Structure-activity relationships of (1'S)-1'-acetoxychavicol acetate, a major constituent of a Southeast Asian condiment plant Languas galanga, on the inhibition of tumor-promoter-induced Epstein-Barr virus activation

Article abstract of DOI:10.1021/jf990528r

The structure-activity relationships of (1'S)-1'-acetoxychavicol acetate (ACA), a cancer chemopreventive agent of food origin, were investigated in an inhibitory test of tumor promoter teleocidin B-4-induced Epstein-Barr virus (EBV) activation in Raji cel

Full text of DOI:10.1021/jf990528r

1518
J. Agric. Food Chem. 2000, 48, 1518−1523
Str u ctu r e-Activity Rela tion sh ip s of (1′S)-1′-Acetoxych a vicol Aceta te,
a Ma jor Con stitu en t of a Sou th ea st Asia n Con d im en t P la n t La n gu a s
ga la n ga , on th e In h ibition of Tu m or -P r om oter -In d u ced
Ep stein -Ba r r Vir u s Activa tion
Akira Murakami,† Kazuo Toyota,† Shin Ohura,‡ Koichi Koshimizu,† and Hajime Ohigashi*,‡
Department of Biotechnological Science, Faculty of Biology-Oriented Science and Technology,
Kinki University, Iwade-Uchita, Wakayama 649-6493, J apan, and Division of Applied Life Sciences,
Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, J apan
The structure-activity relationships of (1′S)-1′-acetoxychavicol acetate (ACA), a cancer chemopre-
ventive agent of food origin, were investigated in an inhibitory test of tumor promoter teleocidin
B-4-induced Epstein-Barr virus (EBV) activation in Raji cells. Through a test of 16 derivatives,
the structural factors regulating activity were found to be as follows: (1) the absolute configuration
at the 1′-position does not affect activity; (2) hydrogenation of the terminal methylene group abolishes
activity; (3) both the phenolic and alcoholic hydroxyl groups are compulsorily acetylated, and it is
necessary that the former is oriented only at the position para to the side chain; (4) an additional
acetoxyl group is allowed to locate at the ortho or meta position; and (5) substitution of the hydrogen
atom at the 1′-position by a methyl group reduces activity. Upon esterase blockade in Raji cells,
(1′R,S)-ACA suppressed EBV activation, the extent of which was the same as tested in the control,
suggesting that ACA bearing two acetoxyl groups is an intracellular structure prerequisite for activity
exhibition. The present study suggests that nucleophilic attack to the 3′-position is important and
involved in the interaction of ACA with an unidentified target molecule(s) participating in the process
of EBV activation.
Keyw or d s: 1′-Acetoxychavicol acetate; Epstein-Barr virus; structure-activity relationships; chemo-
prevention; teleocidin B-4
INTRODUCTION
et al., 1993). The rhizomes are ingested widely in
Southeast Asia as a flavor or condiment and are
occasionally used as a folk medicine for stomach health
(J acquat, 1990). Also, it is interesting to note that 1 was
previously reported as an antiulcer (Mitsui, 1985) and
antitumor agent (Itokawa, 1987). Thereafter, there have
been a considerable number of reports indicating the
effectiveness of ACA on the inhibition of chemically
induced carcinogenesis or the expression of tumor
markers in the initiation and postinitiation (promotion)
phases in multistage carcinogenesis models (Murakami
et al., 1996b; Ohnishi et al., 1996; Tanaka et al.,
1997a,b; Kobayashi et al., 1998). To address the action
mechanisms by which ACA suppresses oncogenic pro-
cesses, it is important to note the biochemical and
biological activities of ACA thus far found: (1) inhibition
of xanthine oxidase activity (Noro et al., 1988); (2)
suppression of superoxide (O2-) generation in leukocytes
(Murakami et al., 1996b); (3) antilipid peroxidation
(Murakami et al., 1996b); (4) inhibition of induction of
proliferation markers such as silver-stained nucleolar
organizer region’s protein and 5-bromo-2′-deoxyuridine
labeling (Ohnishi et al., 1996); (5) inhibition of polyamine
synthesis (Ohnishi et al., 1996); (6) inhibition of orni-
thine decarboxylase activity (Ohnishi et al., 1996); and
(7) induction of both glutathione S-transferase (GST)
and quinone reductase (QR) activities (Tanaka et al.,
1997b). The ability of xenobiotic enzymes to be induced
may be important as the action mechanism in the
initiation phase. On the other hand, we have recently
One of the biological effects of tumor promoters is the
activation of Epstein-Barr virus (EBV), a herpesvirus
causative for African Burkitt’s lymphoma (Klein and
Kelin, 1985), cervical carcinoma (Young and Sixbey,
1988), nasopharyngeal carcinoma (NPC) (Kumar and
Mahanta, 1998), and gastric cancer in part (Leoneini
et al., 1993). Some tumor promoters induce EBV activa-
tion partly through the activation of protein kinase C
(PKC) (Natsukari et al., 1997), a major cellular receptor
of 12-O-tetradecanoylphorbol-13-acetate (TPA)-type tu-
mor promoters and mitogen-activated protein kinase
(Panousis et al., 1997). An EBV-infected lymphocyte is
partly characterized by its high expression level of the
oncogene c-myc (Nishikura et al., 1985). To date, we
have been conducted inhibitory tests of tumor promoter-
induced EBV activation for the estimation of the anti-
tumor-promoting properties of edible plants as well as
for the isolation of their active constituents [for reviews,
see Murakami et al. (1996a, 1998)].
