Antioxidant and antiviral activities of plastoquinones from the brown alga Sargassum micracanthum, and a new chromene derivative converted from the plastoquinones

Article abstract of DOI:10.1248/bpb.28.374

Two plastoquinones were isolated from the methanolic extract of the brown alga Sargassum micracanthum, and these were identified as a known 2-geranylgeranyl-6-methylbenzoquinone and its hydroquinone, respectively, based on spectroscopic analysis. The abso

Full text of DOI:10.1248/bpb.28.374

374
Notes
Biol. Pharm. Bull. 28(2) 374—377 (2005)
Vol. 28, No. 2
Antioxidant and Antiviral Activities of Plastoquinones from the Brown
Alga Sargassum micracanthum, and a New Chromene Derivative
Converted from the Plastoquinones
Makoto IWASHIMA,a Jun MORI,a,b Xiang TING,a Takayuki MATSUNAGA,c Kyoko HAYASHI,d
Daishi SHINODA,a Haruo SAITO,b Ushio SANKAWA,a,1) and Toshimitsu HAYASHI*,a
a Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University; d Faculty of Medicine, Toyama
Medical and Pharmaceutical University; 2630 Sugitani, Toyama 930–0194, Japan: b Department of Pharmaceutical
Research, Lead Chemical Co., Ltd.; 77–3 Himata, Toyama 930–0912, Japan: and c Toyama Prefectural Institute for
Pharmaceutical Research; 17–1 Naka-taikoyama, Kosugi-machi, Imizu, Toyama 939–0363, Japan.
Received June 28, 2004; accepted October 18, 2004
Two plastoquinones were isolated from the methanolic extract of the brown alga Sargassum micracanthum,
and these were identified as a known 2-geranylgeranyl-6-methylbenzoquinone and its hydroquinone, respectively,
based on spectroscopic analysis. The absolute configuration of the secondary hydroxyl group was determined by
the modified Mosher’s method using the new chromene derivative converted from plastoquinones. One of the
plastoquinones and the chromene exhibited significant antioxidant activities, such as an inhibitory effect on lipid
peroxidation and a radical scavenging effect on 1,1-diphenyl-2-picrylhydrazyl (DPPH). The benzoquinone-type
compound and the chromene derivative were found to have potent antiviral activity against human cy-
tomegalovirus (HCMV).
Key words plastoquinone; antioxidant activity; lipid peroxidation; antiviral activity; Sargassum micracanthum; chromene
Brown algae of the genus Sargassum, (Sargassaceae, Fu- 77.0 ppm) as the internal standard (s, singlet; d, doublet; t,
cales) are known to contain structurally unique secondary triplet; q, quartet; m, multiplet; br, broad). Mass spectra were
metabolites, such as plastoquinones,2—5) chromanols,6—8)
a
taken with a JEOL JMS-AX505HAD spectrometer. Column
cyclopentenone,9) and polysaccharides.10) These compounds chromatography was carried out on Kanto Chemical silica
show various biological activities due to their unique struc- gel 60 N (100—210 mm). Normal and reversed phase flash
ture. We previously reported that the methanolic extract of column chromatography was performed on Kanto Chemical
the brown alga, Sargassum micracanthum (KUETZING) silica gel 60 N (40—100 mm, normal phase) and Fuji Silysia
ENDLICHER, “Togemoku” in Japanese, showed strong antioxi- Chemical ODS DM1021T (100—200 mesh, reversed phase),
dant activity.11) In the course of investigations on the biologi- respectively. Medium-pressure liquid chromatography
cally active metabolites from this alga, two known plasto- (MPLC) was carried out with a Tosoh SC-8020 apparatus
quinones, 1 and 2,12) were isolated as major constituents. using a CIG prepack column (silica gel, CPS-HS-221-05).
This paper describes their isolation, determination of the ab- HPLC with a recycling loop was conducted with a YMC-
solute configuration via a new chromene derivative 3,13) and Pack SIL-06 column (silica gel, SH-043-5-06, normal phase)
evaluation of the antioxidant and antiviral activities of these and a YMC-Pack ODS-AM column (ODS silica gel, SH-
compounds.
