Synthesis of biotinylated xestoquinone that retains inhibitory activity against Ca2+ ATPase of skeletal muscle myosin

Article abstract of DOI:10.1016/S0968-0896(03)00276-1

Xestoquinone isolated from a marine sponge binds to skeletal muscle myosin and inhibits its Ca²⁺ ATPase activity. In this study, we first examined xestoquinone and its analogues to assess the relationships between structure and myosin Ca²⁺ ATPase inhibitory activity. On the basis of the resultant data, we then designed a biotinylated xestoquinone analogue. Xestoquinone and its analogues were derived from extracts of the marine sponge Xestospongia sapra. Four xestoquinone analogues with a quinone structure significantly inhibited Ca²⁺ ATPase activity. In contrast, four xestoquinone analogues in which the quinone structure was converted to a quinol dimethyl ether did not inhibit Ca²⁺ ATPase activity. This suggests that the quinone moiety is essential for inhibitory activity. Then, we synthesized a biotinylated xestoquinone in which a biotin tag was introduced to a site far from the quinone moiety, and this molecule exhibited stronger inhibitory activity than that of xestoquinone. This biotinylated xestoquinone could be useful as a probe in studies of the xestoquinone-myosin binding mode.

Full text of DOI:10.1016/S0968-0896(03)00276-1

Bioorganic & Medicinal Chemistry 11 (2003) 3077–3082
Synthesis of Biotinylated Xestoquinone That Retains Inhibitory
Activity Against Ca ATPase of Skeletal Muscle Myosin
2+
y
Mitsuhiro Nakamura, Takahiko Kakuda, Yuichi Oba,* Makoto Ojika
{
and Hideshi Nakamura
Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
Received 24 December 2002;accepted 11 April 2003
Abstract—Xestoquinone isolated from a marine sponge binds to skeletal muscle myosin and inhibits its Ca2+ ATPase activity. In
2
+
this study, we first examined xestoquinone and its analogues to assess the relationships between structure and myosin Ca ATPase
inhibitory activity. On the basis of the resultant data, we then designed a biotinylated xestoquinone analogue. Xestoquinone and its
analogues were derived from extracts of the marine sponge Xestospongia sapra. Four xestoquinone analogues with a quinone
2
+
structure significantly inhibited Ca ATPase activity. In contrast, four xestoquinone analogues in which the quinone structure was
2
+
converted to a quinol dimethyl ether did not inhibit Ca ATPase activity. This suggests that the quinone moiety is essential for
inhibitory activity. Then, we synthesized a biotinylated xestoquinone in which a biotin tag was introduced to a site far from the
quinone moiety, and this molecule exhibited stronger inhibitory activity than that of xestoquinone. This biotinylated xestoquinone
could be useful as a probe in studies of the xestoquinone-myosin binding mode.
#
2003 Elsevier Science Ltd. All rights reserved.
Introduction
Sakamoto et al. studied, in detail, the binding mode of 1
4
2+
and myosin. They found that reduction of Ca
Marine sponges contain several compounds with var-
ious bioactivities, including antibacterial, antifungal and
ATPase activity by 1 was abolished by dithiothreitol,
and that the positions C-14 and C-15 of 1 were modified
by two molecules of 2-mercaptoethanol, indicating that
modification of the thiol groups of myosin by 1 causes
1
cytotoxic activity. Xestoquinone (1), halenaquinone
3
2
3
(
2), halenaquinol (3) and halenaquinol sulfate (4)
2
+
4
were isolated from the marine sponge Xestospongia
sapra or Xestospongia exigua (Fig. 1, Scheme 1). Each
compound was found to have unique biological activ-
ities, despite their structural similarity: 1 exhibits pow-
inhibition of myosin Ca ATPase. A myosin molecule
contains over 40 thiol residues, with 12 or 13 residues
1
0
located on each myosin head. A myosin head contains
two thiol groups (SH and SH ) that can be favorably
modified by maleimide reagents. N-Ethylmaleimide can
1
2
1
2+
erful cardiotonic activity, inhibits the Ca
+
and
K (EDTA) ATPase activities of myosin, activates the
4
4
2+
ATPase of actomyosin, and releases Ca from skele-
5
tal muscle sarcoplasmic reticulum; 2 inhibits protein
6
,7
8
tyrosine kinase and PI3 kinase; 3 inhibits protein
,7
6
tyrosine kinase; 4 inhibits the membrane fusion events
9
of echinoderm gametes. However, the only systematic
study of the structure–activity relationships of xesto-
quinone and its analogues have dealt with inhibition of
6
,7
protein tyrosine kinase.
