Antibacterial quinone metabolites from the brown alga, sargassum sagamianum

Article abstract of DOI:10.1246/bcsj.81.1125

Antibacterial sargaquinoic acid derivatives were isolated from the brown alga Sargassum sagamianum. The structures of these quinone metabolites were confirmed by NMR and mass spectroscopy, and by comparison with published data. The relationship between th

Full text of DOI:10.1246/bcsj.81.1125

ꢀ 2008 The Chemical Society of Japan
Bull. Chem. Soc. Jpn. Vol. 81, No. 9, 1125–1130 (2008) 1125
Antibacterial Quinone Metabolites from the Brown Alga,
Sargassum sagamianum
ꢀ
Shohei Horie, Shinsuke Tsutsumi, Yuuki Takada, and Junji Kimura
Department of Chemistry and Biological Science, College of Science and Engineering,
Aoyama Gakuin University, 5-10-1 Fuchinobe, Sagamihara 229-8558
Received March 12, 2008; E-mail: kimura@chem.aoyama.ac.jp
Antibacterial sargaquinoic acid derivatives were isolated from the brown alga Sargassum sagamianum. The struc-
tures of these quinone metabolites were confirmed by NMR and mass spectroscopy, and by comparison with published
data. The relationship between their structures and antibacterial activities are briefly discussed.
Plastoquinones in plants are widely known to have many bi-
ological activities such as antioxidant activity, insect growth
inhibition, cytotoxicity, and electron transport in the photosys-
tem.1–7 From the family of Sargassaceae algae, often visible
about the coast of Japan, Japanese researchers have obtained
plastoquinones such as sargaquinoic acid (6) and hydroxysar-
gaquinone, and their derived chromenes such as sargachrome-
nol (7) and hydroxysargaol (sargadiol-I).8–12 Recently, plasto-
quinones and chromenes having antioxidant and antiviral
activities were obtained from the same species.13,14 These plas-
toquinones were easily converted into the corresponding chro-
menes.3,8
In our investigation of antibacterial compounds from the al-
ga Sargassum sagamianum, five new sargaquinoic acid deriv-
atives 1–5 were isolated. We describe herein the structures of
these metabolites and discuss plausible pathways for their
formation (Figure 1).
protons at ꢀ 5.57 (H-3), 6.25 (H-4), 6.32 (H-5), and 6.48 (H-7),
resembling those of the chromenol skeleton in 7, and two
methyl protons at ꢀ 1.22 (H-140) and 1.28 (H-130) similar to
the terminal isoprene unit of 1 (H-170 and H-160). Thus, 2
proved to be a chromenol derivative produced by the cycliza-
tion of 1. Actually 1 was more unstable than 2, and 1 was easi-
ly isomerized in pyridine at room temperature for 8 h to afford
2 in 30% yield together with starting material 1 (20%).13
Compound 3 showed a molecular formula of C27H34O4
(m=z 422.2460 [M]þ ꢀ +0.3 mmu) by HREIMS which indi-
cates a dehydration compound from 1 or 2. Also, a peak char-
acteristic of a fragment of the quinone skeleton (m=z 175) was
detected and the 1H NMR spectrum showed end methylene
signals at ꢀ 4.95 and 5.04 (H-160, d, J ¼ 1:2 Hz). Compound
3 proved to be 150-methylenesargaquinolide, which was
formed by dehydration between the 150-hydroxy group and a
neighboring methyl hydrogen in 1.
The MeOH/CHCl3 (1:1 v/v) extract of the algae was sub-
jected to silica gel column chromatography using a stepwise
gradient of EtOAc/hexane. An antibacterial fraction of the
EtOAc/hexane extracts was separated repeatedly by silica
gel PLC and HPLC to afford five new sargaquinoic acid deriv-
atives 1–5 together with known compounds, including sarga-
quinoic acid (6), sargachromenol (7), yezoquinolide, and 2-
methyl-6-phytyl-1,4-benzoquinone, which were identified by
comparison with NMR and MS spectra in the literature.8,9
The EIMS spectrum of 1 showed a characteristic base ion
peak at m=z 175 ½M ꢁ C16H25O3ꢂþ indicating the plastoqui-
none skeleton, and a molecular ion peak at m=z 440.2537
(C27H36O5, [M]þ ꢀ ꢁ2:6 mmu). The 1H and 13C NMR spectra
of 1 were very similar to the corresponding spectra of 6, except
for the 13C chemical shifts were at ꢀc 85.8 (C-140) and 71.3 (C-
150) in 1 instead of the corresponding shifts at 123.4 and 132.1
in 6, and the 1H chemical shift was at ꢀ 4.03 (H-140) instead of
ꢀ 5.09. It was revealed to be the product (1 was named 150-hy-
droxysargaquinolide) formed by the lactonization via oxida-
tion of the end double bond of isoprene units in 6.
