Inhibitory effects of 3-O-acyl-(+)-catechins on Epstein-Barr virus activation

Article abstract of DOI:10.1248/cpb.51.1448

In the course of an exploratory investigation of antitumor-promoting catechins, 3-O-acyl-(+)-catechins of varying carbon lengths from C₄ to C₁₈ were assessed for inhibitory effects on the activation of the Epstein-Barr virus early an

Full text of DOI:10.1248/cpb.51.1448

1
448
Notes
Chem. Pharm. Bull. 51(12) 1448—1450 (2003)
Vol. 51, No. 12
Inhibitory Effects of 3-O-Acyl-(؉)-catechins on Epstein-Barr Virus
Activation
,
a,b
a
a
a,b
c
Shinichi UESATO,* Keisuke TANIUCHI, Ayako KUMAGAI, Yasuo NAGAOKA, Ryota SETO,
c
d
d
Yukihiko HARA, Harukuni TOKUDA, and Hoyoku NISHINO
a
b
Department of Biotechnology, Faculty of Engineering, Kansai University; Organization for Research and Development
c
of Innovative Science and Technology, Kansai University; Suita, Osaka 564–8680, Japan: Food Research Laboratories,
Tokyo Food Techno Co., Ltd.; Fujieda, Shizuoka 426–0133, Japan: and Department of Biochemistry and Molecular
d
Biology, Kyoto Prefectural University of Medicine; Kyoto 602–0841, Japan.
Received August 11, 2003; accepted September 17, 2003
In the course of an exploratory investigation of antitumor-promoting catechins, 3-O-acyl-(؉)-catechins of
varying carbon lengths from C to C were assessed for inhibitory effects on the activation of the Epstein-Barr
4
18
virus early antigen. Like 3-O-acyl-(؊)-epigallocatechins, the (؉)-catechin derivatives showed promising effects
with the C-3 acyl chain of C –C carbon atoms.
8
11
Key words (ϩ)-catechin; 3-O-acyl-(ϩ)-catechin; 3-O-acyl-(Ϫ)-epigallocatechin; Epstein-Barr virus early antigen (EBV-EA)
activation; Uncaria gambir
As an exploratory investigation of antitumor-promoting Chemistry
catechins, we tried to synthesize the (Ϫ)-epigallocatechins
(ϩ)-Catechin (1) was reacted with straight-chain acid
1
,2)
(
EGCs) possessing an acyl group. An acyl group was in- chlorides of C to C in tetrahydrofuran in the presence of
4
18
1
)
troduced at the C-3 hydroxy group of (Ϫ)-EGC (2), which is trifluoroacetic acid. Preparative HPLC (GS-320 column) of
6
)
less responsible for radical-scavenging action than the phe- the reaction mixtures gave 3-O-acyl-(ϩ)-catechins: 3, 4, 5,
3
,4)
6)
7)
nolic hydroxyl groups, to improve their pharmacokinetic 6, 7, 8, 9, and 10 in 7.7% to 16.8% yields. Furthermore,
profile such as cell membrane and tissue permeability. The the (ϩ)-catechin derivative 11 possessing the (RS)-2-methyl-
EGCs with the C-3 acyl chain of C –C carbon atoms octanoyl chain was synthesized by the same method as above
8
11
showed marked antitumor-promoting activities both in the in the expectation that it would avoid the enzyme-catalyzed
Epstein-Barr virus early antigen (EBV-EA) activation test hydrolytic cleavage of the ester bond in the body as in the
2
)
and in the two-stage mouse skin carcinogenesis test; 3-O- case of the corresponding (Ϫ)-EGC derivatives.
octanoyl-(Ϫ)-EGC (12), 3-O-[(RS)-2-methyloctanoyl]-(Ϫ)-
EGC (13), and 3-O-[(S)-2-methyloctanoyl]-(Ϫ)-EGC (14) Results and Disscussion
were found to be especially promising candidates for cancer
The antitumor-promoting efficacy of the synthetic 3-O-
1,2)
chemoprevention. In the present work, we examined the acyl-(ϩ)-catechins was estimated by measuring the inhibi-
inhibitory effects of 3-O-acyl derivatives of (ϩ)-catehin (1) tory effects against the activation of EBV-EA in Raji cells
against the activation of the EBV-EA since 1 is available at induced by 12-O-tetradecanoylphorbol-13-acetate (TPA).
much lower price from Gambir (Asenyaku in Japanese, nat- The assays were performed in triplicate for each compound.
ural medicine, Uncaria gambir ROXB. listed in the Japanese No sample exhibited significant toxicity against Raji cells.
