Anticancer activity of 3-O-acyl and alkyl-(-)-epicatechin derivatives

Article abstract of DOI:10.1016/j.bmcl.2004.07.063

We report the synthesis and anticancer effects of 3-O-acyl and alkyl-(-)-epicatechin derivatives.

Full text of DOI:10.1016/j.bmcl.2004.07.063

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5189–5192
Anticancer activity of 3-O-acyl and
alkyl-(ꢀ)-epicatechin derivatives
b
a
c
d
Ki Duk Park,a, Sul Gi Lee, Sung Uk Kim, Sung Han Kim, Won Suck Sun,
*
Sung Jin Chod and Do Hyeon Jeongc
aLaboratory of Cellular Function Modulator, Korea Research Institute of Bioscience and Biotechnology,
Yusung, Taejon 305-333, Korea
bSamyang Central R&D center, 63-2 Hwaam-dong, Yusung, Taejon, Korea
cNutrex Technology, Seongnam, Kyounggi-do, Korea
dDepartment of Biotechnology, College of Engineering, Yonsei University, Seoul, Korea
Received 8October 2003; accepted 26 July 2004
Available online 1 September 2004
Abstract—By changing the structure or replacing the gallate group of (ꢀ)-ECG, 3-O-acyl and alkyl-(ꢀ)-epicatechin derivatives were
synthesized to be screen as anticancer agents using the MTT assay in vitro against cancer cell lines (PC3, SKOV3, U373MG). 3-O-
Acyl and alkyl-(ꢀ)-epicatechin derivatives (4–25) exhibited better anticancer activity than (ꢀ)-ECG and specially, compounds 6–8,
17–19, which were modified aliphatic chains with moderate sizes (C8–C12) showed strong anticancer activity (IC50 = 6.4–31.2lM).
The introduction of an alkyloxy group on 3-O-hydroxyl instead of an acyloxy group significantly enhanced inhibitory activity. Con-
sequently, the compound that showed the most potency as anticancer agents were 3-O-decyl-(ꢀ)-epicatechin (18) (IC50 = 8.9, 7.9,
6.4lM against PC3, SKOV3, U373MG, respectively), which modified the appropriate lipophilic group on the C-3 hydroxyl as
an alkyloxy group.
Ó 2004 Elsevier Ltd. All rights reserved.
Recently, much attention has been paid to tea, Camellia
sinensis, for the beneficial biological activities of its com-
pounds, catechins, which have been reported to possess
antimutagenic, antibacterial, antioxidant, antitumor
and cancer preventive properties.1–4 The constituents
of the catechins of green tea are epigallocatechin-3 gal-
late (EGCG), epigallocatechin (EGC), epicatechin-3 gal-
late (ECG) (1), epicatechin (EC) (2) (Fig. 1).5 Among
catechins, (ꢀ)-EGCG and (ꢀ)-ECG exhibited better
anticancer activity than (ꢀ)-EGC and (ꢀ)-EC.1,6
Because (ꢀ)-EGCG and (ꢀ)-ECG was postulated to
prevent human cancer by inhibiting enzymes, such as
urokinase and 5a-reductase, that are crucial for cancer
OH
OH
OH
OH
OH
OH
O
HO
O
HO
O
O
OH
OH
OH
OH
OH
OH
EGC
EGCG
OH
OH
OH
OH
O
HO
O
2
HO
O
O
OH
OH
OH
growth and development, though it might be one of
OH
OH
7–9
many ways of cancer inhibition by green tea.
How-
ever, Hiipakka et al. discovered that (ꢀ)-EGCG and
(ꢀ)-ECG exerted potent inhibition of human 5a-reduc-
tase in cell-free but not in whole-cell assays. The lack
1
OH
Figure 1. Chemical structures of major catechins of tea.
of activity in whole cells may be due to an inability of
these catechins to cross the cell membrane or to enzy-
matic or nonenzymatic changes in the structure of these
catechins in assay using whole-cell culture.9
Keywords: 3-O-Acyl-(ꢀ)-epicatechin; 3-O-Alkyl-(ꢀ)-epicatechin; Anti-
cancer activity.
*
Corresponding author. Tel.: +82 42 860 4564; fax: +82 42 860
2675; e-mail: pkduck75@hanmail.net
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.07.063

