’’Me (2) was achieved
via equimolar condensation of catechin and gallate derivatives. The anti-inflammatory
activities of 1 and 2 were investigated. The activities of these compounds were higher
than those of indomethacin and glycyrrhetinic acid, the normally used anti-inflamma-
tory agents. The MeO group at C(3’’) seems to play an important role for the activity.
Experimental Part
General. (þ)-Catechin was purchased from Nacalai Co., Ltd. and (ꢀ)-epicatechin from Sigma-
Aldrich Co., Ltd. TPA was purchased from Sigma-Aldrich Co., Ltd. HPLC: Shimadzu LC-10AT VP
pump and Shimadzu SPD-10A VP UV/VIS detector, with YMC Pack Pro C18 RS 250ꢁ10-mm I.D. S-
5 mm, 8-nm column. Column chromatography (CC): Merck silica gel 60 (70–230 mesh). Prep. TLC:
Merck silica gel 60 PF254. Cartridge CC: Waters Sep-Pakꢀ Plus C18 cartridges. M.p.: Yanako MP-J3;
uncorrected. Optical rotations: Jasco DIP 1000 polarimeter. IR: Jasco FT-IR 480 Plus IR spectropho-
tometer; in cmꢀ1. 1H- and 13C-NMR: Bruker Avance DRX 500 spectrometer; at 500.1 MHz for 1H (rel. to
Me4Si (¼0 ppm)) and 125 MHz for 13C (rel. to CHCl3 (¼ 77.0 ppm) or Me4Si (¼0 ppm, in case of
CD3OD)); in CDCl3 or CD3OD; d in ppm, J in Hz. MS: Jeol JMS-700 spectrometer; in m/z; NOBA¼3-
nitrobenzyl alcohol.
3,4-Bis(benzyloxy)-5-methoxybenzaldehyde (5). To a stirred soln. of 3,4-dihydroxy-5-methylbenz-
aldehyde (2.00 g, 12.0 mmol) in DMF (20 ml) was added K2CO3 (5.00 g, 35.7 mmol) under Ar. After
stirring for 30 min, BnBr (3.60 ml) was added dropwise, and the mixture was stirred overnight. The
reaction was quenched with sat. aq. NH4Cl, and the mixture was extracted with AcOEt. The org. phase
was washed with H2O and brine, and dried (MgSO4). Filtration, concentration, and CC (silica gel;
hexane/AcOEt 5 :1) afforded 4.63 g (13.3 mmol, quant.) of 5. Colorless solid. M.p. 85.5–87.58.
Spectroscopic data of 5 were in good agreement with those reported in [14].
3,4-Bis(benzyloxy)-5-methoxybenzoic acid (6). To a stirred soln. of 5 (520 mg, 1.49 mmol) in H2O
(250 ml) was added NaOH (30.0 g, 750 mmol). The mixture was cooled to 08, and Ag2O (693 mg,
2.98 mmol) was added. After the mixture had been stirred for 24 h, the mixture was neutralized with aq.
HCl and filtered. The filtrate was extracted with AcOEt, and the org. phase was washed with H2O and
brine, and dried (MgSO4). Filtration, concentration, and purification by prep. TLC (hexane/AcOEt 1:2)
afforded 326 mg (0.896 mmol, 60%) of 6. Colorless powder. M.p. 149.0–152.08. IR: 3031, 2938, 2643,
1686, 1587, 1423, 1328, 1232, 1125, 970, 914, 865, 736, 696. 1H-NMR: 3.90 (s, 3 H); 5.14 (d, J¼7.0, 4 H);
7.26–7.44 (m, 12 H). 13C-NMR: 56.2; 75.0; 107.0; 109.2; 124.2; 127.5; 128.0 (2 C); 128.2; 128.4; 128.5;
136.5; 137.3; 142.4; 152.3; 153.5; 155.7; 171.7 [15].
(2R,3R)-5,7-Bis(benzyloxy)-2-[3,4-bis(benzyloxy)phenyl]-3,4-dihydro-2H-1-benzopyran-3-yl 3,4-
Bis(benzyloxy)-5-methoxybenzoate (7). To a soln. of 6 (16.5 mg, 0.045 mmol) in CH2Cl2 (2 ml) was
added 4-(dimethylamino)pyridine (DMAP; 0.6 mg, 5 mmol), and the mixture was stirred for 10 min.