In 1993, we isolated (1′S)-1′-acetoxychavicol acetate
[(1′S)-ACA) (1; Figure 1) from the rhizomes of a sub-
tropical ginger, Languas galanga Stuntz (Zingiber-
aceae), as a potent inhibitor of EBV activation (Kondo
* Author to whom correspondence should be addressed
(telephone +81-75-753-6281; fax +81-75-753-6284; e-mail
ohigashi@kais.kyoto-u.ac.jp).
† Kinki University.
‡ Kyoto University.
10.1021/jf990528r CCC: $19.00 © 2000 American Chemical Society
Published on Web 04/14/2000

Inhibition of Epstein−Barr Activation by ACA
J. Agric. Food Chem., Vol. 48, No. 5, 2000 1519
Nozaki of Takasago International Corp. (1′R,S)-1′-Acetoxy-
chavicol acetate (3), (1′R,S)-1′-hydroxychavicol (4), (1′R,S)-1′-
hydroxychavicol acetate (5), (1′R,S)-2′,3′-dihydro-ACA (6),
(1′R,S)-1-phenyl-2-propene-1-acetate (7), chavicol acetate (8),
and S,S,S-tributyl phosphorotrithioate (DEF) were synthesized
as previously reported (Kondo et al., 1993; Irie et al., 1985).
Vinylmagnesium bromide was obtained from Aldrich (Mil-
waukee, WI). Raji cells and high-titer early antigen (EA)-
positive sera from nasopharyngeal carcinoma patients (NPC)
for the EBV assay were a kind gift from Dr. S. Imai of
Hokkaido University. Fluorescence isothiocyanate (FITC)-
labeled anti-human IgG was obtained from Dako Co. Ltd.
(Glostrup, Denmark). Other chemicals were obtained from
Wako Pure Chemical Industries Co., Ltd. (Osaka, J apan) and
were of special grade.
P r ep a r a tion of Der iva tives. (1′R,S)-1′-Acetoxy-1′-meth-
ylchavicol Acetate (9). A solution of p-hydroxyacetophenone
(108 mg) in 40 mL of dry THF was added to 1.0 M vinylmag-
nesium bromide in 100 mL of dry THF under N2 atmosphere
for 2 h. After evaporation of THF, the reaction mixture was
partitioned between ethyl acetate (EtOAc) and NH4Cl-
saturated water to give 1′-hydroxy-1′-methylchavicol, which
was then acetylated with 4-(dimethylamino)pyridine and acetic
anhydride. The reaction mixture was purified by using pre-
parative TLC (toluene/acetone, 9:1) and HPLC (µBondasphere
C18 19 mm × 15 cm column, Waters; mobile phase of 40%
acetonitrile in water; flow rate ) 7.0 mL; detection at 254 nm)
to give 9 (24 mg): 1H NMR (300 MHz, CDCl3) δ 1.87 (3H, s,
1′-Me), 2.06 (3H, s, 1′-OAc), 2.28 (3H, s, 4-OAc), 5.24 (1H, dd,
J ) 10.8, 0.5 Hz, H-3′a), 5.26 (1H, dd, J ) 17.4, 0.5 Hz, H-3′b),
6.23 (1H, dd, J ) 17.4, 10.8 Hz, H-2′), 7.05 (2H, d, J ) 8.7 Hz,
H-3,5), 7.38 (2H, d, J ) 8.7 Hz, H-2,6); APCI-MS, m/z 249
[(MH)+, C14H17O4].
(1′R,S)-1-(2-Acetoxyphenyl)-2-propene-1-acetate (10). A solu-
tion of p-hydroxybenzaldehyde (1.5 g) in 5 mL of dry THF was
added to 1.0 M vinylmagnesium bromide in 25 mL of dry THF
under N2 atmosphere for 3.5 h. After evaporation of THF, the
reaction mixture was partitioned between EtOAc and NH4Cl-
saturated water to give 1-(2-hydroxyphenyl)-2-propen-1-ol,
which was then acetylated with pyridine and acetic anhydride.