5
)
Pharmacopoeia XIV) than green tea catechins and thus As shown in Table 1, the (ϩ)-catechin derivatives 11, 5, and
could be a substitute for (Ϫ)-EGC (2) as a synthetic starting 6 with an acyl chain of carbon atoms C –C had more effect
8
11
material of catechins with a 3-O-acyl group.
(percentage EBV-EA activationsϭ7.2, 8.8, and 10.7%, re-
spectively) than the original (ϩ)-catechin (1) (21.6%) at
Fig. 1. Structures of Catechins and 3-O-Acyl-(ϩ)-catechins
∗
To whom correspondence should be addressed. e-mail: uesato@ipcku.kansai-u.ac.jp
© 2003 Pharmaceutical Society of Japan

December 2003
1449
ϩ
Table 1. Inhibitory Effects of 3-O-Acyl-(ϩ)-catechins against EBV-EA Jϭ7.6 Hz, H-3Ј), 6.78 (1H, s, H-6Ј). FAB-MS m/z: 389.2 [MϩH] . HR-
a)
ϩ
Activation
FAB-MS m/z: 389.1578 ([MϩH] , Calcd for C H O : 389.1600).
21 25 7
2
0
3
-O-Octanoyl-(؉)-catechin (5) 12.9% yield. [a]D ϩ5.2° (cϭ0.4,
EtOH). IR (KBr) cm : 3310, 2928, 2856, 2359, 1734, 1622, 1607, 1528,
1518, 1475, 1389, 1300, 1254, 1150, 1057, 1028, 964, 829, 731, 669. H-
Ϫ1
EBV-EA-positive cells (% viability)
Compound concentration (mol ratio/32 pmol TPA)
1
Compound
NMR (270 MHz) d: 0.89 (3H, t, Jϭ6.7 Hz, –COCH CH (CH ) CH ),
2
2
2
4
3
1
000
500
100
1
1.12—1.33 (8H, m, –COCH CH (CH ) CH ), 1.39—1.49 (2H, m, –COCH -
2 2 2 4 3 2
CH (CH ) CH ), 2.20 (2H, t, Jϭ7.2 Hz, –COCH CH (CH ) CH ), 2.59 (1H,
2
2
4
3
2
2
2
4
3
1
3
4
5
6
7
8
9
21.6 (60)
19.3 (60)
12.9 (60)
8.8 (60)
10.7 (60)
16.4 (60)
19.8 (60)
22.6 (60)
25.1 (60)
7.2 (60)
46.2 (Ͼ80)
44.0 (Ͼ80)
41.8 (Ͼ80)
39.5 (Ͼ80)
41.9 (Ͼ80)
46.9 (Ͼ80)
46.0 (Ͼ80)
47.3 (Ͼ80)
49.6 (Ͼ80)
38.0 (Ͼ80)
75.9 (Ͼ80) 100 (Ͼ80)
dd, Jϭ7.2, 16.2 Hz, H-4), 2.81 (1H, dd, Jϭ5.6, 16.2 Hz, H-4) , 5.16—5.23
(1H, m, H-3), 5.88 (1H, d, Jϭ2.4 Hz, H-6 or H-8), 5.94 (1H, d, Jϭ2.2 Hz,
H-8 or H-6), 6.67 (1H, dd, Jϭ1.9, 8.2 Hz, H-2Ј), 6.73 (1H, d, Jϭ8.2 Hz, H-
73.4 (Ͼ80)
71.8 (Ͼ80)
69.7 (Ͼ80)
71.2 (Ͼ80)
96.4 (Ͼ80)
94.9 (Ͼ80)
91.9 (Ͼ80)
93.6 (Ͼ80)
ϩ
3Ј), 6.79 (1H, d, Jϭ1.9 Hz, H-6Ј). FAB-MS m/z: 417.2 [MϩH] . HR-FAB-
ϩ
MS m/z: 417.1906 ([MϩH] , Calcd for C H O : 417.1914).