Then, a soln. of EDCI (17.3 mg, 0.090 mmol) was added to the mixture at 08, and the mixture was stirred
for 10 min. A soln. of 3 (29.3 mg, 0.045 mmol) in CH2Cl2 (2 ml) was added to the mixture at 08, and the
mixture was stirred for 48 h. The mixture was diluted with H2O and extracted with AcOEt. The extract
was washed with H2O and brine, dried (MgSO4), filtered, and concentrated in vacuo. The resulting
mixture was purified by prep. TLC (silica gel; toluene/AcOEt 80 :1) to give 7 (30.7 mg, 0.031 mmol,

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
525
68%). [a]1D9 ¼ ꢀ68 (c¼0.64, CH2Cl2). IR: 2935, 1715, 1591, 1498, 1328, 1215, 1117, 1027, 734, 696.
1H-NMR: 3.06 (dd, J¼2.5, 18.0, 1 H); 3.11 (dd, J¼4.5, 18.0, 1 H); 3.77 (s, 3 H); 4.73 (d, J¼11.5, 1 H);
4.84 (d, J¼12.0, 1 H); 4.95–5.13 (m, 13 H); 5.60 (s, 1 H); 6.30 (d, J¼2.5, 1 H); 6.34 (d, J¼2.5, 1 H); 6.84
(d, J¼8.0, 1 H); 6.94 (dd, J¼1.8, 8.3, 1 H); 7.07 (d, J¼2.0, 1 H); 7.16 (d, J¼1.5, 1 H); 7.19–7.42 (m,
30 H). 13C-NMR: 26.0; 56.2; 68.7; 70.0; 70.1; 71.0; 71.2; 71.3; 75.0; 77.5; 93.9; 94.6; 100.9; 107.3; 108.8;
113.8; 114.7; 120.0; 125.1; 127.2 (2 C); 127.4; 127.5; 127.7; 127.9; 128.0 (2 C); 128.2; 128.3 (2 C); 128.4;
128.5; 128.6 (2 C); 131.1; 136.5; 136.8 (2 C); 137.0; 137.1; 137.4; 141.9; 148.9; 148.9; 152.1; 153.4; 155.7;
158.0; 158.8; 165.1. HR-FAB-MS (NOBA): 997.3947 ([MþH]þ , C65H57O1þ0 ; calc. 997.3952).
(2R,3S)-5,7-Bis(benzyloxy)-2-[3,4-bis(benzyloxy)phenyl]-3,4-dihydro-2H-1-benzopyran-3-yl 3,4-
Bis(benzyloxy)-5-methoxybenzoate (8). To a soln. of 6 (180 mg, 0.495 mmol) in CH2Cl2 (20 ml) was
added DMAP (6.0 mg, 0.049 mmol), and the mixture was stirred for 10 min. A soln. of DCC (102 mg,
0.495 mmol) was added to the mixture at 08, and the mixture was stirred for 10 min. A soln. of 4 (326 mg,
0.495 mmol) in CH2Cl2 (20 ml) was added to the mixture at 08, and the mixture was stirred for 24 h. The
mixture was diluted with H2O and extracted with CH2Cl2. The extract was washed with H2O and brine,
dried (MgSO4), filtered, and concentrated in vacuo. The resulting mixture was purified by CC (silica gel;
hexane/AcOEt/CH2Cl2 30 :1:15) to give 8 (379 mg, 0.381 mmol, 77%). Colorless solid. M.p. 114.5–
116.38. [a]1D9 ¼ þ53 (c¼0.65, CH2Cl2). IR: 2935, 1716, 1591, 1498, 1421, 1331, 1214, 1122, 1024, 734, 696.
1H-NMR: 2.83 (dd, J¼7.3, 16.8, 1 H); 3.08 (dd, J¼5.3, 16.8, 1 H); 3.78 (s, 3 H); 4.96–5.16 (m, 13 H); 5.45
(dd, J¼6.5, 12.5, 1 H); 6.28 (dd, J¼2.0, 7.0, 2 H); 6.86 (d, J¼8.5, 1 H); 6.92 (dd, J¼1.5, 8.5, 1 H); 7.02 (d,
J¼2.0, 1 H); 7.11 (d, J¼1.5, 1 H); 7.17 (d, J¼1.5, 1 H); 7.24–7.48 (m, 30 H). 13C-NMR: 24.4; 56.2; 69.9;
70.0; 70.1; 71.1; 71.2; 71.4; 75.0; 78.4; 93.8; 94.4; 101.4; 107.2; 108.8; 113.5; 114.8; 120.0; 125.0; 127.2 (2 C);
127.4; 127.5; 127.6; 127.8; 127.9 (2 C); 128.0; 128.1; 128.4 (2 C); 128.5 (2 C); 128.6; 131.1; 136.5; 136.8
(2 C); 136.9; 137.1; 137.4; 141.8; 149.0; 149.1; 152.1; 153.3; 155.0; 157.7; 158.9; 165.2. HR-FAB-MS
(NOBA): 997.3951 ([MþH]þ , C65H57O1þ0 ; calc. 997.3952).