5190
K. D. Park et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5189–5192
The purpose of the present study was to synthesize 3-O-
acyl-(ꢀ)-epicatechin derivatives, which might enhance
anticancer activities on whole cell assay. By replacing
the gallate group of (ꢀ)-ECG with lipophilic group, var-
ious aromatic group and aliphatic chains, for increasing
the lipophilicity, 3-O-acyl-(ꢀ)-epicatechin derivatives
were synthesized (4–14). In order to prevent the acyloxy
group at the C-3 hydroxyl group from enzymatic or
non-enzymatic cleavage, the synthesized compounds
were modified with an alkyloxy group instead of an acyl-
oxy group (15–25).
For the synthesis of 3-O-acyl-(ꢀ)-epicatechin deriva-
tives, the phenolic hydroxyl group of 2 was benzylated
by treatment with benzyl bromide and K2CO3 to give
5,7,30,40-tetra-O-benzyl-(ꢀ)-epicatechin (3) in 74%
yield.10 The treatment of 3 with various acyl chloride
and triethylamine (TEA) gave the 5,7,30,40-tetra-O-
benzyl-3-O-acyl-(ꢀ)-epicatechins. The debenzylation of
5,7,30,40-tetra-O-benzyl-3-O-acyl-(ꢀ)-epicatechins was
carried out with Pd/C in presence of H2 to give com-
pounds 4–14 (Scheme 1). To obtain the 3-O-alkyl-(ꢀ)-
epicatechin derivatives, 3 was treated with cesium
OBn
OBn
OH
OBn
OBn
3'
OH
2'
TEA
4'
BnBr
BnO
O
B
8
BnO
O
2
3
7
O
HO
O
K2CO3
A
O
6
OH
Cl
OH
R
OBn
5
4
O
R
OBn
OH
2
3
Pd/C H2
R
OH
4. CH3 CH2 CH2-
11.
12.
13.
14.
OH
F
CH3O
CH3O
CH3O
O
HO
5. CH3 (CH2)3CH2-
6. CH3 (CH2)5CH2-
7. CH3 (CH2)7CH2-
8. CH3 (CH2)9CH2-
9. CH3 (CH2)11CH2-
10. CH3 (CH2)13CH2-
F
F
O
OH
O
R
F
CF3O
F
Scheme 1. Synthesis of 3-O-acyl epicatechins.
OBn
OH
OBn
OBn
OH
OBn
HO
O
O
BnO
CsOH/ TBAI
Br
Pd/C
H2
BnO
O
O
O
R1
OH
OH
OBn
R
1
R1
OBn
3
R1
15. CH3 CH2CH2-
22.
23.
24.
CH3O
CH3O
CH3O
F
F
16. CH3 (CH2)3CH2-
17. CH3 (CH2)5CH2-
18. CH3 (CH2)7CH2-
19. CH3 (CH2)9CH2-
20. CH3 (CH2)11CH2-
21. CH3 (CH2)13CH2-
F
25.
F
CF3O
F
Scheme 2. Synthesis of 3-O-alkyl epicatechins.