The HREIMS spectrum of 4 gave C27H34O6, which indi-
cates the molecular ion of 1 or 2 plus an oxygen atom. 1H
and 13C NMR spectra revealed the presence of a lactone ring
in the side chain and quinone skeleton as in 1 with the excep-
tion of having ꢀ 1.67 and 1.89 as methylene signals next to the
chromenol skeleton as in 2, instead of the corresponding signal
at ꢀ 3.13 as those of the quinone skeleton in 1. It also showed a
signal at ꢀc 82.9 of a methine carbon neighboring an oxygen
like the chromenol skeleton in 2. Detailed NMR analysis re-
vealed that the skeleton of 4 was chromenquinone and that
its side chain was a tetraprenyl group containing a lactone ring
as in 1 and 2; 4 was thus named chromequinolide.15 Com-
pound 4 might be postulated to be formed by the epoxidation
of the 30-position of the side chain followed by cyclization and
subsequent oxidation.
The MS spectrum of 5 showed a molecular ion peak at m=z
258.1269 (C16H18O3, [M]þ, ꢀ +1.3 mmu) and a base ion peak
at m=z 175, which is characteristic of the quinone skeleton. 1H
and 13C NMR spectra showed characteristic signals of a broad
doublet at ꢀ 2.94 (H-40, J ¼ 6:9 Hz, ꢀc 42.4), and two double
triplets at ꢀ 6.75 (H-50, J ¼ 16:0, 6.9 Hz, ꢀc 145.0) and at ꢀ
6.09 (H-60, J ¼ 16:0, 1.4 Hz, ꢀc 132.5), which were connected
by COSY analysis. Further analysis of the HMBC spectrum
The HREIMS spectrum of 2 showed the same characteristic
peaks at m=z 175 and 440 (m=z 440.2552: C27H36O5, [M]þ
ꢀ
1
ꢁ1:1 mmu) as 1. The H NMR spectrum showed four olefinic
Published on the web September 11, 2008; doi:10.1246/bcsj.81.1125

1126 Bull. Chem. Soc. Jpn. Vol. 81, No. 9 (2008)
Antibacterial Quinone Metabolites
18' 14'
O
9
20'
O
15' 11'
10
16'
O
O
O
7
1'
OH
1
O
4
7
OH
2
1'
3
HO
1
2
O
O
O
O
COOMe
O
OH
OR
2b : R = (S)-MTPA
RO
2a : R = H
3
O
O
O
O
4
11'
O
9
16'
O
2
OH
7
O
1'
O
O
10
5
4
COOR
COOR
O
O
HO
6
: R = H
7
: R = H
O
6a : R = Me
7a : R = Me
Figure 1. Sargaquinoic acid derivatives from Sargassum sagamianum.
connected C-50 and C-70 (cross peaks between H-50/C-70), and
C-40 and C-30 (cross peaks between H-40/C30, H-40/C-90).
These data revealed that 5 was (20E,50E)-2-methyl-6-(70-
oxo-30-methylocta-20,50-dienyl)-1,4-benzoquinone, which was
formed by oxidative cleavage of the 70–80 position of a qui-
none derivative such as 1 or 3 (Tables 1 and 2).
10 mg mLꢁ1
.
The activity of these compounds is about the
18
same as that of chromene derivatives from plastquinone,13
but weaker than that of cyclic depsipeptides reported in our
laboratory.19
The brown algae, Sargassaceae, possess a bonanza of qui-
none metabolites, and the investigation of other bioactivities
of these compounds and the detection of other new quinone
derivatives from these algae are now in progress. Furthermore,
the total synthesis of 5 is being attempted because of its
detailed activities.
The chromenes such as 2 and 4 have a chiral center at C-2.