The reaction mixture was purified by using preparative
TLC (toluene/acetone, 9:1) and HPLC (µBondasphere C18 3.9
mm × 15 cm column, Waters; mobile phase of 40% acetonitrile
in water; flow rate ) 1.0 mL; detection at 254 nm) to give 10
(800 mg): 1H NMR (300 MHz, CDCl3) δ 2.03 (3H, s, 1′-OAc),
2.28 (3H, s, 4-OAc), 5.29 (1H, dd, J ) 10.8, 1.0 Hz, H-3′a), 5.34
(1H, dd, J ) 17.5, 1.0 Hz, H-3′b), 6.10 (1H, dd, J ) 17.5, 10.8
Hz, H-2′), 6.55 (1H, br d, J ) 8.9 Hz, H-1′), 7.39 (4H, m, H-2-
5); APCI-MS, m/z 235 [(MH)+, C13H15O4].
(1′R,S)-1-(3-Acetoxyphenyl)-2-propene-1-acetate (11). A solu-
tion of m-hydroxybenzaldehyde (1.5 g) in 5 mL of dry THF
was added to 1.0 M vinylmagnesium bromide in 25 mL of dry
THF under N2 atmosphere for 3.5 h. After evaporation of THF,
the reaction mixture was partitioned between EtOAc and NH4-
Cl-saturated water to give 1-(3-hydroxyphenyl)-2-propen-1-ol,
which was acetylated with pyridine and acetic anhydride. The
reaction mixture was purified by using preparative TLC
(toluene/acetone, 9:1) and HPLC (µBondasphere C18 3.9
mm × 15 cm column, Waters; mobile phase of 40% acetonitrile
in water; flow rate ) 1.0 mL; detection at 254 nm) to give 11
(780 mg): 1H NMR (300 MHz, CDCl3) δ 1.87 (3H, s, 1′-Me),
2.06 (3H, s, 1′-OAc), 2.28 (3H, s, 4-OAc), 5.24 (1H, dd, J )
10.4, 1.0 Hz, H-3′a), 5.26 (1H, dd, J ) 17.5, 1.0 Hz, H-3′b),
5.93 (1H, ddd, J ) 17.0, 10.0, 5.8 Hz, H-2′), 6.23 (1H, d, J )
5.8 Hz, H-1′), 7.05 (2H, d, J ) 8.7 Hz, H-3,5), 7.38 (2H, d, J )
8.7 Hz, H-2,6); APCI-MS, m/z 235 [(MH)+, C13H15O4].
F igu r e 1. Structures of (1′S)-1′-acetoxychavicol acetate and
its derivatives.
found that ACA inhibits inducible nitric oxide synthase
expression by suppressing the degradation of Iκ-B in
mouse macrophage RAW 264.7 cells (Ohata et al., 1998).
Furthermore, ACA can suppress double-TPA applica-
tion-induced hydrogen peroxide formation and other
biochemical parameters related to inflammation in
mouse skin (Nakamura et al., 1998). Because oxidative
stress is closely associated with carcinogenesis (Kensler
et al., 1989), we regard the inhibition of oxidative
damage in inflammation by ACA as the most acceptable
and major action mechanism in the postinitiation phase.
The intracellular target molecule(s) of ACA, with
which it directly interacts, has (have) yet to be discov-
ered. As one of the approaches to elucidate the action
mechanism of ACA, we examined the structure-activity
relationships of ACA on the inhibition of tumor-promoter-
induced EBV activation using 16 ACA derivatives.
MATERIALS AND METHODS
1
An a lytica l In str u m en ts. H and 13C NMR [Bruker DRX-
300, tetramethylsilane (TMS) as an internal standard] and
APCI-MS (MH-1200, Hitachi Co. Ltd., Tokyo, J apan) were
recorded. HPLC (LC10AS and Chromatopac C-R6A, Shimadzu,
Kyoto, J apan) was also used.
Ch em ica ls. (1′S)-1′-Acetoxychavicol acetate (ACA) [(1′S)-
ACA, 1] and teleocidin B-4 were isolated from Languas
galanga and Streptoverticillium blastmyceticum NA 34-17,
respectively, as previously reported (Kondo et al., 1993; Irie
et al., 1984). (1′R)-ACA (2) was a gift from Drs. Suzuki and
(1′R,S)-1-(2,4-Diacetoxyphenyl)-2-propene-1-acetate (12): A
solution of o,p-dihydroxybenzaldehyde (1.5 g) in 5 mL of dry
THF was added to 1.0 M vinylmagnesium bromide in 25 mL
of dry THF under N2 atmosphere for 3.5 h. After evaporation
of THF, the reaction mixture was partitioned between EtOAc
and NH4Cl-saturated water to give 1-(2,4-dihydroxyphenyl)-
2-propen-1-ol, which was acetylated with pyridine and acetic
anhydride. The reaction mixture was purified by using pre-
parative TLC (toluene/acetone, 9:1) and HPLC (µBondasphere

1520 J. Agric. Food Chem., Vol. 48, No. 5, 2000
Murakami et al.