*Corresponding author. Tel./fax: +81-52-789-4284;e-mail: oba@agr.
nagoya-u.ac.jp
y
Present address: Department of Applied Physics and Chemistry, Uni-
versity of Electro-Communications, Chofu, Tokyo, 182-8585, Japan.
{
Deceased, 9th November, 2000.
Figure 1. Xestoquinone and its analogues.
0
968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00276-1

3078
M. Nakamura et al. / Bioorg. Med. Chem. 11 (2003) 3077–3082
Ca2+ ATPase activity
Myosin Ca2+ ATPase activity was measured by released
phosphoric acid, which was detected using malachite
1
7
green. A large excess (2.64 mM) of each quinone analo-
2
+
gue (1, 2, 10 and 11) inhibited the myosin Ca ATPase,
but no dimethyl ether analogue (6, 7, 8 and 9) inhibited the
2
+
myosin Ca ATPase (Fig. 2). This suggests that the qui-
none structure is necessary for inhibition of the myosin
2
+
Ca ATPase, and that the A-ring structure far from the
quinone moiety is not important.
When the well was washed twice with the assay buffer
after incubation with 1, inhibition was retained (Fig. 2).
Scheme 1. Preparation of marine sponge extract, and derivatization of
hydroquinone components to dimethyl ethers.
modify both the SH and SH groups, even after modi-
1
2
fication of 2 mol of SH groups by 1, suggesting that the
binding sites of 1 are SH groups distinct from the SH
1
4
and SH of myosin. Because modification of myosin by
2
1
causes changes in tryptophan fluorescence intensity
and the circular dichroism spectrum, it is presumed that
induces a conformational change via modification of
1
4
myosin SH groups. However, the site at which 1 binds
to myosin has remained unidentified. In the present
study, in an attempt to identify the site at which 1 binds
to myosin, we first examined relationships between
structures of xestoquinone analogues and their myosin
Ca ATPase inhibitory activity. Then, on the basis of
the resultant data, we synthesized a biotinylated xesto-
quinone analogue.
2
+
Results and Discussion
Derivatization of xestoquinone analogues
We examined the structure–activity relationship for
xestoquinone (1) and its analogues, to determine the
most suitable position at which a biotin tag could be
attached. Rather than perform total synthesis (which
requires many steps), we derived the tested compounds
from halenaquinol (3) and xestoquinol (5), which were
obtained from extracts of the marine sponge X.
1
1ꢀ15
sapra.
The acetone extract was partitioned between
EtOAc and water. The EtOAc solution that contained 3
and 5 was methylated with iodomethane in the presence
of potassium carbonate to give dimethyl ether (6 and 7).
The major product 7 was selectively reduced with
Figure 2. Effects of xestoquinone (1) and its analogues on myosin
Ca ATPase activity: (A) test at substance concentration of 132 mM;
B) test at substance concentration of 528 mM;(C) test at substance
concentration of 2.64 mM. ‘Wash’ indicates that the well was washed
with the assay buffer after incubation. The ordinate gives the relative
phosphoric acid value;the phosphoric acid value corresponding to
lack of inhibitor was designated as 100% (dashed line). Values are
given as meansꢁSE (n=4). Values significantly different from the
control value (without inhibitor, filled bar) are indicated by asterisks
(Student’s t-test, *p<0.05, **p<0.01, ***p<0.005).
2+
(
1
1
NaBH in the presence of CeCl
(
to give alcohol 8
Scheme 2). Alcohol 8 was dehydrated in the presence of
4
3
an acid catalyst to give olefin-dimethyl ether 9. The
resulting dimethyl ethers 6, 7, 8 and 9 were oxidized by
1
1
ceric ammonium nitrate (CAN), giving quinones 1,
1
2
2
,
10 and 11, respectively.

M. Nakamura et al. / Bioorg. Med. Chem. 11 (2003) 3077–3082
3079
Scheme 2. Derivatization of various xestoquinone analogues.