They were considered to be artifacts from the corresponding
plastoquinones because of their lack of optical activity.3 Com-
pound 2 has another chiral carbon at the C-110 position besides
C-2. After methanolysis of the lactone moiety with sodium
methoxide, attempts at MTPA esterification of the product af-
forded diastereomers. Additionally, 1 and 2 have the same
small optical rotation value; therefore it seems that the chiral
carbon of the 110-position in 2 exists in a racemic mixture as
for kuhistanol.16 Stereochemistry of the corresponding posi-
tions in 3 and 4 could not be determined because complex mix-
tures were obtained by the process of MTPA esterification.
Compounds 1, 2, and 6 show about 30% inhibition against
Bacillus subtilis and Staphylococcus aureus as compared to
a standard sample of vancomycin.17 In contrast, 5 shows a
strong inhibition of 80%. Thus, we conducted a detailed
examination between the relationship of the compounds’
structures and antibacterial activities by MIC measurements
(Table 3). These compounds (especially 5) proved to have
strong activities against S. aureus. Compared to known com-
pounds 6 and 7 with corresponding methyl esters 6a and 7a,
the free carboxylic groups of 6 and 7 indicate very strong
activity. However, 2 shows a reverse tendency. Additionally,
it could be considered that the action mechanism of 7 is differ-
ent from that of the others by MBC measurement results.
Compound 5 exhibits cytotoxicity against Hela S3 cells with
an IC50 of 4.0 mg mLꢁ1, and 1 and 2 show an IC50 of
Experimental
General Experimental Procedures. NMR spectra were mea-
sured on a JEOL ECP-500 spectrometer operating at 500 MHz for
1H and 125 MHz for 13C in CDCl3. Optical rotation was deter-
mined on a Perkin-Elmer 341 digital polarimeter. Mass spectra
were obtained with a JEOL MS-700 mass spectrometer. HPLC
was performed on a Shimadzu LC-10 equipped with an RI or
UV detector using a GL Science column (Inertsil prep-SIL, Inert-
sil prep-ODS, 10 ꢃ 250 or 20 ꢃ 250 mm2). The solvents were
distilled prior to use.
Plant Material. Sargassum sagamianum Yendo was collected
in July 2005 at Manazuru in Kanagawa prefecture. The alga is
often seen on the coast of Japan and was identified by Mr. A.
Takahashi. A voucher specimen has been deposited at the Depart-
ment of Chemistry and Biological Science, Aoyama Gakuin Uni-
versity.
Extraction and Isolation. An air-dried sample (320 g) was
extracted with MeOH/CHCl3 (1:1, 3 ꢃ 3 L) for 1 day. The com-
bined extracts were concentrated and partitioned between CHCl3
and H2O (650 mL each). The CHCl3 extract (6.1 g) was subjected
to silica gel column chromatography (Fuji Silysia, BW-300
7:0 ꢃ 47:0 cm2) using a stepwise gradient of EtOAc/hexane to
afford roughly ten fractions. Checking against characteristic

Table 1. NMR Data for 1, 3, and 5
1
3
5