C18 3.9 mm × 15 cm column, Waters; mobile phase of 40%
acetonitrile in water; flow rate ) 1.0 mL; detection at 254 nm)
to give 12 (500 mg): 1H NMR (300 MHz, CDCl3) δ 2.10 (3H, s,
1′-OAc), 2.28 (6H, s, 2- and 3-OAc), 5.27 (1H, d, J ) 10.6 Hz,
H-3′a), 5.32 (1H, d, J ) 17.6 Hz, H-3′b), 5.96 (1H, ddd, J )
17.6, 10.6, 5.8 Hz, H-2′), 6.26 (1H, d, J ) 5.8 Hz, H-1′), 7.12
(1H, d, J ) 2.1 Hz), 7.21 (2H, m); APCI-MS, m/z 293 [(MH)+,
N2 atmosphere for 2 h. After evaporation of THF, the reaction
mixture was partitioned between EtOAc and NH4Cl-saturated
water to give (1R,S)-1-(4-chrolophenyl)-2-propen-1-ol, which
was acetylated with pyridine and acetic anhydride. The
reaction mixture was purified by using preparative HPLC
(µBondasphere C18 19 mm × 15 cm column, Waters; mobile
phase of 60% acetonitrile in water; flow rate ) 7.0 mL;
detection at 254 nm) to give 17 (320 mg): 1H NMR (300 MHz,
CDCl3) δ 2.11 (3H, s, 1′-OAc), 5.27 (2H, dd, J ) 10.1, 1.0 Hz,
H-3′a), 5.30 (1H, dd, J ) 17.1, 1.0 Hz, H-3′b), 5.98 (1H, ddd,
J ) 17.1, 10.1, 5.8 Hz, H-2′), 6.22 (1H, d, J ) 5.8 Hz, H-1′),
6.94 (2H, d, J ) 8.5 Hz, H-3,5), 7.80 (2H, d, J ) 8.5 Hz, H-2,6);
EIMS, m/z 211 [(MH)+, C11H12O2Cl].
EBV Activa tion Test. The inhibitory assay of EBV activa-
tion, using EBV genome-carrying human B lymphoblastoid
Raji cells, was done basically in the same manner as reported
previously (Kondo et al., 1993). Raji cells (5 × 105) were
incubated in 1 mL of RPMI 1640 medium (supplemented with
10% fetal bovine serum, 200 units/mL penicillin, and 250 µg/
mL streptomycin) containing sodium n-butyric acid (3 mM),
teleocidin B-4 (50 nM), and the test compound dissolved in 5
µL of DMSO at 37 °C under 5% CO2 atmosphere for 48 h. The
cytotoxicity of the test compound was evaluated by cell
viability, which was measured by staining the cells with trypan
blue. After smears were made from the cell suspension, EA-
positive cells were detected by a conventional indirect immu-
nofluorescence technique with high-titer EA-positive sera from
an NPC patient, followed by FITC-labeled IgG. The ordinary
average percentage of EA-induced cells in the control experi-
ment (with only n-butyrate, teleocidin B-4, and DMSO) was
∼40%. Each experiment was done in triplicate, and the data
were expressed as mean ( standard deviations (m ( SD).
Ester a se Block a d e. The influence of S,S,S-tributyl phos-
phorotrithioate (DEF) on the inhibition of ACA toward EBV
activation was examined as previously reported (Irie et al.,
1985) with some modifications. Raji cells (5 × 105 cells/mL)
were preincubated with or without DEF (10 or 50 µM) at 37
°C under a 5% CO2 atmosphere. After 24 h, extracellular DEF
was removed by centrifugation at 2000g for 5 min followed by
two washings with PBS, and the cells were suspended in a
fresh medium in a density of 5 × 105 cells/mL. The cells were
incubated with teleocidin B-4 (50 nM), n-butyrate (3 mM), and
(1′R,S)-ACA (2.5 µM) at 37 °C for 48 h. The following procedure
to measure the EBV activation was the same as described
above.
C
15H17O6].
(1′R,S)-1-(3,4-Diacetoxyphenyl)-2-propene-1-acetate (13). A
solution of m-hydroxybenzaldehyde (1.5 g) in 5 mL of dry THF
was added to 1.0 M vinylmagnesium bromide in 25 mL of dry
THF under N2 atmosphere for 3.5 h. After evaporation of THF,
the reaction mixture was partitioned between EtOAc and NH4-
Cl-saturated water to give 1-(3,4-dihydroxyphenyl)-2-propen-
1-ol, which was acetylated with pyridine and acetic anhydride.