343-5AM, reversed phase).
Material The brown alga, Sargassum micracanthum
(KUETZING) ENDLICHER (order Sargassaceae, family Fucales),
was collected from the Toyama Bay coastline in the middle
MATERIALS AND METHODS
Instruments Optical rotations were measured with a area of the Sea of Japan, Toyama Prefecture, Japan, in Sep-
JASCO DIP-1000 automatic polarimeter. IR spectra were tember 2001, at a depth of 2—5 m. A voucher specimen (No.
recorded with a Perkin–Elmer FT-IR 1600 spectrophotome- SM-0109) has been on deposit at Lead Chemical Co., Ltd.,
ter, and UV spectra with a JASCO V-530 spectrophotometer. Himata, Toyama, Japan.
NMR spectra were recorded with a Varian Unity-500 (1H,
Extraction and Isolation Wet specimens (2.3 kg) were
500 MHz; 13C, 125 MHz) in CDCl3. Chemical shifts are immersed in methanol (3ꢀ3 l). After filtration, the combined
given on a d (ppm) scale with CHCl3 (1H, 7.26 ppm; 13C, extracts were concentrated under reduced pressure. The
Fig. 1. The Structures of Compounds 1, 2, and 3
∗ To whom correspondence should be addressed. e-mail: hayashi9@ms.toyama-mpu.ac.jp
© 2005 Pharmaceutical Society of Japan

February 2005
375
methanol extract (442 g) was dissolved into a mixture of
chloroform–methanol (3 : 1, 0.8 l), then the precipitate was
filtered with a sintered glass filter to remove high polar com-
pounds and salts. The precipitate on the glass filter was
rinsed with a small amount of a 3 : 1 mixture of chloro-
form–methanol. The combined filtrate was concentrated
under reduced pressure to give a chloroform–methanol (3 : 1)
soluble portion (203 g). An aliquot of this portion (10.0 g)
was chromatographed on a silica gel column (300 g). Step-
wise elution with chloroform–methanol (99 : 1, 9 : 1 and 1 : 1,
400 ml of each) gave three fractions. The second fraction
(2.2 g) eluted with chloroform–methanol (9 : 1), contained
terpenoids and glycerides, along with color pigments such as
chlorophylls, according to the NMR analysis. The second
fraction (2.15 g out of 2.2 g) was subjected to ODS silica gel
flash column chromatography [H2O–methanol (1 : 19),
methanol, and then methanol–acetone (3 : 1)] to obtain a
mixture of plastoquinones. The separation and purification of
the plastoquinone fraction by MPLC [hexane–ethyl acetate
(5 : 2)] and HPLC [hexane–ethyl acetate (7 : 3)] gave 1
(1.28 g) and 2 (113 mg). These two compounds were identi-
fied by comparison of their physical properties with those re-
ported in the patent,12) however, some data, such as UV, IR,
and the NMR assignments were missing. Their data were
shown to be as follows.
H-9ꢄ), 2.06 (1H, m, one of H-12ꢄ), 2.07 (1H, m, one of H-
5ꢄ), 2.18 (1H, m, one of H-12ꢄ), 3.10 (2H, br d, Jꢂ7.3 Hz, H-
1ꢄ), 3.33 (1H, dd, Jꢂ1.5, 9.8 Hz, H-14ꢄ), 5.07 (1H, dt, Jꢂ0.9,
6.8 Hz, H-6ꢄ), 5.14 (1H, dt, Jꢂ0.8, 6.8 Hz, H-10ꢄ), 5.27 (1H,
dt, Jꢂ0.9, 7.3 Hz, H-2ꢄ), 6.46 (1H, d, Jꢂ3.0 Hz, H-3), 6.49
(1H, d, Jꢂ3.0 Hz, H-5). 13C-NMR (125 MHz, CDCl3) d
ppm: 15.8 (q, 3ꢄ-Me), 15.93 (q, 7ꢄ-Me), 15.97 (q, 6-Me),
16.02 (q, 11ꢄ-Me), 23.2 (q, 15ꢄ-Me), 26.2 (t, C-5ꢄ), 26.3 (q,
15ꢄ-Me), 26.5 (t, C-9ꢄ), 27.5 (t, C-1ꢄ), 29.5 (t, C-13ꢄ), 36.7 (t,
C-12ꢄ), 39.54 (t, C-8ꢄ), 39.56 (t, C-4ꢄ), 72.9 (s, C-15ꢄ), 78.1
(s, C-14ꢄ), 117.9 (d, C-2ꢄ), 123.8 (d, C-6ꢄ), 124.9 (d, C-10ꢄ),
132.2 (s, C-3), 133.1 (d, C-5), 135.16 (s, C-7ꢄ), 135.23 (s, C-
11ꢄ), 139.8 (s, C-3ꢄ), 145.9 (s, C-6), 148.5 (s, C-2), 187.96
(s, C-4), 188.02 (s, C-1). EI-MS m/z: 428 (Mꢁ), 410
[(MꢃH2O)ꢁ]. HR-FAB-MS m/z: 451.2807 [Calcd for
C27H40O4Na: 451.2824 (M)ꢁ].