#
C
H
J/Hz
HMBC
H!C
C
H
J/Hz
HMBC
H!C
C
H
J/Hz
HMBC
H!C
1
2
3
4
5
188.0
148.5
132.3
188.0
133.2
145.9
16.0
188.0
148.5
132.3
188.0
133.2
145.9
16.0
187.8
147.8
132.4
187.8
133.2
146.0
16.0
6.46
6.55
dt, 2.7, 1.8
dq, 2.7, 1.4
4
4
6.46
6.54
dt, 2.7, 1.8
dq, 2.7, 1.4
6.47
6.57
m
4, 5, 10
4
dq, 1.8, 1.4
3, 4
6
7
2.06
3.13
5.15
d, 1.4
d, 7.8
tq, 7.3, 1.3
1, 5, 6
2, 3, 20, 30
10
2.06
3.13
5.15
d, 1.4
d, 7.3
m
1, 5, 6
2, 20, 30
2.07
3.16
5.24
d, 1.4
br d, 7.3
tq, 7.3, 1.4
1, 5, 6
1, 2, 3, 20, 30
10
10
20
30
40
50
60
70
80
90
100
110
120
130
27.5
27.5
27.8
118.0
139.8
39.6
118.0
139.8
38.8
121.2
136.2
42.4
145.0
132.5
198.3
27.1
2.10
2.10
5.12
m
50
40
2.06
2.11
5.13
m
t, 7.1
m
50
90
2.94
6.75
6.09
br d, 6.9
dt, 16.0, 6.9
dt, 16.0, 1.4
20, 30, 50, 60, 90
70
26.4
m
tq, 6.9, 1.3
26.4
124.6
134.6
38.8
124.6
134.6
39.6
2.10
2.70
6.06
m
90
100
2.11
2.72
6.03
t, 7.1
br q, 6.8
tt, 7.3, 1.8
2.26
1.67
s
br s
70
28.1
q, 7.3
tt, 7.3, 1.8
28.0
16.4
20, 30, 40
147.8
124.6
28.7
147.6
124.3
28.4
2.56
1.70
1.98
4.03
m
2.55
1.83
1.99
4.65
m
23.5
ddt, 13.7, 11.9, 6.4
ddt, 13.7, 5.5, 2.8
dd, 11.9, 2.8
27.5
dddd, 13.7, 10.1, 9.6, 6.9
ddt, 13.7, 5.0, 3.2
dd, 10.1, 3.2
140
150
160
85.8
71.3
25.7
82.0
142.6
112.9
1.28
1.23
br s
br s
5.04
4.95
1.78
d, 1.2
d, 1.2
s
170
180
190
200
24.2
165.5
15.9
18.1
1.60
1.62
br s
br s
15.9
16.1
1.60
1.62
br s
br s
16.1

Table 2. NMR Data for 2, 2a, and 4
2
2a
4
#
C
H
J/Hz
HMBC
H!C
C
H
J/Hz
C
H
J/Hz
HMBC
H!C
2
3
4
4a
5
6
7
8
8a
9
77.8
130.7
122.9
121.3
110.2
148.5
117.0
126.3
144.8
15.5
77.8
130.7
122.9
121.3
110.3
148.6
117.1
126.3
145.0
15.5
82.9
128.6
115.7
115.5
184.5
145.4
131.3
181.3
150.8
15.9
5.57
6.25
d, 10.1
d, 10.1
10
8a
5.52
6.25
d, 9.6
d, 9.6
5.55
6.50
d, 10.1
d, 10.1
2, 4
2
6.32
6.48
d, 3.2
8a
6.32
6.48
d, 3.2
d, 3.2
br d, 3.2
5, 6, 8a
6.47
q, 1.4
6, 8a
2.14
1.36
1.66
s
s
m
7, 8, 8a
2, 3, 10
20
2.14
1.36
1.66
s
s
m
2.05
1.46
1.67
1.89
d, 1.4
s
7, 8, 8a
2, 3, 10
20
10
10
25.7
40.7
26.0
40.7
27.4
41.4
m
ddd, 14.2, 9.6, 6.4
20
30
40
50
60
70
80
90
22.6
125.0
134.2
38.8
2.08
5.12
m
br t, 7.3
22.6
125.0
134.3
39.1
2.12
5.12
m
br t, 7.3
22.5
124.1
135.1
38.7
2.03–2.14
5.11
m
br t, 7.3
20
60
20
2.08
2.68
6.01
m
2.05
2.58
5.92
t, 7.3
dt, 7.3, 7.3
t, 7.3
2.03–2.14
2.69
6.03
m
60
50
27.9
dt, 7.3, 7.2
tt, 7.3, 1.8
28.0
27.9
dt, 7.3, 7.2
tq, 7.3, 1.3
147.9
124.2
28.7
143.0
131.2
31.5
147.5
124.5
28.7
2.52
m
2.47
2.30
1.42
1.59
3.33
m
2.55
m
ddt, 16.0, 8.2, 2.8
dddd, 13.3, 10.6, 8.2, 5.0
m
100
23.5
1.66
1.97
4.01
m
31.4
23.5
1.69
1.98
4.02
m
ddt, 13.7, 6.0, 2.7
ddd, 11.9, 2.7, 0.9
ddt, 13.7, 5.5, 2.7
dd, 11.9, 2.7
110
85.7
71.3
25.9
24.1
77.5
73.0
26.4
23.3
168.8
51.4
15.7
dd, 10.5, 1.8
85.7
71.4
25.7
24.3
120
130
1.28
1.22
s
s
110, 120, 140
110, 120, 130
1.19
1.14
s
s
1.28
1.23
s
s
110, 120, 140
110, 120, 130
140
150
165.5
–OCH3
160
3.73
1.56
s
s
15.7
1.57
s
30, 40, 50
15.9
1.56
s
30, 40, 50