The reaction mixture was purified by using preparative TLC
(toluene/acetone, 9:1) and HPLC (µBondasphere C18 3.9
mm × 15 cm column, Waters; mobile phase of 40% acetonitrile
in water; flow rate ) 1.0 mL; detection at 254 nm) to give 13
(380 mg): 1H NMR (300 MHz, CDCl3) δ 2.05 (3H, s, 1′-OAc),
2.25 (3H, s, 2- or 3-OAc), 2.28 (3H, s, 2- or 3-OAc), 5.23 (1H, d,
J ) 10.4 Hz, H-3′a), 5.25 (1H, d, 17.2 Hz, H-3′b), 5.95 (1H,
ddd, J ) 17.2, 10.4, 5.8 Hz, H-2′), 6.40 (1H, d, J ) 5.8 Hz,
H-1′), 6.97 (1H, d, J ) 2.2 Hz, H-2), 6.99 (2H, m, H-5,6); APCI-
MS, m/z 293 [(MH)+, C15H17O6].
(1′R,S)-1′-Acetoxychavicol Methyl Ether (14). p-Methoxy-
benzaldehyde (1.2 mL) was added to 1.0 M vinylmagnesium
bromide in 15 mL of dry THF under N2 atmosphere for 2 h.
After evaporation of THF, the reaction mixture was partitioned
between EtOAc and NH4Cl-saturated water to give 1′-hy-
droxychavicol methyl ether, which was acetylated with pyri-
dine and acetic anhydride. The reaction mixture was purified
by using preparative TLC (toluene/acetone, 10:1) and HPLC
(µBondasphere C18 19 mm × 15 cm column, Waters; mobile
phase of 80% methanol in water; flow rate ) 7.0 mL; detection
at 254 nm) to give 14 (400 mg): 1H NMR (300 MHz, CDCl3) δ
2.08 (3H, s, 1′-OAc), 3.79 (3H, s, 4-OMe), 5.22 (1H, dd, J )
10.8, 1.0 Hz, H-3′a), 5.26 (1H, dd, J ) 17.5, 1.0 Hz, H-3′b),
6.00 (1H, ddd, J ) 17.5, 10.8, 6.1 Hz, H-2′), 6.22 (1H, br d,
J ) 6.1 Hz, H-1′), 6.85 (2H, d, J ) 8.9 Hz, H-3,5), 7.32 (2H, d,
J ) 8.9 Hz, H-2,6); APCI-MS, m/z 207 [(M)+, C12H15O3].
(1′R,S)-1′-Methoxychavicol Acetate (15). 3-Methoxybenzal-
dehyde (200 mg) in 2.5 mL of dry THF was added to 1.0 M
vinylmagnesium bromide in 15 mL of dry THF under N2
atmosphere for 3 h. After evaporation of THF, the reaction
mixture was partitioned between EtOAc and NH4Cl-saturated
water to give (1R,S)-1-(3-methoxy)-2-propen-1-ol, which was
acetylated with pyridine and acetic anhydride. The reaction
mixture was purified by using preparative HPLC (µBonda-
sphere C18 19 mm × 15 cm column, Waters; mobile phase of
40% acetonitrile in water; flow rate ) 7.0 mL; detection at 254
nm) to give 15 (72 mg): 1H NMR (300 MHz, CDCl3) δ 2.29
(3H, s, 4-OAc), 3.78 (3H, s, 1′-OMe), 5.22 (1H, dd, J ) 10.8,
1.0 Hz, H-3′a), 5.26 (1H, dd, J ) 17.5, 1.0 Hz, H-3′b), 6.00 (1H,
ddd, J ) 17.5, 10.8, 6.1 Hz, H-2′), 6.22 (1H, d, J ) 6.1 Hz,
H-1′), 6.85 (2H, d, J ) 8.9 Hz, H-3,5), 7.32 (2H, d, J ) 8.9 Hz,
H-2,4); APCI-MS, m/z 207 [(MH)+, C12H15O3].
RESULTS
Str u ctu r e-Activity Rela tion sh ip s of ACA. We
first examined the inhibitory activities of (1′S)-ACA (1)
and its 16 derivatives toward teleocidin B-4-induced
EBV activation in Raji cells. Teleocidin B-4 is an indole
alkaloid-type of tumor promoter in mouse skin (Irie et
al., 1984). EBV activation was measured by the induc-
tion rate of EBV early antigen (EA)-positive cells. The
structures and activities of the test compounds are
shown in Figure 1 and Table 1, respectively.