Oxidation of 1 to 2 To a mixture of 1 (51 mg,
119 mmol) in acetonitrile (2 ml) was added a solution of am-
monium cerium (IV) nitrate (CAN, 80 mg, 146 mmol) in
H2O (0.8 ml) at 0 °C. The reaction mixture was stirred for
30 min at 0 °C, a Na2S2O3 solution (5 drops) was added to
quench active CAN, and the product was concentrated under
reduced pressure. The residue was extracted twice with ethyl
acetate (2ꢀ20 ml), and the combined extracts were washed
with a saturated NaCl solution, dried over MgSO4, filtered,
and concentrated under reduced pressure. The oily residue
was purified by passing over a small plug of silica gel
[hexane–ethyl acetate (2 : 1)], followed by MPLC separation
[normal phase, hexane–acetone (3 : 1)] to afford 2 (37 mg,
72% yield) as pale yellowish oil: [a]D25 ꢁ12.2° (cꢂ0.26,
CHCl3). The spectral data obtained for the product were
identical to those of isolated 2.
Compound 1 Pale yellowish oil. [a]D25 ꢁ10.3° (cꢂ0.53,
CHCl3). UV lmax (EtOH) nm (log e): 230 (4.0), 265 (3.6),
334 (3.4). IR (dry film) cmꢃ1: 3400 (br), 2923, 1470, 1197.
1H-NMR (500 MHz, CDCl3) d ppm: 1.16 (3H, s, one of 15ꢄ-
Me), 1.19 (3H, s, one of 15ꢄ-Me), 1.42 (1H, m, one of H-
13ꢄ), 1.53 (1H, m, one of H-13ꢄ), 1.57 (3H, s, 7ꢄ-Me), 1.59
(3H, s, 11ꢄ-Me), 1.74 (3H, s, 3ꢄ-Me), 1.98 (1H, m, one of H-
8ꢄ), 2.00 (1H, m, one of H-4ꢄ), 2.04 (1H, m, one of H-12ꢄ),
2,05 (1H, m, one of H-5ꢄ), 2.05 (1H, m, one of H-9ꢄ), 2.06
(1H, m, one of H-6ꢄ), 2.08 (1H, m, one of H-8ꢄ), 2.10 (1H,
m, one of H-5ꢄ), 2.10 (1H, m, one of H-9ꢄ), 2.17 (3H, s, 6-
Me), 2.23 (1H, m, one of H-12ꢄ), 3.27 (2H, br d, Jꢂ7.3 Hz,
H-1ꢄ), 3.37 (1H, dd, Jꢂ1.7, 9.7 Hz, H-14ꢄ), 5.07 (1H, br t,
Jꢂ6.9 Hz, H-6ꢄ), 5.12 (1H, br t, Jꢂ7.3 Hz, H-2ꢄ), 5.15 (1H,
dt, Jꢂ0.9, 7.3 Hz, H-10ꢄ), 6.44 (1H, d, Jꢂ3.8 Hz, H-3), 6.52
(1H, d, Jꢂ3.8 Hz, H-5). 13C-NMR (125 MHz, CDCl3) d
ppm: 15.7 (q, 7ꢄ-Me), 15.9 (q, 11ꢄ-Me), 16.05 (q, 3ꢄ-Me),
16.09 (q, 6-Me), 23.1 (q, 15ꢄ-Me), 26.13 (q, 15ꢄ-Me), 26.19
(t, C-5ꢄ), 26.21 (t, C-9ꢄ), 29.4 (t, C-13ꢄ), 29.7 (t, C-1ꢄ), 36.8