(2R,3R)-3,4-Dihydro-5,7-dihydroxy-2-(3,4-dihydroxyphenyl)-2H-1-benzopyran-3-yl 3,4-Dihydroxy-
5-methoxybenzoate (1; ECG3’’Me). A suspension of 7 (45.0 mg, 0.046 mmol) and Pd(OH)2 on activated
charcoal (20 wt-%, 50 mg) in THF/MeOH/H2O 20 :1:1 (15 ml) was stirred for 24 h under H2. The
mixture was filtered, and the filtrate was concentrated. The residue was purified by HPLC (ODS;
MeOH/H2O 4 :1) to give 1 (15.3 mg, 0. 034 mmol, 74%). Colorless waxy solid. [a]1D8 ¼ ꢀ133 (c¼0.130,
1
EtOH). IR: 3200, 2923, 2852, 2359, 1685, 1463, 1259. H-NMR (CD3OD): 2.88 (dd, J¼2.5, 17.5, 1 H);
3.00 (dd, J¼4.5, 17.5, 1 H); 3.81 (s, 3 H); 5.03 (s, 1 H); 5.51 (s, 1 H); 5.97 (dd, J¼2.3, 7.3, 2 H); 6.70 (d, J¼
8.0, 1 H); 6.80 (dd, J¼2.0, 8.0, 1 H), 6.95 (d, J¼2.0, 1 H), 7.01 (d, J¼2.0, 1 H), 7.07 (d, J¼2.0, 1 H).
13C-NMR: 26.7; 56.7; 58.4; 70.4; 78.6; 95.9; 96.7; 99.4; 106.4; 112.0; 115.1; 116.1; 119.3; 121.6; 131.6; 146.0;
146.1 (2 C); 149.1; 157.3; 157.9; 158.0; 167.6. HR-FAB-MS (NOBA): 457.1141 ([MþH]þ , C23H21O1þ0
;
calc. 457.1135).
(2R,3S)-3,4-Dihydroxy-5,7-dihydroxy-2-(3,4-dihydroxyphenyl)-2H-1-benzopyran-3-yl 3,4-Dihy-
droxy-5-methoxybenzoate (2; CG3’’Me). A suspension of 8 (51.3 mg, 0.052 mmol) and Pd(OH)2 on
activated charcoal (20 wt-%, 50 mg) in THF/MeOH/H2O 20 :1:1 (15 ml) was stirred for 24 h under H2.
The mixture was filtered, and the filtrate was concentrated. The residue was purified by cartridge CC
(ODS; MeOH/H2O 3 :7) to give 2 (25.0 mg, 0.052 mmol, quant.). Colorless waxy solid. [a]1D8 ¼ þ70 (c¼
0.17, EtOH). IR: 3200, 2923, 1684, 1606, 1517, 1456, 1337, 1223, 1139. 1H-NMR (CD3OD): 2.71 (dd, J¼
6.8, 16.3, 1 H); 2.92 (dd, J¼5.0, 16.5, 1 H); 3.82 (s, 3 H); 5.02 (d, J¼6.5, 1 H); 5.31 (dd, J¼5.5, 6.5, 1 H);
5.93 (d, J¼2.5, 1 H); 5.96 (d, J¼2.0, 1 H); 6.71–6.76 (m, 2 H); 6.86 (d, J¼2.0, 1 H); 6.99 (d, J¼1.5, 1 H);
7.06 (d, J¼2.0, 1 H). 13C-NMR: 25.2; 56.1; 56.7; 58.4; 70.6; 71.7; 79.8; 95.6; 96.6; 99.8; 106.2; 111.9; 114.8;
116.3; 119.5; 131.5; 146.2; 146.4; 149.1; 156.7; 157.7; 158.2; 167.5. HR-FAB-MS (NOBA): 457.1134 ([Mþ
H]þ , C23H21Oþ10 ; calc. 457.1135).
Anti-Inflammatory Test. A sample (200 mg) was applied on one mouse ear and, after 30 min, TPA
(0.5 mg) was applied on both ears of the mouse. The edema was evaluated after 7 h. The inhibitory effect
is expressed as percentage ratio of the edema. Five mice were used for each experiment [12]. This
experiment complied with the regulations concerning animal experimentation and the care of
experimental animals of the Faculty of Agriculture at Shinshu University.

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
The authors thank the Regional Science Promotion Program, Nagano Techno Foundation, for
financial support. Our thanks are also due to Mrs. Teiko Yamada for the FAB-MS data.
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Received June 26, 2008