(1′R,S)-1-(4-Nitrophenyl)-2-propene-1-acetate (16). p-Nitro-
benzaldehyde (800 mg) in 2.5 mL of dry THF was added to
1.0 M vinylmagnesium bromide in 15 mL of dry THF under
N2 atmosphere for 3 h. After evaporation of THF, the reaction
mixture was partitioned between EtOAc and NH4Cl-saturated
water to give (1R,S)-1-(4-nitrophenyl)-2-propen-1-ol, which was
acetylated with pyridine and acetic anhydride. The reaction
mixture was purified by using preparative HPLC (µBonda-
sphere C18 19 mm × 15 cm column, Waters; mobile phase of
40% acetonitrile in water; flow rate ) 7.0 mL; detection at 254
nm) to give 16 (72 mg): 1H NMR (300 MHz, CDCl3) δ 2.15
(3H, s, 1′-OAc), 5.34 (2H, m, H-3′ab), 5.96 (1H, ddd, J ) 17.1,
10.4, 6.1 Hz, H-2′), 6.31 (1H, d, J ) 6.1 Hz, H-1′), 7.52 (2H, d,
J ) 8.5 Hz, H-3,5), 8.22 (2H, d, J ) 8.5 Hz, H-2,6); APCI-MS,
m/z 222 [(MH)+, C11H12O4N].
Naturally occurring 1 completely inhibited EBV ac-
tivation at a concentration of 5 µM (IC50 ) 1.0 µM) and
exhibited a marked cytotoxicity (1% > cell viability) at
g50 µM. No optical separation of the racemate (3) or
other derivatives (4-7, 9-17) was attempted because
(1′R)-ACA (2) and (1′R,S)-ACA (3) showed activities
(IC50 ) 1.5 and 1.2 µM, respectively) very similar to that
of 1. Hydrogenated ACA was weakly active (6, IC50
)
29 µM). Replacement of the 1′-H by a methyl group
resulted in a drastic activity decrease (9, IC50 ) 24 µM).
It is of great interest that two orientational isomers of
ACA (10, 11), in which a phenolic acetoxyl group is
placed at the ortho and meta positions to the alkyl C3
side chain, respectively, possessed no significant activity
(IC50 ) 57 and >100 µM, respectively). The derivatives
(1′R,S)-1-(4-Chlorophenyl)-2-propene-1-acetate (17). p-Chlo-
robenzaldehyde (800 mg) in 2.5 mL of dry THF was added to
1.0 M vinylmagnesium bromide in 15 mL of dry THF under

Inhibition of Epstein−Barr Activation by ACA
J. Agric. Food Chem., Vol. 48, No. 5, 2000 1521
Ta ble 1. In h ibitor y Activities of (1′S)-ACA a n d Its Der iva tives tow a r d Tu m or -P r om oter -In d u ced EBV Activa tion in
Ra ji Cells
% inhibition (% cell viability) at a concentration of
compound
100 µM
50 µM
5 µM
100 (63)
100 (68)
100 (63)
0 (<80)
0 (>80)
15 ( 4 (>80)
0 (>80)
0 (>80)
1.5 µM
0.5 µM
IC50 (µM)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
NDa (1>)
NDa (1>)
NDa (1>)
NDa (1>)
32 ( 6 (>80)
0 (>80)
80 ( 1 (>80)
24 ( 8 (>80)
0 (>80)
80 ( 1 (>80)
44 ( 2 (>80)
14 ( 8 (>80)
NDa (1>)
NDa (1>)
0 (>80)
60 ( 5 (>80)
50 ( 5 (>80)
55 ( 5 (>80)
0 (>80)
36 ( 8 (>80)
32 ( 6 (>80)
35 ( 8 (>80)
0 (>80)
1.0
1.5
1.2
NDa (1>)
NDa (1>)
48 ( 8 (>80)
8 ( 4 (>80)
85 ( 6 (>80)
34 ( 3 (>80)
5 ( 3 (>80)
85 ( (>80)
85 ( 2 (>80)
24 ( 3 (>80)
NDa (1>)
>100
>100
29
>100
>100
24
0 (>80)
0 (>80)
0 (>80)
0 (>80)
0 (>80)
0 (>80)
0 (>80)
0 (>80)
15 ( 5 (>80)
29 ( 9 (>80)
0 (>80)
0 (>80)
0 (>80)
0 (>80)
0 (>80)
57
>100
0 (>80)
0 (>80)
81 ( 11 (>80)
70 ( 4 (>80)
0 (>80)
28 ( 6 (>80)
47 ( 12 (>80)
0 (>80)
23 ( 4 (>80)
12 ( 6 (>80)
0 (>80)
2.9
1.9
NDa (1>)
15 ( 5 (>80)
0 (>80)
>100
>100
>100
>100
0 (>80)
17 ( 8 (>80)
0 (>80)
0 (>80)
0 (>80)
0 (>80)
35 ( 8 (>80)
15 ( 5 (>80)
0 (>80)
0 (>80)
0 (>80)
0 (>80)
0 (>80)
0 (>80)
a
Not detected due to cytotoxicity.