(t, C-12ꢄ), 39.4 (t, C-8ꢄ), 39.6 (t, C-4ꢄ), 73.3 (s, C-15ꢄ), 78.2
(s, C-14ꢄ), 113.8 (d, C-3), 115.3 (d, C-5), 121.7 (s, C-2ꢄ),
123.9 (d, C-6ꢄ), 125.3 (d, C-10ꢄ), 125.5 (s, C-6), 127.6 (s, C-
2), 134.7 (s, C-11ꢄ), 135.2 (s, C-7ꢄ), 138.6 (s, C-3ꢄ), 149.08
(s, C-4), 149.10 (s, C-1). EI-MS m/z: 430 (Mꢁ), 412
[(MꢃH2O)ꢁ]. HR-FAB-MS m/z: 453.2960 [Calcd for
C27H42O4Na: 453.2981 (MꢁNa)ꢁ]. 78.3 (d, C-11),
Conversion of 2 to 3 Compound 2 (56 mg, 130 mmol)
was treated with pyridine (1 ml) at room temperature under
N2 atmosphere. After standing for more than 12 h, pyridine
was removed under reduced pressure. The residue was puri-
fied with silica gel column chromatography [hexane–acetone
(3 : 1)] to provide the chromene derivative 3 (49 mg, 97%
yield).
Compound 39) Pale yellowish oil. [a]D25 ꢁ11.0° (cꢂ2.0,
CHCl3). UV lmax (EtOH) nm (log e): 230 (4.0), 265 (3.6),
1
334 (3.4). IR (dry film) cmꢃ1: 3400 (br), 1590, 1240. H-
NMR (500 MHz, CDCl3) d ppm: 1.16 (3H, s, one of 12ꢄ-
Me), 1.20 (3H, s, one of 12ꢄ-Me), 1.36 (3H, s, 2-Me), 1.41
(1H, m, one of H-10ꢄ), 1.56 (1H, m, one of H-10ꢄ), 1.57 (3H,
s, 4ꢄ-Me), 1.59 (3H, s, 8ꢄ-Me), 1.66 (2H, m, H-1ꢄ), 1.96 (2H,
m, H-5ꢄ), 2.02 (1H, m, one of H-9ꢄ), 2.07 (2H, m, H-6ꢄ),
2.11 (2H, m, H-2ꢄ), 2.14 (3H, s, 8-Me), 2.22 (1H, m, one of
H-9ꢄ), 3.36 (1H, dd, Jꢂ2.1, 10.5 Hz, H-11ꢄ), 5.10 (1H, br t,
Jꢂ6.1 Hz), 5.15 (1H, br t, Jꢂ6.3 Hz), 5.58 (1H, d,
Jꢂ9.9 Hz), 6.24 (1H, d, Jꢂ9.9 Hz), 6.33 (1H, d, Jꢂ2.7 Hz),
6.49 (1H, d, Jꢂ2.7 Hz). 13C-NMR (125 MHz, CDCl3) d ppm:
15.4 (q, 8-Me), 15.5 (q, 4ꢄ-Me), 15.9 (q, 8ꢄ-Me), 22.5 (t, C-
2ꢄ), 23.3 (q, 12ꢄ-Me), 25.8 (q, 2-Me), 26.4 (q, 12ꢄ-Me), 26.4
(t, C-6ꢄ), 29.6 (t, C-10ꢄ), 36.8 (t, C-9ꢄ), 39.5 (t, C-5ꢄ), 40.7 (t,
C-1ꢄ), 73,2 (s, C-12ꢄ), 77.8 (s, C-2), 78.3 (d, C-11ꢄ), 110.3 (d,
C-5), 117.1 (d, C-7), 121.3 (s, C-4a), 122.9 (d, C-4), 124.2 (d,
C-3ꢄ), 125.0 (d, C-7ꢄ), 126.3 (s, C-8), 130.7 (d, C-3), 134.8
(s, C-8ꢄ), 135.0 (s, C-4ꢄ), 144.7 (s, C-8a), 148.7 (s, C-6). EI-
MS m/z: 428 (Mꢁ), 410 [(MꢃH2O)ꢁ]. HR-EI-MS m/z:
428.2945 [Calcd for C27H40O4: 428.2927 (M)ꢁ].