2
3
29
7
2
0
75.7 (Ͼ80) 100.0 (Ͼ80)
78.1 (Ͼ80) 100.0 (Ͼ80)
77.1 (Ͼ80) 100.0 (Ͼ80)
81.4 (Ͼ80) 100.0 (Ͼ80)
3-O-Decanoyl-(؉)-catechin (6) 16.0% yield. [a]D ϩ13.4° (cϭ0.4,
EtOH). IR (KBr) cm : 3352, 2922, 2852, 1711, 1632, 1518, 1468, 1359,
1245, 1140, 1063, 818, 419. H-NMR (400 MHz) d: 0.07 (3H, t, Jϭ6.8 Hz,
–COCH CH (CH ) CH ), 0.32—0.49 (12H, m, –COCH CH (CH ) CH ),
Ϫ1
1
1
0
1
2
2
2
6
3
2
2
2
6
3
1
67.9 (Ͼ80)
90.1 (Ͼ80)
0.58—0.65 (2H, m, –COCH CH (CH ) CH ), 1.37 (2H, t, Jϭ7.0 Hz,
2 2 2 6 3
–
COCH CH (CH ) CH ), 1.76 (1H, dd, Jϭ7.0, 16.6 Hz, H-4), 1.98 (1H, dd,
2 2 2 6 3
a) Mol ratio/TPA (32 pmolϭ20 ng/ml), 1000 mol ratioϭ32 nmol, 500 mol
ratioϭ16 nmol, 100 mol ratioϭ3.2 nmol, and 10 mol ratioϭ0.32 nmol. Values are EBV-
EA activation (%) in the presence of the test compound relative to the positive control
Jϭ5.4, 16.6 Hz, H-4), 4.35—4.39 (1H, m, H-3), 5.06 (1H, s, H-6 or H-8),
.11 (1H, s, H-8 or H-6), 5.82—5.86 (1H, m, H-2Ј), 5.90 (1H, d, Jϭ7.6 Hz,
5
ϩ
H-3Ј), 5.96 (1H, s, H-6Ј). FAB-MS m/z: 445.2 [MϩH] . HR-FAB-MS m/z:
(
0
100%). Values in parentheses represent the viability % of Raji cells measured using
.25% trypan blue dye staining. At least 60% viability of Raji cells 2 d after treatment
with compounds is required under normal conditions.
ϩ
4
45.2260 ([MϩH] , Calcd for C H O : 445.2227).
25 33 7
2
0
3
-O-Dodecanoyl-(؉)-catechin (7) 14.5% yield. [a]D ϩ1.5° (cϭ0.5,
Ϫ1
EtOH). IR (KBr) cm : 3609, 3560, 3302, 2924, 2328, 1713, 1659, 1518,
452, 1286, 1140, 1016, 665, 517. H-NMR (400 MHz) d: 1.04 (3H, t,
Jϭ6.6 Hz, –COCH CH (CH ) CH ), 1.29—1.52 (16H, m, –COCH CH -
1
1
2
2
2
8
3
2
2
3
1
ϫ10 mol ratios/TPA although they were slightly less effec- (CH ) CH ), 1.57—1.60 (2H, m, –COCH CH (CH ) CH ), 2.34 (2H, t,
2 8 3 2 2 2 8 3
tive than the corresponding (Ϫ)-EGC derivatives (percentage Jϭ7.4 Hz, –COCH CH (CH ) CH ), 2.74 (1H, dd, Jϭ7.0, 16.2 Hz, H-4),
2 2 2 8 3
1
)
2.95 (1H, dd, Jϭ5.0, 16.2 Hz, H-4), 5.33—5.35 (1H, m, H-3), 6.03 (1H, s,
H-6 or H-8), 6.08 (1H, s, H-8 or H-6), 6.80—6.83 (1H, m, H-2Ј), 6.87 (1H,
activationsϭ5.0—9.3%). Furthermore, either shortening C
(
8
in 5) or lengthening C10 (in 6) led to a reduction in the in-
hibitory activity as in the case of 3-O-acyl-(Ϫ)-EGCs. There-
fore the (ϩ)-catechin derivatives possessing an acyl chain of
carbon atoms C to C could be substitutes for the corre- EtOH), IR (KBr) cm : 3612, 2922, 2853, 2357, 1715, 1651, 1520, 1456,
sponding 3-O-acyl-(Ϫ)-EGCs.