S. Horie et al.
Bull. Chem. Soc. Jpn. Vol. 81, No. 9 (2008) 1129
Table 3. Antibacterial Activity against S. aureus
90-H), 3.56 and 3.68 (s, –OCH3), 3.70 (s, –COOCH3), 4.98 (dd,
J ¼ 7:5, 2.5 Hz, 110-H), 5.79 (t, J ¼ 7:4 Hz, 70-H): 2b0 (rt
18.8 min): 1.11 (s, 130-CH3), 1.14 (s, 140-CH3), 2.50 (dt, J ¼
7:5, 7.5 Hz, 90-H), 3.59 and 3.68 (s, –OCH3), 3.71 (s, –COOCH3),
4.98 (dd, J ¼ 7:5, 2.5 Hz, 110-H), 5.86 (t, J ¼ 7:4 Hz, 70-H).
Methylation of 6 to 6a: To a solution of 6 (6.1 mg, 14 mmol)
in methanol (0.3 mL) and hexane (0.4 mL) was added TMSCH2N2
(144 mmol) in hexane (72 mL) at room temperature. After the reac-
tion mixture was stirred for 10 min at room temperature and blown
down with nitrogen, TMSCH2N2 was evaporated under reduced
pressure. The oily residue was purified by passing over a small
plug of silica gel (10% EtOAc in hexane), followed by SIL-HPLC
separation (7% EtOAc in hexane) to afford 6a (1.5 mg, 24% yield)
as a pale yellowish oil. 1H NMR (500 MHz, CDCl3): ꢀ 1.57 (s, H-
160), 1.60 (s, H-190), 1.62 (s, H-200), 1.67 (s, H-170), 2.03–2.13 (m,
H-40, 50, 80, 130), 2.06 (d, J ¼ 1:4 Hz, H-7), 2.24 (t, J ¼ 7:3 Hz, H-
120), 2.51 (dt, J ¼ 7:3, 7.3 Hz, H-90), 3.12 (br d, J ¼ 7:3 Hz, H-10),
3.73 (s, 180-OMe), 5.08 (m, H-140), 5.12 (m, H-60), 5.15 (m, H-20),
5.85 (br t, J ¼ 7:3 Hz, H-100), 6.46 (dt, J ¼ 1:8, 2.8 Hz, H-3), 6.54
(dq, J ¼ 2:8, 1.4 Hz, H-5). 13C NMR (125 MHz, CDCl3): ꢀ 15.9
(q, C-190), 16.0 (q, C-7), 16.2 (q, C-200), 17.7 (q, C-160), 25.7
(q, C-170), 26.5 (t, C-50), 27.6 (t, C-10), 27.9 (t, C-130), 28.0
(t, C-90), 34.7 (t, C-120), 39.2 (t, C-80), 39.6 (t, C-40), 51.1 (q,
180-OMe), 118.0 (d, C-20), 123.5 (d, C-140), 124.5 (d, C-60),
131.5 (s, C-110), 132.1 (s, C-150), 132.3 (d, C-3), 133.2 (d, C-5),
134.7 (s, C-70), 139.9 (s, C-30), 142.1 (d, C-100), 145.9 (s, C-6),
148.5 (s, C-2), 168.5 (s, C-180), 188.0 (s, C-1, 4). HREIMS m=z
438.2782 [M]þ (calcd for C28H38O4: 438.2771).