Compound 2 Pale yellowish oil. [a]D25 ꢁ11.3° (cꢂ0.59,
CHCl3). UV lmax (EtOH) nm (log e): 234 (3.3), 285 (5.2),
310 (3.9). IR (dry film) cmꢃ1: 3400 (br), 2925, 1680, 1158.
1H-NMR (500 MHz, CDCl3) d ppm: 1.13 (3H, s, one of 15ꢄ-
Me), 1.17 (3H, s, one of 15ꢄ-Me), 1.40 (1H, m, one of H-
13ꢄ), 1.56 (1H, m, one of H-13ꢄ), 1.57 (3H, s, 7ꢄ-Me), 1.58
(3H, s, 11ꢄ-Me), 1.60 (3H, s, 3ꢄ-Me), 1.97 (1H, m, one of H-
4ꢄ), 1.97 (1H, m, one of H-8ꢄ), 1.97 (1H, m, one of H-9ꢄ),
2.03 (3H, s, 6-Me), 2,03 (1H, m, one of H-4ꢄ), 2.03 (1H, m,
one of H-8ꢄ), 2.04 (1H, m, one of H-5ꢄ), 2.05 (1H, m, one of

376
Vol. 28, No. 2
Conversion of 3 to an Enantiomeric Pair of MTPA Di- plaque assay at 24-h incubation for HSV-1, HSV-2, mumps
esters To a mixture of 3 (21 mg, 49 mmol) in pyridine virus, measles virus, influenza virus, poliovirus and cox-
(1 ml) was added (S)-(ꢁ)-a-methoxy-a-(trifluoromethyl)- sackie virus, and at 5-d incubation for HCMV. The 50% in-
phenylacetyl chloride [(S)-MTPA-Cl, 37 mg, 147 mmol] in hibitory concentration (IC50) was obtained from dose–re-
the presence of a catalytic amount of 4-dimethylaminopyri- sponse curves. For anti-adenovirus assay, HeLa cells were in-
dine (DMAP) at 0 °C. The reaction mixture was stirred for fected with virus at 100 TCID50 (50% tissue culture infec-
6 h at room temperature. After diluting the mixture with ethyl tious dose) and incubated in maintenance medium for 3 d.
acetate (20 ml), the organic phase was washed in turn with a Production of adenovirus was determined by cytopathic ef-
saturated solution of CuSO4, H2O and brine, dried over fects, and TCID50s were calculated.
MgSO4, filtered, and concentrated under reduced pressure.
The oily residue was purified with silica gel column chro- RESULTS AND DISCCUSION
matography [hexane–ethyl acetate (5 : 1)] to provide the (R)-
1
MTPA diester (33 mg, 78% yield) as colorless oil. H-NMR
The chloroform–methanol (3 : 1) soluble portion of the
(500 MHz, CDCl3) d: 1.15 (3H, s), 1.21 (3H, s), 1.38 (3H, s), hot methanol extract of S. micracanthum, collected at the
1.51 (3H, s), 1.55 (2H, m), 1.57 (3H, s), 1.69 (2H, m), 1.86 Toyama Bay coastline in the middle area of the Sea of Japan,
(2H, m), 1.95 (2H, m), 2.03 (2H, m), 2.12 (2H, m), 2.17 (3H, was repeatedly purified with a silica gel column and an ODS
s), 3.57 (3H, s), 3.68 (3H, s), 4.98 (1H, dd, Jꢂ2.1, 10.2 Hz), silica gel column, followed by HPLC separation to obtain
5.02 (1H, br t, Jꢂ6.7 Hz), 5.11 (1H, br t, Jꢂ6.9 Hz), 5.60 plastoquinones 1 and 2. Although the gross structures of 1
(1H, d, Jꢂ10.2 Hz), 6.28 (1H, d, Jꢂ10.2 Hz), 6.60 (1H, d, and 2 were already reported as biologically active compo-
Jꢂ2.6 Hz), 6.72 (1H, d, Jꢂ2.6 Hz), 7.38—7.63 (10H, m).