ϩ
d, Jϭ8.0 Hz, H-3Ј), 6.94 (1H, s, H-6Ј). FAB-MS m/z: 473.3 [MϩH] . HR-
ϩ
FAB-MS m/z: 473.2548 ([MϩH] , Calcd for C H O : 473.2540).
2
7
37
7
2
0
3-O-Myristoyl-(؉)-catechin (8) 8.6% yield. [a] ϩ1.0° (cϭ0.7,
D
Ϫ1
8
11
1
1
–
362, 1142, 1061, 816, 419. H-NMR (400 MHz) d: 0.08 (3H, t, Jϭ6.6 Hz,
COCH CH (CH ) CH ), 0.43—0.53 (20H, m, –COCH CH (CH ) CH ),
2
2
2
10
3
2
2
2
10
3
Experimental
0.62—0.65 (2H, m, –COCH CH (CH ) CH ), 1.38 (2H, t, Jϭ7.4 Hz,
2 2 2 10 3
General Procedures IR spectra were recorded on Shimadzu FTIR-8400 –COCH CH (CH ) CH ), 1.79 (1H, dd, Jϭ7.4, 16.0 Hz, H-4), 2.00 (1H, dd,
2
2
2
10
3
infrared spectrophotometer. Optical rotations were measured with JASCO Jϭ5.2, 16.0 Hz, H-4), 4.38—4.41 (1H, m, H-3), 5.01 (1H, s, H-6 or H-8),
MODEL PTC-102 polarimeter. Low resolution (LR)- and high-resolution 5.13 (1H, s, H-8 or H-6), 5.84—5.88 (1H, m, H-2Ј), 5.92 (1H, d, Jϭ8.0 Hz,
ϩ
(
7
HR)-FAB-MS spectra were recorded on a JEOL Tandem MStation JMS- H-3Ј), 5.98 (1H, s, H-6Ј). FAB-MS m/z: 501.3 [MϩH] . HR-FAB-MS m/z:
1
ϩ
00. H-NMR spectra were recorded on JEOL EX-270 (270 MHz) and 501.2861 ([MϩH] , Calcd for C H O : 501.2853).
29 41 7
2
0
JEOL EX-400 (400 MHz) instruments using CD OD and tetramethylsilane
(
3-O-Palmitoyl-(؉)-catechin (9) 7.7% yield. [a]D ϩ16.4° (cϭ0.5,
Ϫ1
3
TMS) as an internal standard. Analytical TLC was performed using Silica EtOH); IR (KBr) cm : 3736, 2918, 2851, 2498, 1747, 1606, 1521, 1474,
1
gel 60 F254 (Merck, 0.25 mm). Preparative HPLC was performed with an 1362, 1254, 1144, 1057, 814, 419. H-NMR (400 MHz) d: 0.08 (3H, t,
LC-908 (Japan Analytical Industry, Co. Ltd.) using a GS-320 column Jϭ6.8 Hz, –COCH CH (CH ) CH ), 0.45—0.52 (24H, m, –COCH CH -
2
2
2
12
3
2
2
(
21.5 mm i.d.ϫ500 mm) and MeOH as an eluent.
(CH ) CH ), 0.61—0.65 (2H, m, –COCH CH (CH ) CH ), 1.38 (1H, t,
2 12 3 2 2 2 12 3
General Procedure for the Synthesis of 3-O-Acyl-(؉)-catechins (ϩ)-
Jϭ7.2 Hz, –COCH CH (CH ) CH ), 1.78 (1H, dd, Jϭ7.0, 16.2 Hz, H-4),
2 2 2 12 3
Catechin (1) (purified from Gambir (super grade) containing 1 at ca. 40 1.98—2.02 (1H, m, H-4), 4.37—4.39 (1H, m, H-3), 5.07 (1H, s, H-6 or H-
wt%) (3.51 mmol), acid chloride (1.70 mmol), and trifluoroacetic acid 8), 5.13 (1H, s, H-8 or H-6), 5.83—5.87 (1H, m, H-2Ј), 5.91 (1H, d,
ϩ
(
3.50 mmol) were dissolved in tetrahydrofuran (10 ml), and the solution was Jϭ8.0 Hz, H-3Ј), 5.78 (1H, s, H-6Ј). FAB-MS m/z: 529.3 [MϩH] . HR-
ϩ
stirred for 24 h under Ar gas. The reaction mixture was diluted with FAB-MS m/z: 529.3128 ([MϩH] , Calcd for C H O : 529.3166).