Methylation of 7 to 7a: Similar to the synthesis of 6a, 7a was
obtained in a 79% yield as a colorless oil. 1H NMR (500 MHz,
CDCl3): ꢀ 1.35 (s, H-10), 1.57 (s, H-160), 1.58 (s, H-130), 1.65
(m, H-10), 1.67 (s, H-140), 2.04 (t, J ¼ 7:3 Hz, H-50), 2.07 (dt,
J ¼ 7:3, 7.3 Hz, H-100), 2.08 (m, H-20), 2.14 (s, H-9), 2.24 (t,
J ¼ 7:4 Hz, H-90), 2.50 (dt, J ¼ 7:3, 7.3 Hz, H-60), 3.73 (s, 150-
OMe), 5.08 (br t, J ¼ 7:3 Hz, H-110), 5.13 (br t, J ¼ 7:3 Hz,
H-30), 5.58 (d, J ¼ 9:6 Hz, H-3), 5.83 (br t, J ¼ 7:3 Hz, H-70),
6.24 (d, J ¼ 10:1 Hz, H-4), 6.32 (d, J ¼ 3:2 Hz, H-5), 6.48 (d,
J ¼ 3:2 Hz, H-7). 13C NMR (125 MHz, CDCl3): ꢀ 15.5 (q, C-9),
15.7 (q, C-160), 17.7 (q, C-130), 22.6 (t, C-20), 25.7 (q, C-10),
25.7 (q, C-140), 27.8 (t, C-100), 28.0 (t, C-60), 34.7 (t, C-90),
39.1 (t, C-50), 40.8 (t, C-10), 51.1 (q, 150-OMe), 77.8 (s, C-2),
110.3 (d, C-5), 117.0 (d, C-7), 121.3 (s, C-4a), 122.9 (d, C-4),
123.5 (d, C-110), 124.8 (d, C-30), 126.3 (s, C-8), 130.7 (d, C-3),
131.4 (s, C-80), 132.1 (s, C-120), 134.4 (s, C-40), 142.1 (d, C-70),
144.9, (s, C-8a) 148.6 (s, C-6), 168.6 (s, C-150). HREIMS m=z
438.2784 [M]þ (calcd for C28H38O4: 438.2771).
MIC
MBC
MIC
/mg mLꢁ1
Compound
Compound
/mg mLꢁ1 /mg mLꢁ1
1
2
5
16
128
2
128
>256
64
2a
32
6
32
8
1
256
8
1
6a
7a
>256
>256
7
Vancomycin
1H NMR signals (around ꢀ 6.2–6.5) and/or antibacterial activities,
the sixth and seventh fractions (60–80% EtOAc/hexane, 3.3 g)
were re-chromatographed by silica gel column and/or PLC, and
new quinone metabolites 1–5 were purified by repeated HPLC
using EtOAc/hexane; 1 (5.5 mg, 1:7 ꢃ 10ꢁ3%), 2 (15.8 mg,
4:9 ꢃ 10ꢁ3%),
3
(0.7 mg, 2:3 ꢃ 10ꢁ4%),
4
(4.7 mg, 1:5 ꢃ
10ꢁ3%), and 5 (2.4 mg, 7:6 ꢃ 10ꢁ4%), together with known 6
(28.4 mg, 8:9 ꢃ 10ꢁ3%), 7 (510 mg, 1:6 ꢃ 10ꢁ1%), yezoquinolide
(1.1 mg, 3:5 ꢃ 10ꢁ4%), and 2-methyl-6-phytyl-1,4-benzoquinone
(0.9 mg, 2:9 ꢃ 10ꢁ4%).
Compound 1 (150-Hydroxysargaquinolide): Yellowish oil
20
½ꢁꢂD ¼ ꢁ5ꢄ (c 0.025, CHCl3); IR (KBr) ꢂmax 3460, 2926,
1716, 1653, 1457, 1381, 1294, 1180, 914, 680 cmꢁ1; HREIMS
m=z 440.2537 [M]þ (calcd for C27H36O5, 440.2563); 1H and
13C NMR, see Table 1.
Compound 2 (110-Hydroxysargachromelide): Yellowish oil
20
½ꢁꢂD ¼ ꢁ5ꢄ (c 0.042, CHCl3); IR (KBr) ꢂmax 3425, 2926, 1715,
1700, 1636, 1385, 1250, 1097, 758 cmꢁ1; HREIMS m=z 440.2552
[M]þ (calcd for C27H36O5, 440.2563); 1H and 13C NMR, see
Table 2.
Compound 3 (150-Methylenesargaquinolide): Yellowish oil
20
½ꢁꢂD ¼ þ10ꢄ (c 0.037, CHCl3); IR (KBr) ꢂmax 3425, 2926, 1716,
1653, 1380, 1294, 1192, 908 cmꢁ1; HREIMS m=z 422.2460 [M]þ
(calcd for C27H34O4, 422.2457); H and 13C NMR, see Table 1.