nents from other Sargassum species,12) each absolute config-
The (S)-MTPA diester was prepared by the same proce- uration of the C-14ꢄ hydroxyl group was not yet determined.
dure as above except that the reagent used (R)-MTPA-Cl in- Its configuration was deduced by the modified Mosher’s
stead of (S)-MTPA-Cl. The yield and NMR data is as fol- method.13) Standard conditions for the esterification of 2 did
lows: 34 mg, 79% yield as colorless oil from 21 mg of not work, and a complex mixture was obtained. In this case,
1
chromene 3, H-NMR (500 MHz, CDCl3) d: 1.13 (3H, s), pretreatment with pyridine to convert a new chromene deriv-
1.17 (3H, s), 1.38 (3H, s), 1.55 (3H, s), 1.57 (3H, s), 1.65 ative 314) afforded a good result. After preparing each enan-
(2H, m), 1.69 (2H, m), 1.93 (2H, m), 1.97 (2H, m), 2.04 (2H, tiomeric pair of MTPA diesters of 3 at C-11ꢄ (corresponding
m), 2.11 (2H, m), 2.17 (3H, s), 3.58 (3H, s), 3.68 (3H, s), to the C-14ꢄ in 1 and 2) hydroxyl and phenol, 1H-NMR spec-
4.99 (1H, dd, Jꢂ1.7, 9.8 Hz), 5.07 (1H, br t, Jꢂ6.8 Hz), 5.11 tra of each were measured, and the dD values (dS-MTPAꢃ
(1H, br t, Jꢂ6.7 Hz), 5.60 (1H, d, Jꢂ10.2 Hz), 6.28 (1H, d,
Jꢂ10.2 Hz), 6.60 (1H, d, Jꢂ2.7 Hz), 6.72 (1H, d, Jꢂ2.7 Hz), concluded that the absolute configuration at C-14ꢄ in 1 and 2
dR-MTPA) were obtained as depicted in Fig. 2. These findings
7.38—7.63 (10H, m).
was determined as R.
Animal Male Wistar rats (5 weeks of age) were pur-
Compounds 1, 2 and 3 displayed various bioactivities, es-
chased from Sankyo Labo Co., Ltd. The animals were main- pecially significant antioxidant activities, such as an in-
tained in a thermostatically controlled room at 24ꢅ2 °C dur- hibitory effect on NADPH-dependent lipid peroxidation in
ing the experimental period.
rat liver microsomes16) and a radical scavenging effect on
Antioxidant Activity Antioxidant activities, such as ef- 1,1-diphenyl-2-picrylhydrazyl (DPPH)17) (Table 1). The in-
fects on lipid peroxidation in rat liver homogenates15) and hibitory effect of compounds 1, 2 and 3 on lipid peroxidation
radical reductive activities on DPPH16) were evaluated ac- were observed at IC50 0.11, 1.0 and 0.28 mg/ml, respectively,
cording to the corresponding literature.
showing them to be more than a ten to hundredfold stronger
Cells and Viruses Vero, human embryonic lung (HEL), than that of the positive controls, a-tocopherol and L-ascor-
HeLa 229 and MDCK cells were grown in Eagle minimum
essential medium (MEM) supplemented with 5% fetal
bovine serum (FBS). HSV-1 (HF and ACV-resistant A4-3
strains), HSV-2 (UW 268 strain), mumps virus (EY strain),
measles virus (Toyoshima strain), poliovirus type 1 (Sabin
strain), and coxsackie virus type B-1 (Conn-5 strain) were
propagated on Vero cells, HCMV (Towne strain) on HEL
cells, adenovirus type 2 on HeLa 229, and influenza A virus
(NWS strain, H1N1) on MDCK cells.