This is consistent with the findings of Sakamoto et al.,4
who suggested that the quinone moiety of 1 was mod-
ified by the thiol groups of the Cys residues of myosin.
Interestingly, only the olefinic analogue 11 exhibited
strong inhibitory activity at a low concentration (528
mM);this novel molecule may be useful in studies of the
ATPase activity of myosin.
with epimerization at the position C-3 (Scheme 2). The
ester 12 was treated with biotin-PEAC maleimide, and
5
then oxidized by CAN, to give the biotinylated xesto-
quinone analogue 13. Reversed-phase high-perfor-
mance liquid chromatography (RP-HPLC) analysis of
13 gave two major and one minor peaks (asterisks in
Fig. 3), and each fraction gave identical pseudomole-
cular ions at m/z 971.4 in MALDI-TOF-MS. Further-
more, the major peaks (29.6 and 30.4 min in Fig. 3) and
the minor one (24.8 min in Fig. 3) were equilibrated
each other within 18 h (data not shown), indicating that
they were all isomers of 13. We examined inhibition of
myosin ATPase by the biotinylated analogue 13, and
found that its inhibitory activity was higher than that of
1 at a concentration of 2.64 mM (Fig. 2). This is further
evidence that biotinylation at the position C-3 had little
Derivatization of a biotinylated analogue and its Ca2+
ATPase inhibitory activity
Because modifications at the A-ring structure far from
the quinone moiety of 1 were not expected to affect
2
+
myosin Ca
ATPase activity (Fig. 2) (as discussed
above), a biotin tag was introduced at the position C-3
of 1. Condensation of alcohol 8 with mercaptopropionic
acid in the presence of an acid catalyst gave 12 and 9
2
+
effect on inhibition of myosin Ca ATPase, and again

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M. Nakamura et al. / Bioorg. Med. Chem. 11 (2003) 3077–3082
5774). Silica gel for column chromatography was obtained
from Wakogel C-300 (Wako chemicals). RP-HPLC was
performed using a JASCO 1500 system with a Develosil
ODS-HG-5 column (4.6ꢂ250 mm, Nomura Chemicals).
Preparative RP-HPLC was performed using a Develosil
ODS-5 column (20ꢂ250 mm, Nomura Chemicals).
Extraction of crude material containing halenaquinol (3)
and xestoquinol (5), and derivatization of xestoquinol di-
methyl ether (6) and halenaquinol dimethyl ether (7). X.
sapra was collected on 9 November 2000, at Akajima,
Okinawa, Japan. The sponge was frozen immediately,
Figure 3. HPLC analysis of biotinylated analogue (13), which was
eluted using a linear gradient of 20–80% acetonitrile containing 0.1%
TFA in 60 min. Peaks were detected at 220 nm. Three peaks corre-
sponding to each isomers of 13 are marked with an asterisk.
ꢃ
and then stored at ꢀ30 C until used. The sponge (739
g) was homogenized in a blender with acetone (1000
mL), and then filtered. The residue was washed twice
with acetone (500 mLꢂ2). The filtrates were combined
and the solvent was then evaporated. The aqueous resi-
due was extracted with EtOAc (500 mLꢂ2, 200 mLꢂ3),
and the combined organic layers were dried over anhy-
suggests that the quinone moiety of 1 is the structure that is
essential for this inhibitory activity. Biotinylated com-
pounds that retain original bioactivities are sometimes
very useful in biochemical and physiological analysis. The
novel biotinylated xestoquinone 13 may be useful as a
probe in studies of myosin–xestoquinone interaction, par-
ticularly for determining the site where 1 binds to myosin.
drous MgSO . Evaporation of the solvent in vacuo gave
4
a crude pitch-black oil, which contained 3 and 5 (3.9 g).