K. D. Park et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5189–5192
5191
hydroxide (CsOH), tert-butyl ammonium iodide (TBAI)
and various alkyl bromide to give 5,7,30,40-tetra-O-ben-
zyl-3-O-alkyl-(ꢀ)-epicatechins, as shown in Scheme 2.
Compounds 15–25 were obtained with the deprotection
of the benzyl group in the same way as Scheme 1.
or nonenzymatic cleavage, which in turn may make
them more stable in whole-cell culture. The compound
18 exerted the strongest anticancer activities with a
IC50 values of 8.9, 7.9 and 6.4lM against PC3, SKOV3
and U373MG, respectively. This result indicates that the
introduction of moderate sized aliphatic chain at the C-3
hydroxyl with an alkyloxy group can lead to increasing
of permeability and stability, which significantly im-
prove inhibitory effect of cancer cell growth.
All the above compounds were tested for their in vitro
anticancer activity against PC3, SKOV3, U373MG cells
using the MTT assay, which was performed in 96-well
plates essentially as described by Mosmann.11 The
IC50 concentration represents the concentration, which
results in a 50% decrease in cell growth after 3days incu-
bation. The given values are the mean values of the three
experiments.
Acknowledgements
We thank Nutrex Technology for the financial support.
The pharmacological activities against the PC3,
SKOV3, U373MG cells are summarized in Table 1. In
effect, 3-O-acyl and alkyl-(ꢀ)-epicatechin derivatives
(4–25) were synthesized to support the assumption that
increasing their lipophilicity and the permeability of the
cell membrane would enhance the anticancer action of
ECG. Among the new compounds (4–25), the replace-
ment of aliphatic chains with moderate sizes (C10–
C12) (7–8, 18–19)12 exhibited great enhancement of anti-
cancer activity and too short (C4) or too long (C16)
displayed little increase of anticancer effect. These results
suggests that the presence of lipophilic substituents with
moderate sizes might be crucial for the optimal antican-
cer activity. The most significant structural change lead-
ing to enhance activity was the introduction of an alkyl
group at the C-3 hydroxyl in place of an acyloxy group.
3-O-(4-Trifluoromethoxy benzyl)-(ꢀ)-epicatechin (25)12
showed three times the activity than 3-O-(4-trifluoro-
methoxy benzoyl)-(ꢀ)-epicatechin (14).12 This modifica-
tion is likely to prevent the compounds from enzymatic
References and notes
1. Paschka, A. G.; Butler, R.; Young, C. Y. F. Cancer Lett.
1998, 130, 1.
2. Kondo, K.; Kurihara, M.; Miyata, N.; Suzuki, T.;
Toyoda, M. Free Radical Biol. Med. 1999, 27, 855.
3. Hu, Z. Q.; Zhao, W. H.; Hara, Y.; Shimamura, T. J.
Antimicrob. Chemother. 2001, 48, 361.
4. Kohri, T.; Matsumoto, N.; Yamakawa, M.; Nanjo, F.;
Oku, N.; Hara, Y. Abstracts of Papers, p98 Chemistry and
Health Promotion, 2nd International Conference on Food
Factors, Kyoto, Japan, Dec 1999; 12–17.
5. Kuroda, Y.; Hara, Y. Mutation Res. 1999, 436, 69.
6. Uesato, S.; Kitagawa, Y.; Hara, Y.; Tokuda, H.; Okuda,
M.; Mou, M. O.; Mou, X. Y.; Mukainaka, T.; Nishino, H.
Bioorg. Med. Chem. Lett. 2000, 10, 1673.
7. Jankun, J.; Selman, S. H.; Swiercz, R.; Skrzypczak-
jankun, E. Nature 1997, 387, 561.
8. Yang, C. S. Nature 1997, 389, 134.
9. Hiipakka, R. A.; Zhang, H. Z.; Dai, W.; Dai, Q.; Liao, S.
Biochem. Pharm. 2002, 63, 1165.
10. Typical procedure: to a stirred solution of (ꢀ)-epicatechin
(2.63g, 9.1mmol) in 13mL of DMF, benzyl bromide
(4.30mL, 36.3mmol) and potassium carbonate (7.50g,
54.4mmol) were added and the resulting solution was
stirred at room temperature for 20h. The reaction mixture
was diluted with ethyl acetate and washed successively
with water and brine. The organic layer was dried over
MgSO4, and the solvent was evaporated in vacuo to yield
a brown oil, which was applied on a silica gel short column
(CH2Cl2) to remove high polar products. Compound 3
was crystallized from MeOH/ether to give colorless
crystals. 1H NMR (CDCl3, 500MHz) d 7.20–7.60 (m,
20H), 6.95, 7.01 (2s, 3H, aromatic proton (B-ring)), 6.20,
7.01 (2d, 2H, aromatic proton (A-ring)), 5.16 (s, 4H,
–Bzl), 4.99, 5.02 (2s, 4H, –Bzl), 4.62 (d, 1H, C2–H), 3.97
(m, 1H, C3–H), 3.10 (dd, 1H C4–Hequatorial), 2.61 (dd, 1H,
C4–Haxial); MS (positive ESI mode) m/z: 650.3.
Table 1. In vitro anticancer effects against the PC3, SKOV3 and
U373MG cell lines
Compounds
IC50 (lM)
PC3
168.2
>500
95.6
SKOV3
U373MG
1
185.4
>500
103.5
68.2
31.2
20.819.2
22.4
34.7
48.1
61
157
>500
107.3
71.2
2
4
5
67.2
24.2
14.6
23.1
33.7
43.1
71.9
58.7
51.3
45.2
98.3
35.6
14.3
8.9
6
29.7
7
8
24.3
36.7
42.9
59.2
64.5
57.2
50.3
77.2
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
78.2
50.1
47.3
78.3
27.823.4
15.5
7.9
11. Mosmann, T. J. Immunol. Meth. 1983, 65, 55.
12. Spectral data for compound 4–14; 7; 1H NMR (CDCl3,
500MHz) d 8.89–9.35 (m, 4H, OH (A,B-ring)), 6.73, 6.892
(2s, 3H, aromatic proton (B-ring)), 5.76, 5.88 (2d, 2H,
aromatic proton (A-ring)), 5.36 (d, 1H, C2–H), 5.05 (m,
1H, C3–H), 2.77 (dd, 1H C4–Hequatorial), 2.62 (dd, 1H, C4–
12.3
6.4
9.3
8.6
7.1
H
axial), 2.50 (2H, t, COCH2(CH2)7CH3 at C3–O), 1.38
17.5
29.1
21.827.2
33.2
32.5
14.6
18.2
30.3
22.3
41.7
38.2
19.1
16.9
22.5
(14H, m, COCH2(CH2)7CH3 at C3–O), 0.85 (3H, t,
COCH2(CH2)7CH3 at C3–O); MS (positive ESI mode)
m/z: 444.3. 8; 1H NMR (CDCl3, 500MHz) d 8.84–9.35 (m,
4H, OH (A,B-ring)), 6.62, 6.84 (2s, 3H, aromatic proton
(B-ring)), 5.83, 5.92 (2d, 2H, aromatic proton (A-ring)),
5.37 (d, 1H, C2–H), 5.08(m, 1H, C 3–H), 2.83 (dd, 1H
25.2
29.8
11.2