Fig. 2. Absolute Configuration of the Secondary Hydroxyl Group
Cytotoxicity For cell growth inhibition studies, subcon-
fluent Vero, HeLa 229 and MDCK cells were treated with in-
creasing amounts of the compound for 3 d. HEL cells were
treated for 5 d in the presence of the compound. The 50% in-
hibitory concentration (CC50) was calculated from dose–re-
sponse curves.
Antiviral Activity In the antiviral assay for the viruses,
except for adenovirus, cell monolayers were infected with
virus at 0.1 plaque-forming unit (PFU) per cell for 1 h at
room temperature and incubated in maintenance medium
(MEM plus 2% FBS). Virus production was determined by
Table 1. Antioxidative Activity
Inhibitory effect on
Reductive effect
on DPPHa)
Samples
lipid peroxidationa)
MeOH ext.
0.7
0.11
1.0
0.28
34
34
11.0
400
10.7
10.0
2.5
Compound 1
Compound 2
Compound 3
a-Tocopherol
L-Ascorbic acid
28
a) IC50 values (mg/ml).

February 2005
377
Table 2. Antiviral Spectra of 1, 2, and 3a)
1
2
3
Virus
(strain)
Host
cell
IC50 (mM)
CC50/IC50
IC50 (mM)
CC50/IC50
IC50 (mM)
CC50/IC50
CC50
CC50
CC50
(mM)
(mM)
(mM)
A
B
A
B
A
B
A
B
A
B
A
B
HSV-1
HSV-2
HCMV
Mumps virus
Measles virus
Adeno virus
Influenza virus
Poliovirus
Vero
8.9
8.9
7.7
8.9
8.9
8.2
8.1
8.9
8.9
9.3
9.1
1.9
8.9
3.1
9.4
9.4
1.8
9.6
2.9
0.96
0.99
4.2
1.0
2.9
0.53
1.0
0.94
0.96
0.95
0.97
4.4
0.93
3.1
0.53
0.83
0.95
0.99
35
35
32
35
35
29
32
35
35
18
19
2.0
19
22
22
24
19
21
19
18
2.6 16
2.0
1.9
1.8
1.9
12
1.9
1.7
1.9
1.2
1.7
1.6
17
17
22
17
17
24
24
17
17
2.8
2.6
0.49
7.6
2.7
3.5
2.7
0.67 47
8.7
2.8
18
4.8
9.4
7.5
6.5
6.7
4.9
6.3
35
2.0
6.0
1.6
5.0
1.8
2.3
Vero
HEL
Vero
Vero
HeLa
MDCK
Vero
Vero
18
1.9
1.6
1.3
1.3
1.8
1.7
2.2
21
18
26
21
23
6.4
2.0
5.5
1.9
2.5
18
18
14
8.1
9.2
9.4
9.7
9.4
9.0
4.4
9.0
6.8
Coxsackievirus
a) A: Sample was added to the medium during viral infection and through the incubation. B: Sample was added to the medium immediately after viral incubation.
Toyama International Health Complex, 151 Tomosugi, Toyama
939–8224, Japan.
2) Kusumi T., Shibata Y., Ishitsuka M., Kinoshita T., Kakisawa H., Chem.
Lett., 1979, 277—278 (1979).
3) Kikuchi T., Mori Y., Yokoi T., Nakazawa S., Kuroda H., Masada Y.,
Kitamura K., Kuriyama K., Chem. Pharm. Bull., 31, 106—113 (1983).
4) Segawa M., Shirahama H., Chem. Lett., 1987, 1365—1366 (1987).
5) Amico V., Neri P., Oriente G., Piattelli M., Phytochemistry, 28, 215—
219 (1989).
6) Ishitsuka M., Kusumi T., Nomura Y., Konno T., Kakisawa H., Chem.
Lett., 1979, 1269—1272 (1979).
7) Numata A., Kanbara S., Takahashi C., Fujiki R., Yoneda M., Fujita E.,
Nabeshita Y., Chem. Pharm. Bull., 31, 106—113 (1983).