The pitch-black oil (3.9 g) was dissolved in acetone (60
mL), and anhydrous potassium carbonate (9.7 g) and
iodomethane (36 mL) were then added under a nitrogen
atmosphere. After being refluxed in dim light for 14 h,
the reaction mixture was diluted with ethyl acetate (250
Conclusion
We firstly identified the importance of the quinone
moiety of xestoquinone (1) in inhibition of myosin
ATPase, by analyzing structure–activity relationships of
mL), washed with aqueous NaHCO and brine, and
3
then dried over anhydrous MgSO . Evaporation in
4
vacuo and purification by chromatography on silica gel
(CH Cl /Et O 9:1) afforded xestoquinol dimethyl ether
(6, 30 mg, brown-orange powder) and halenaquinol
1
and its derived analogues. Based on the resultant data,
2
2
2
we then designed a biotin-tagged xestoquinone (13), in
which the biotin tag was introduced at the position C-3
of 1, far from the essential quinone moiety. The syn-
thesized 13 exhibited myosin ATPase inhibitory activity
that was equal to or stronger than that of 1. We also
found that the olefin 11 exhibited much stronger myosin
ATPase inhibitory activity than 1. The two novel com-
pounds 11 and 13 may be useful for further studies of
xestoquinone–myosin interaction.
1
1
dimethyl ether (7, 332 mg, lemon-yellow powder). 6:
1
H NMR (300MHz, CDCl ) d 1.54 (3H, s), 1.82 (1H, td,
3
J=13, 4 Hz), 2.10–2.23 (2H, m), 2.57–2.67 (2H, m), 2.88
(1H, dd, J=17, 7 Hz), 3.98 (6H, s), 6.70 (1H, d, J=8 Hz),
6.82 (1H, d, J=8 Hz), 7.48 (1H, t, J=1.4 Hz), 8.28 (1H, s)
1
8 1
and 9.28 (1H, s). 7: H NMR (300MHz, CDCl ) d 1.69
3
(3H, s), 2.34 (1H, td, J=5, 13 Hz), 2.79–3.10 (3H, m), 4.00
(6H, s), 6.74 (1H, d, J=8 Hz), 6.86 (1H, d, J=8 Hz), 8.23
(1H, s), 8.32 (1H, s) and 9.31 (1H, s).
3-Hydroxyxestoquinol dimethyl ether (8). Halenaquinol
dimethyl ether (7, 607 mg, 1.68 mmol) was dissolved in
dichloromethane (50 mL) and methanol (50 mL), fol-
Experimental
General procedures
.
lowed by addition of CeCl 7H O (6.4 g, 17.2 mmol).
3
2
Melting points were measured with a Yanaco MP-J3
micro melting point apparatus and uncorrected. IR
spectra were obtained using a JASCO FT/IR-8300
spectrometer. H NMR spectra were obtained using a
Varian Gemini-2000 (300 MHz), Bruker ARX-400
This mixture was stirred at room temperature for 10
min. Sodium borohydride (16.4 mg, 0.43 mmol) was
then added, followed by stirring for 20 min, which was
repeated three times. This reaction mixture was poured
into water and extracted with ethyl acetate. The organic
layer was washed with brine and evaporated in vacuo. The
crude product was purified by column chromatography
on silica gel (hexane/EtOAc 1:2) to give dimethyl ether 8
1
(
400 MHz) or Bruker AMX-600 (600 MHz) spectro-
meter. Chemical shifts (d) were given in parts per mil-
lion, relative to tetramethylsilane (d 0.00) as an internal
standard, and coupling constants (J) in Hz. MALDI-
TOF mass spectrometry (MS) was performed by an
Applied Biosystems Voyager DE Pro. High-resolution
1
8 1
(373 mg, 61%, bright yellow powder):
H NMR
(300MHz, CDCl /CD OD=9:1) d 1.63 (3H, s), 1.94 (1H,
3
3
td, J=13, 4 Hz), 2.20–2.30 (1H, m), 2,55 (1H, m), 2.63
(1H, dt, J=4, 13 Hz), 3.98 (6H, s), 5.00 (1H, brt, J=8 Hz),
6.71 (1H, d, J=8 Hz), 6.83 (1H, d, J=8 Hz), 7.78 (1H, d,
J=0.6 Hz), 8.24 (1H, s) and 9.27 (1H, s).