5192
K. D. Park et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5189–5192
C4–Hequatorial), 2.62 (dd, 1H, C4–Haxial), 2.50 (2H, t, COCH2-
(–CH2(CH2)8CH3 at C3–O)); MS (positive ESI mode) m/z:
430.3. 19; 1H NMR (CDCl3, 500MHz) d 8.85–9.34 (m,
4H, OH (A,B-ring)), 6.71, 6.84 (2s, 3H, aromatic proton
(B-ring)), 5.84, 5.93 (2d, 2H, aromatic proton (A-ring)),
4.64 (d, 1H, C2–H), 3.68(m, 1H, C 3–H), 3.03, 3.26
(–CH2(CH2)10CH3 at C3–O), 2.86 (dd, 1H C4–Hequatorial),
2.68(dd, 1H, C 4–Haxial), 1.29 (16H, m, (–CH2(CH2)10CH3
at C3–O)), 0.85 (3H, t, (–CH2(CH2)10CH3 at C3–O)); MS
(positive ESI mode) m/z: 458.2. 25; 1H NMR (CDCl3,
500MHz) d 8.84–9.24 (m, 4H, OH (A,B-ring)), 6.9–7.33
(m, 4H aromatic proton (Ar–CH2– at C3–O)), 6.62, 6.72
(2s, 3H, aromatic proton (B-ring)), 5.72, 5.91 (2d, 2H,
aromatic proton (A-ring)), 4.76 (d, 1H, C2–H), 4.35, 4.5
(dd, 2H Ar–CH2– at C3–O), 3.79 (m, 1H, C3–H), 2.89 (dd,
1H C4–Hequatorial), 2.65 (dd, 1H, C4–Haxial); MS (positive
ESI mode) m/z: 464.1.
(CH2)7CH3 at C3–O), 1.35 (14H, m, COCH2(CH2)9CH3 at
C3–O), 0.85 (3H, t, COCH2(CH2)9CH3 at C3–O); MS
(positive ESI mode) m/z: 472.2. 14; 1H NMR (CDCl3,
500MHz) d 8.83–9.32 (m, 4H, OH (A,B-ring)), 7.46–7.68
(m, 4H aromatic proton (Ar–CO– at C3–O)), 6.66, 6.89
(2s, 3H, aromatic proton (B-ring)), 5.86, 5.95 (2d, 2H,
aromatic proton (A-ring)), 5.31 (d, 1H, C2–H), 5.05 (m,
1H, C3–H), 2.82 (dd, 1H C4–Hequatorial), 2.69 (dd, 1H, C4–
H
1
axial); MS (positive ESI mode) m/z: 478.1. 18; H NMR
(CDCl3, 500MHz) d 8.85–9.36 (m, 4H, OH (A,B-ring)),
6.71, 6.86 (2s, 3H, aromatic proton (B-ring)), 5.85, 5.95
(2d, 2H, aromatic proton (A-ring)), 4.68(d, 1H, C –H),
2
3.67 (m, 1H, C3–H), 3.08, 3.28 (–CH2(CH2)8CH3 at C3–
O), 2.82 (dd, 1H C4–Hequatorial), 2.67 (dd, 1H, C4–Haxial),
1.31 (16H, m, (–CH2(CH2)8CH3 at C3–O)), 0.84 (3H, t,