CHCl –MeOH (3 : 1) and washed five times with water. The organic layer
3
1
45
7
2
0
3-O-Stearoyl-(؉)-catechin (10) 14.8% yield. [a]D ϩ10.4° (cϭ0.5,
was concentrated in vacuo to give a residue, which was purified by prepara- EtOH). IR (KBr) cm : 3927, 3562, 2851, 2355, 1730, 1614, 1518, 1470,
3
Ϫ1
1
tive HPLC with MeOH as an eluent, followed by freeze-drying, giving a 1142, 1061, 887, 719, 598, 419. H-NMR (400 MHz) d: 0.40 (3H, t,
white powder.
-O-Butyryl-(؉)-catechin (3) 14.0% yield. [a] ϩ7.8° (cϭ0.5, EtOH).
IR (KBr) cm : 3707, 2607, 2326, 1697, 1504, 1454, 1140, 1013, 833, 781,
4
1
Jϭ6.6 Hz, –COCH CH (CH ) CH ), 0.75—0.88 (28H, m, –COCH CH -
2 2 2 14 3 2 2
2
0
3
(CH ) CH ), 0.94—0.97 (2H, m, –COCH CH (CH ) CH ), 1.71 (2H, t,
2 14 3 2 2 2 14 3
Jϭ7.4 Hz, –COCH CH (CH ) CH ), 2.11 (1H, dd, Jϭ7.0, 16.6 Hz, H-4),
D
Ϫ1
2 2 2 14 3
1
19. H-NMR (400 MHz) d: 0.79 (3H, t, Jϭ7.4 Hz, –COCH CH CH ), 2.32 (1H, dd, Jϭ5.0, 16.6 Hz, H-4), 4.70—4.73 (1H, m, H-3), 5.40 (1H, s,
2 2 3
.45—1.53 (2H, m, –COCH CH CH ), 2.13—2.19 (2H, m, –COCH - H-6 or H-8), 5.44 (1H, s, H-8 or H-6), 6.16—6.20 (1H, m, H-2Ј), 6.24 (1H,
2
2
3
2
ϩ
CH CH ), 2.58—2.62 (1H, m, H-4), 2.78—2.82 (1H, m, H-4), 5.17—5.21 d, Jϭ8.0 Hz, H-3Ј), 6.30 (1H, s, H-6Ј). FAB-MS m/z: 557.3 [MϩH] . HR-
2
3
ϩ
(
(
1H, m, H-3), 5.88 (1H, s, H-6 or H-8), 5.93 (1H, s, H-8 or H-6), 6.65—6.68 FAB-MS m/z: 557.3457 ([MϩH] , Calcd for C H O : 557.3479).
1H, m, H-2Ј), 6.72 (1H, d, Jϭ8.0 Hz, H-3Ј), 6.78 (1H, s, H-6Ј). FAB-MS:
33 49 7
20
3-O-[(RS)-2-methyloctanoyl]-(؉)-catechin (11) 14.9% yield. [a]
D
Ϫ1
ϩ
ϩ
m/z 361.1 [MϩH] . HR-FAB-MS m/z: 361.1285 ([MϩH] , Calcd for ϩ24.6° (cϭ0.8, EtOH); IR (KBr) cm : 3310, 2928, 2856, 2349, 1742,
C H O : 361.1287).