(
HR) ESI-TOF-MS was performed using an Applied
Biosystems Mariner Biospectrometry Workstation,
using an angiotensin I/neurotensin mixture as a stan-
dard for internal calibration. Reactions were monitored
using TLC (60F-254, Merck, Art 5715). Preparative
TLC was performed using 60F-254 plates (Merck, Art
3,4-Dehydroxestoquinol dimethyl ether (9). Hydroxy-
dimethyl ether (8, 14.3 mg, 0.039 mmol) and p-TsOH (1

M. Nakamura et al. / Bioorg. Med. Chem. 11 (2003) 3077–3082
3081
ꢃ
25
mg, 5 mmol) in toluene (2 mL) were refluxed for 15 min
under an argon atmosphere. The crude products were
purified by preparative TLC (hexane/EtOAc 1:1) to give
olefin dimethyl ether (9, 13 mg, 95%, yellowish pow-
a dark yellow powder: mp 213–215 C, [a] D ꢀ55 (c
0.39, CHCl );UV (MeOH) lmax 242 (e 23,500), 295
(10,200) and 339 nm (sh, 6400);IR (KBr) n
1772, 1670, 1653, 1559 and 1541 cm ; H NMR
(400MHz, CDCl ) d 1.53 (3H, s, C-20), 2.60 (1H, brd,
3
3071, 2361,
1
max
ꢀ
1
1
2 1
der). 9:
H NMR (300 MHz, CDCl ) d 1.53 (3H, s),
3
3
2
3
6
.67 (1H, brd, J=17 Hz), 3.17 (1H, dd, J=17, 6 Hz),
.99 (6H, s), 6.12 (1H, m), 6.62 (1H, dd, J=10, 2 Hz),
.72 (1H, d, J=8 Hz), 6.83 (1H, d, J=8 Hz), 7.58 (1H,
J=16.6 Hz, H-5), 3.10 (1H, dd, J=16.6, 6.2 H , H-5), 6.12
2
(1H, ddd, J=9.7, 6.2, 2.1 Hz, H-4), 6.64 (1H, dd, J=9.7,
3.0 Hz, H-3), 7.04/7.07 (1H each, d, J=10.6 Hz, H-14 and
15), 7.62 (1H, s, H-1), 8.22 (1H, s, H-18) and 9.04 (1H, s,
s), 8.27 (1H, s) and 9.31 (1H, s).
13
H-11); C NMR (100 MHz, CDCl ) d 31.0 (q), 34.3 (t),
3
Xestoquinone (1). Xestoquinol dimethyl ether (6, 8.0 mg,
0.023 mmol) was dissolved in acetonitrile (2 mL), fol-
lowed by addition of a solution of ceric ammonium
36.3 (s), 117.6 (d), 121.0 (s), 123.6 (d), 127.1 (d), 128.1 (d),
130.5 (s), 133.5 (s), 138.0 (s), 138.6 (d), 139.4 (d), 142.5 (d),
143.6 (s), 144.7 (s), 155.0 (s), 170.1 (s), 183.7 (s) and 184.6
(s);HRMS (ESI-TOF) calcd for C H O (M+H)
317.0808, found 317.0794. Anal. calcd for C H O H O:
20 12 4 2
C, 71.85;H, 4.22%;found C, 71.57;H, 3.71%.
nitrate (CAN) (IV) (37.2 mg, 0.0679 mmol) in H O (1
2
20 13 4
ꢃ
This reaction mixture was washed with aqueous
mL) at 0 C, and this mixture was stirred for 10 min.
NaHCO and brine, and then dried over anhydrous
3
MgSO . Evaporation in vacuo gave a crude product,
4
3-(3-Mercaptopropionyloxy)xestoquinol dimethyl ether
(12). A solution of mercaptopropionic acid (92 mL,
1.06 mmol) and hydroxy dimethyl ether (8, 389.7 mg,
1.096 mmol) in toluene (80 mL) was refluxed for 1 h
under an argon atmosphere. The crude products were
purified by preparative TLC (hexane/EtOAc, 2:1), to
give the SH compound (12, 29.5 mg, 12%, lemon-yellow
powder) and olefin-dimethyl ether (9, 93.8 mg, 26%).
The SH compound 12 was used immediately in the
relevant reactions, because of its instability. 12: mp 125–
which was purified by preparative TLC (hexane/EtOAc
1
:1) to give xestoquinone (1, 5.9 mg, 81%, bright brown
1
1 1
powder). 1: H NMR (300 MHz, CDCl ) d 1.54 (3H,
3
s), 1.76 (1H, td, J=13, 5 Hz), 2.17–2.30 (2H, m), 2.55–
2.71 (2H, m), 2.99 (1H, dd, J=17, 8 Hz), 7.04 (1H, d,
J=11 Hz), 7.07 (1H, d, J=11 Hz), 7.55 (1H, t, J=1.5
Hz), 8.25 (1H, s) and 9.06 (1H, s).