Ta ble 2. In flu en ce of Ester a se In h ibitor DEF on th e
P oten cy of (1′R,S)-ACA (3) in th e In h ibition of EBV
Activa tion
% of EBV-EA-positive cellsa
pretreatment
controlb
ACA (3)c
% inhibition
ethanol
DEF (10 µM)
DEF (50 µM)
53 ( 4
57 ( 4
57 ( 1
25 ( 5
26 ( 3
27 ( 6
52
54
52
a
b
Mean ( SD (n ) 3). EBV was activated with teleocidin B-4
(50 nM) and n-butyrate (3 mM). c At a concentration of 2.5 µM.
toward EBV activation in Raji cells in which esterase(s)
was blocked by DEF (10 or 50 µM) 24 h prior to ACA
addition, as compared with the vehicle control.
DISCUSSION
The structural prerequisites of naturally occurring
(1′S)-ACA (1) for EBV activation inhibition are remark-
ably strict. It should be noted that the activity of no
derivatives, except for 2, 3, 12, and 13 in the present
study, was comparable to that of 1 and only four
F igu r e 2. Deduced important structural factors of ACA for
the inhibitory activity toward EBV activation.
lacking the acetoxyl group at the 4-position (7) or 1′-
postion (8) in 1 were also inactive (IC50 > 100 µM each).
Hydrolyzed derivatives of ACA (4 and 5, IC50 > 100 µM
each) or those bearing a methoxyl group in place of an
acetoxyl (14 and 15) were also inactive (IC50 > 100 and
66 µM, respectively). However, substitution of a phenolic
acetoxyl group by the nitro (16) or chroline (17) group
caused a loss of activity. Importantly, the triacetoxyl
derivatives 12 and 13 maintained an inhibitory poten-
tial comparable to that of 1 (IC50 ) 2.9 and 1.9 µM,
respectively). In summary, no derivatives in the present
study showed an activity higher than that of 1, and the
activities of 2, 3, 12, and 13 were all comparable to that
of 1.
In flu en ce of Ester a se Block a d e. It is well-known
that ester groups of organic compounds, in general, are
hydrolyzed by intracellular esterase(s) to allow their
hydrolysates to be trapped within cells. To confirm the
importance of the presence of acetoxyl groups in (1′S)-
ACA (1) for activity exhibition (see Figure 2), an esterase
blockade was executed in the EBV assay. DEF, a potent
potentiator of insecticide, was selected as an esterase
inhibitor because of its low toxicity to Raji cells (Irie et
al., 1985). As shown in Table 2, 1 at a concentration of
2.5 µM completely maintained its inhibitory activity
derivatives (6, 9, 10, and 15) were weakly active (1/57-
1
/
of 1). Because both an enantiomer of 1 (2, IC50 )
24
1.5 µM) and a racemic mixture (3, IC50 ) 1.2 µM)
showed the same activity as 1, the absolute configura-
tion at the 1′-position does not affect inhibitory activity.
The low activity of hydrogenated ACA (6, IC50 ) 29 µM)
indicates that the terminal methylene group is neces-
sary for activity. The lack of activity from 5, 8, and 15
demonstrates that the functional group at the 1′-position
is required to be acetylated. The phenolic hydroxyl group
is also forced to be acetylated and yet is oriented only
at the position para to the C3 alkyl side chain because
both the derivatives, with other functional groups (7,
14, 16, and 17), and orientational isomers (10, 11) had
no notable activity (IC50 ) 57-100 µM). An additional
acetoxyl group to the ortho or meta position to the side
chain did not diminish the activity (IC50 ) 2.9 and 1.9
µM, respectively). The structural factors regulating the
inhibitory activity of 1 are summarized as follows: (1)
the absolute configuration at the 1′-position does not
affect activity; (2) lack of a terminal methylene group
results in marked activity reduction; (3) both phenolic
and alcoholic hydroxyl groups are compulsorily acety-
lated, and the former should be oriented only at the

1522 J. Agric. Food Chem., Vol. 48, No. 5, 2000
Murakami et al.
position para to the C3 alkyl side chain; (4) an additional
acetoxyl group is allowed at the ortho or meta position
for the activity; and (5) substitution of the 1′-H by a
methyl group drastically decreases the activity. Inter-
estingly, the structure-activity relationships described
here have some correlation with those on the antitumor
activity of (1′S)-ACA derivatives (1, 4, and 5) against
Sarcoma 180 ascites in mice, as reported by Itokawa et
al. (1987).