1
20
Compound 4 (Chromequinolide): Reddish oil ½ꢁꢂD ¼ þ3ꢄ
(c 0.104, CHCl3); IR (KBr) ꢂmax 3425, 1722, 1714, 1668, 1651,
1471, 1385, 1192, 921, 756 cmꢁ1; HREIMS m=z 454.2368 [M]þ
(calcd for C27H34O6, 454.2350); H and 13C NMR, see Table 2.
1
Compound 5 [(20E,50E)-2-Methyl-6-(70-oxo-30-methylocta-
20,50-dienyl)-1,4-benzoquinone]: Yellowish oil; IR (KBr) ꢂmax
3425, 2924, 1714, 1659, 1651, 1456, 1361, 1294, 1255, 979,
914, 756 cmꢁ1
;
HREIMS m=z 258.1269 [M]þ (calcd for
C16H18O3, 258.1256);1H and 13C NMR, see Table 1.
Methanolysis of 2 (2a): To a solution of 2 (4.1 mg, 9.3 mmol)
in methanol (0.2 mL), 0.3 M sodium methoxide solution (0.40 mL)
was added and stirred constantly at room temperature. After 2.5 h,
the solution was neutralized by several drops of saturated aqueous
NH4Cl and was passed through an ODS-cartridge. The extract
was purified by SIL-HPLC (60% EtOAc in hexane) to afford the
desired product 2a in a 82% yield.
Antibacterial Assay.20,21 The microdilution method was em-
ployed as an antibacterial activity test against the microorganism,
Staphylococcus aureus (ATCC 6538P) obtained from RIKEN
BioResource Center, Saitama, Japan). The specimen was suspend-
ed in aqueous DMSO at 512 mg mLꢁ1 final concentration, and the
suspension was transferred to a 96-well microplate (100 mL wellꢁ1
except for the first line which contained 200 mL wellꢁ1). Two-fold
dilutions were then prepared in a concentration range from 0.5 to
256 mg mLꢁ1 (for positive control, the vancomycin concentration
range was 0.125–64 mg mLꢁ1) diluted in 100 mL of brain heart in-
fusion (BHI) broth. A 5-mL volume, which contained approxi-
mately 107 cfu mLꢁ1 of S. aureus, was added. Following incuba-
tion of the plates for 24 h at 37 ꢄC in ambient air, the minimum
inhibitory concentration (MIC) values were determined by the
lowest concentration at which observable growth was inhibited.
Minimum bactericidal concentrations (MBC) were determined
20
2a: Colorless oil ½ꢁꢂD ¼ þ3ꢄ (c 0.172, CHCl3); HREIMS m=z
472.2851 [M]þ (calcd for C28H40O6, 472.2826); 1H and
13C NMR, see Table 2.
(S)-MTPA Esterification of 2a (2b):
A solution of 2a
(4.1 mg, 9.3 mmol), (R)-MTPA-Cl (50 mmol), Et3N (16 mL), and
a catalytic amount of DMAP in CH2Cl2 (0.30 mL) was stirred
for 16 h at room temperature. The reaction mixture was added
to N,N-dimethyl-1,3-propanediamine and was blown down with
nitrogen. The residue was subjected to SIL-HPLC (30% EtOAc
in hexane) to give two (S)-MTPA diesters 2b. 2b (rt 18.0 min):
1.13 (s, 130-CH3), 1.19 (s, 140-CH3), 2.50 (dt, J ¼ 7:5, 7.5 Hz,

1130 Bull. Chem. Soc. Jpn. Vol. 81, No. 9 (2008)
Antibacterial Quinone Metabolites
by plating out aliquots of incubation BHI on agar plates in 1 mL
drops (undiluted) of MIC testing mixtures that showed no growth
in the BHI microplates after 24 h incubation. MBC values were
recorded as the lowest concentrations at which no colony of
S. aureus was observed. Experiments were carried out three times.
7
8
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The authors wish to thank Dr. Kikuo Tsukamoto and
Dr. Yoshihiro Inoue of Showa Pharmaceutical University
and Yoichi Nakao of the University of Tokyo for the bioassay.
We are also grateful to Mr. Akiyoshi Takahashi for identifica-
tion of the alga. This work was supported in part by the
Research Institute of Aoyama Gakuin University, and by a
Grant-in-Aid (No. 12640527) for Scientific Research from
the Ministry of Education, Culture, Sports, Science and Tech-
nology, Japan.
15 P. E. Brown, R. A. Lewis, M. A. Waring, J. Chem. Soc.,
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