8) Pérez-Castorena A. L., Arciniegas A., Apan M. T. R., Villaseñor J. L.,
de Vivar A. R., Planta Med., 68, 645—647 (2002).
bic acid, well known as vitamins E and C. On the other hand,
compounds 1 and 3 were found to have a moderate radical
reducing effect on DPPH, with IC50 values of 11.0 and
10.7 mg/ml, respectively (Table 1). Positive controls showed a
similar activity to that of the samples, except for 2.
The strong antioxidant activity of 1 and 3 might be as-
cribed to the hydroquinone and phenol moiety, respectively,
both of which could be converted to a stable phenoxyl radi-
cal, rather than the isoprene side chain, as the precursor of an
allyl radical. Due to the structural similarity between 3 and
a-tocopherol, the structure–activity relationship has been in-
vestigated in detail.
The antiviral activity of 3, together with 1 and 2, against
various viruses was also estimated in vitro, because anti-HIV
active chromene derivatives were reported.18) Cytotoxic con-
centration toward host cells (CC50) and inhibitory activity
against replication of progeny viruses (IC50) were summa-
rized in Table 2. It is generally evaluated to be antivirally ac-
tive when the selectivity index (CC50/IC50) is more than 10.
In the case of compound 1 and a-tocopherol, essentially no
effect (ꢆ10) was found, despite of the structural similarities
to those of 2 and 3. These results suggested that 2 and 3
might be candidates as lead compounds in an anti-HCMV
drug. Their antiviral mechanisms are currently under investi-
gation.
9) Nakayama M., Fukuoka Y., Nozaki H., Matsuo A., Hayashi S., Chem.
Lett., 1980, 1243—1246 (1980).
10) Hoshino T., Hayashi T., Hayashi K., Hamada J., Lee J.-B., Sankawa U.,
Biol. Pharm. Chem., 21, 730—734 (1998).
11) Mori J., Matsunaga T., Takahashi S., Hasegawa C., Saito H., Phytother.
Res., 17, 549—551 (2003).
12) Inaoka K., Nishizawa Y., Tone H., Kamiya T., Jpn. Kokai Tokkyo
Koho, JP H04-49259 (1992).
13) Ohtani I., Kusumi T., Kashman Y., Kakisawa H., J. Am. Chem. Soc.,
113, 4092—4096 (1991).
14) Chromene 3 derived from 1 is an epimeric mixture (1 : 1) at C-2.15)
These diastereomers could not be separated from each other, even
using recycled HPLC. In the NMR (500 MHz) analysis of 3, all the
proton signals were identified as a single compound. In addition, no
twin signal was observed in the NMR spectrum of their MTPA di-
esters.
15) Compound 3 was previously isolated from this alga from another col-
lection in 1995, as a pale yellowish oil, [a]D25 ꢁ13.0° (cꢂ1.0, CHCl3).
The obtained chromene was also found to be an epimeric mixture
(1 : 1) at C-2. Compound 3 could be considered both as a natural prod-
uct and as an artifact.7,8) In the recent collections, 3 was not detected
from the methanol extracts.
16) Pederson T. C., Buege J. A., Aust S. D., J. Biol. Chem., 248, 7134—
7141 (1973).
17) Blois M. S., Nature (London), 181, 1199—1200 (1958).
18) Kashiwada Y., Yamazaki K., Ikeshiro Y., Yamagishi T., Fujioka T., Mi-
hashi K., Mizuki K., Cosentino L. M., Fowke K., Morris-Natschke S.
L., Lee K.-H., Tetrahedron, 57, 1559—1563 (2001).
Acknowledgments The authors express their apprecia-
tion to Dr. N. Nakajima at Toyama Prefectural University for
taking HR-FAB-MS, and to Dr. D. Fujita at Toyama Prefec-
ture Office for collection and identification the alga. This
work was supported in part by a Grant-in-Aid for Scientific
Research (15590097, M. I.) from the Ministry of Education,
Culture, Sports, Science and Technology of Japan.
REFERENCES AND NOTES
1) Present address: International Traditional Medicine Research Center,