esterase(s), forming 4 and 5, which are intracellular
candidates interacting with target molecule(s). (2) In-
versely, the activity of 3 drastically decreases when the
two acetyl groups are hydrolyzed; namely, holding two
acetoxyl groups is an active structure. Intracellular
esterase(s) hydrolyzed two acetoxyl groups at some point
after starting the EBV assay. It is extremely important
to elucidate whether 3 exhibits activity before or after
hydrolyzation of the acetoxyl groups. In the latter case,
the activity potency of 3 should be reduced significantly
by the esterase blockade. (1′R,S)-ACA, however, was
postulated to exhibit activity before intracellular hy-
drolyzation, because 3 at 2.5 µM maintained a complete
inhibitory activity in Raji cells preincubated with an
esterase inhibitor, DEF (10 or 50 µM), for 24 h as
compared with the control experiment (Table 2). Irie et
al. (1985) reported that 10 µM DEF was appropriate for
esterase blockade in Raji cells, and its preincubation
time was optimized at 18-24 h. Thus, the results here,
together with the validity of the experimental condi-
tions, raise the possibility that both acetoxyl groups in
3 are necessary for an exhibition of activity.
In conclusion, the structure-activity relationships of
ACA suggested that the nucleophilic attack to the 3′-
position of ACA, accompanying acetate elimination, may
be involved in the interaction of ACA with an unidenti-
fied target molecule(s) participating in the process of
EBV activation. ACA is postulated to modify them by
forming adducts. Furthermore, the phenolic acetoxyl
group at the para position could play an important role
in the interaction. The present findings may confirm the
action mechanism(s) of ACA after the discovery of its
target molecule(s).
It is important to note that the 3′-carbon is susceptible
to nucleophiles and this may accompany double-bond
migration and acetate elimination, that is, abnormal
bimolecular displacement (SN2′). A low activity of
hydrogenated ACA (6) strongly supports the hypothesis
that the high reactivity of the 3′-carbon with electro-
philes, in part, is an increasing factor for activity and
vice versa. Along a similar line, there is a premise for
the properties of the functional group at C-1′, namely,
the chemical trait of the leaving group. In this regard,
it should be noted that the acetoxyl group was the best
leaving group tested here as compared with the hydro-
gen atom (7), hydroxyl (5), or methoxyl (15) group. They
were conversely inactive (IC50 > 100 µM). Talalay et al.
(1988) reported that the compounds which have an
electrophilic terminal methylene, including Michael
reaction acceptors, are capable of inducing phase II
enzymes such as GST or QR by using a hepatocyte
model. As expected, oral feeding of ACA significantly
induced these xenobiotic enzymes in the liver and colon
of rats (Tanaka et al., 1997b). Thus, the structure-
activity relationships of ACA derivatives in their ability
to induce xenobiotic enzymes remain to be examined in
the future.
Despite the interesting data, there is no clear-cut
explanation for the contrast in activity between (1′R,S)-
ACA (3) (IC50 ) 1.2 µM) and the inactive derivatives 7,
10, 11, 14, 16, and 17 (IC50 ) 57-100 µM), all of which
are considered to be susceptible to both nucleophilic
attack and acetate elimination. It is tempting, however,
to speculate that the phenolic acetoxyl group only at the
para position could appropriately be oriented for inter-
action with the specific target site(s). The hypothesis
that ACA acts specifically toward biological systems
may be indirectly supported by the data showing that
ACA has no inhibition in some in vitro biological
systems related to tumor promotion, for example, PKC
activity, arachidonate release, prostaglandin E2 syn-
thesis, cyclooxygenase, and lipoxygenase activity (data
not shown). If ACA undergoes nonspecific interactions
with any nucleophiles, the above-mentioned specificity
in biological activities may be impossible. In addition,
ACA exhibited no detectable toxicity or marked body
weight retardation in rodents by oral feeding (Ohnishi
et al., 1996; Tanaka et al., 1997a,b). Such activity and
toxicity profiles suggest that the mode of action of ACA
is rather specific to biological systems rather than
simple and nonspecific interactions with any nucleo-
philic groups of cellular components.
ABBREVIATIONS USED
ACA, 1′-acetoxychavicol acetate; APCI-MS, aperture
chemical ionization mass spectrum; DMSO, dimethyl
sulfoxide; EBV, Epstein-Barr virus; EtOAc, ethyl ac-
etate; GST, glutathione S-transferase; HPLC, high-
performance liquid chromatography; PBS, phosphate-
buffered saline; PKC, protein kinase C; QR, quinone
reductase; THF, tetrahydrofuran; TLC, thin-layer chro-
matography; TMS, tetramethylsilane; TPA, 12-O-tet-
radecanolyphorbol-13-acetate; NMR, nuclear magnetic
resonance.
ACKNOWLEDGMENT
We thank Drs. N. Suzuki and M. Nozaki of Takasago
International Corp. for kindly supplying (1′R)-ACA.
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Received for review May 17, 1999. Revised manuscript received
December 1, 1999. Accepted February 29, 2000. This study
was supported in part by a Grant-in-Aid for Scientific Research
on Priority AreassCancer from the Ministry of Education,
Science, and Culture and by subsidy from Takeda Food Co.
Ltd.
J F990528R