1713, 1620, 1605, 1518, 1470, 1454, 1360, 1254, 1144, 1059, 1028, 966,
19
21
7
2
0
1
3
-O-Hexanoyl-(؉)-catechin (4) 16.8% yield. [a]D ϩ4.7° (cϭ0.5,
829, 731, 505. H-NMR (270 MHz) d: 0.89 (3H, t, Jϭ6.9 Hz,
Ϫ1
EtOH). IR (KBr) cm : 3732, 2927, 2358, 1867, 1715, 1605, 1520, 1456,
–COCH(CH )CH (CH ) CH ), 0.96 (1.5H, d, Jϭ7.0 Hz, –COCH(CH )-
3
2
2
4
3
3
1
1
362, 1252, 1140, 1015, 827, 667, 419. H-NMR (400 MHz) d: 0.83 (3H, t, CH (CH ) CH ), 1.00 (1.5H, d, Jϭ6.8 Hz, –COCH(CH )CH (CH ) CH ),
2 2 4 3 3 2 2 4 3
Jϭ7.4 Hz, –COCH CH (CH ) CH ), 1.10—1.23 (4H, m, –COCH CH - 1.18—1.39 (10H, m, –COCH(CH )CH (CH ) CH ), 2.27—2.35 (1H, m,
2
2
2
2
3
2
2
3
2
2
4
3
(
CH ) CH ), 1.41—1.45 (2H, m, –COCH CH (CH ) CH ), 2.18 (2H, t, –COCH(CH )CH (CH ) CH ), 2.58 (1H, dd, Jϭ7.6, 18.4 Hz, H-4), 2.79—
2 2 3 2 2 2 2 3 3 2 2 4 3
Jϭ7.0 Hz, –COCH CH (CH ) CH ), 2.58 (1H, dd, Jϭ6.8, 16.0 Hz, H-4),
2.90 (1H, m, H-4), 5.17 (1H, AB, Jϭ5.4, 7.6 Hz, H-3), 5.87 (1H, s-like, H-6
.79—2.83 (1H, m, H-4), 5.18 (1H, d, Jϭ5.6 Hz, H-3), 5.87 (1H, s, H-6 or or H-8), 5.94 (1H, d, Jϭ2.4 Hz, H-8 or H-6), 6.68 (1H, dd, Jϭ1.9, 8.1 Hz,
H-8), 5.93 (1H, s, H-8 or H-6), 6.63—6.66 (1H, m, H-2Ј), 6.71 (1H, d, H-2Ј), 6.73 (1H, d, Jϭ8.1 Hz, H-3Ј), 6.79 (1H, d, Jϭ1.6 Hz, H-6Ј). FAB-MS
2
2
2
2
3
2

1
450
Vol. 51, No. 12
ϩ
ϩ
m/z: 431.2 [MϩH] . HR-FAB-MS m/z: 431.2096 ([MϩH] , Calcd for
2) Kumagai A., Nagaoka Y., Obayashi T., Terashima Y., Tokuda H., Hara
Y., Okuda M., Mukainaka T., Nishino H., Kuwajima H., Uesato S.,
Bioorg. Med. Chem., 11, 5143—5148 (2003).
3) Nanjo F., Goto K., Seto R., Suzuki M., Sakai M., Hara Y., Free Radi-
cal Biol. Med., 21, 895—902 (1996).
C H O : 431.2070).
EBV-EA Activation Assay 3-O-Acyl-(ϩ)-catechins were assessed for
the inhibitory effects on the EBV-EA activation as reported previously.
The assays were performed in triplicate for each sample. No sample exhib-
ited significant toxicity against Raji cells. The viability of the cells was as-
sayed against treated cells using the trypan blue dye staining method.
24
31
7
8
,9)
4) Nanjo F., Mori M., Goto K., Hara Y., Biosci. Biotechnol. Biochem., 63,
1621—1623 (1999).
5
)
Kariyone T., “Saishin Shouyaku Gaku,” Hirokawa Publishing Co.,
Tokyo, 1965, pp. 295—297.
Acknowledgments We are grateful to Dr. Masanori Morita (Research
Institute for Technology and Science, Kinki University) for measurement of
MS spectra.
6) Zyma S. A., JP 54081274 (1979).
7) Hackett A. M., Griffiths L. A., Xenobiotica, 12, 447—456 (1982).
8
)
Ito C., Itoigawa M., Furukawa H., Tokuda H., Okuda Y., Mukainaka
References and Notes
T., Okuda M., Nishino H., Cancer Lett., 138, 87—92 (1999).
1
)
Uesato S., Kitagawa U., Hara Y., Tokuda H., Okuda M., Mou X.-Y.,
Mukainaka T., Nishino H., Bioorg. Med. Chem. Lett., 10, 1673—1675
9) Ito C., Itoigawa M., Furukawa H., Ichiishi E., Mukainaka T., Okuda
M., Ogata M., Tokuda H., Nishino H., Cancer Lett., 142, 49—54
(1999).
(
2000).