Halenaquinone (2). Halenaquinol dimethyl ether (7, 13
mg) was oxidized with CAN (IV), as described above, to
give halenaquinone (2, 6.2 mg, 52%, bright brown
ꢃ
1
127 C; H NMR (300 MHz, CDCl ) d 1.52 (1.5H, s),
3
1.64 (1.5H, s), 1.99 (0.5H, td, J=13, 3 Hz), 2.17 (0.5H,
td, J=14, 3 Hz), 2.25–2.88 (7H, m), 3.98 (6H, s), 5.92
(0.5H, brt, J=8 Hz), 5.99 (0.5H, d, J=4 Hz), 6.71 (1H,
d, J=8.4 Hz), 6.83 (1H, d, J=8.4 Hz), 7.79 (1H, s), 8.26
(0.5H, s), 8.27 (0.5H, s) and 9.27 (1H, s).
1
2 1
powder). 2: H NMR (300 MHz, CDCl ) d 1.70 (3H,
3
s), 2.29 (1H, dt, J=6, 12 Hz), 2.86 (2H, m), 3.03 (1H,
m), 7.07 (1H, d, J=12 Hz), 7.11 (1H, d, J=12 Hz), 8.29
(
2H, s) and 9.31 (1H, s).
(
3R)-3-Hydroxyxestoquinone (10). 3-Hydroxyxestoquinol
dimethyl ether (8, 10.0 mg) was oxidized with CAN
Biotinylated xestoquinone analogue (13). The SH com-
pound (12, 29.5 mg, 0.065 mmol) was dissolved in
CH CN/H O (1:1, 3 mL) with biotin-PEAC -maleimide
(
8
+
IV), as described above, to give quinone 10 (7.7 mg,
ꢃ
3
2
5
25
4%) as a bright brown powder: mp 225–228 C;[ a]D
27 (c 0.15, CHCl –MeOH 1:9);UV (MeOH) l 217
(38.8 mg, 0.066 mmol, Dojindo Laboratories), and stir-
red at room temperature for 4.5 h. After the mixture
3
max
ꢃ
(107.7 mg, 0.196 mmol) in CH CN/H O (2:1, 0.3 mL)
(e 16,600), 256 (17,500), 292 (12,600) and 341 nm
was allowed to stand at 0 C, a solution of CAN (IV)
(
6700);IR (KBr) n 3536, 2360, 1668, 1671, 1601 and
max
3
2
ꢃ
ꢀ
1
1
1
1
2
559 cm ; H NMR (400 MHz, CDCl –CD OD 9:1) d
was added, and stirred for 20 min at 0 C. This reaction
mixture was filtered and lyophilized. The crude product
was purified by preparative HPLC (eluted with a linear
gradient of 20–80% acetonitrile in 0.1% TFA in 60 min,
5 mL/min) to collect the major peaks to give biotin
compound 13 (18.1 mg, 29%, lemon-yellow powder).
The equilibrium experiments of 13 were performed as
follows. Each HPLC peak (Fig. 3) was lyophilized, dis-
solved in CH CN/H O (1:1), incubated at rt for 18 h,
3
3
.64 (3H, s, H-20), 1.93 (1H, dt, J=3.6, 13.3 Hz, H-5a),
.25 (1H, ddt, J=9.0, 3.6, 13.3 Hz, H-4b), 2.51 (1H,
ddt, J=13.3, 7.4, 3.6 Hz, H-4a), 2.62 (1H, dt, J=13.3,
3
7
.6 Hz, H-5b), 4.93 (1H, dd, J=9.0, 7.4 Hz, H-3), 7.06/
.09 (1H each, d, J=10.8 Hz, H-14 and 15), 7.87 (1H,
d, J=1.0 Hz, H-1), 8.23 (1H, s, H-18) and 9.00 (1H, s,
1
3
H-11); C NMR (100 MHz, CDCl –CD OD 9:1) d
3
1
3
3
0.2 (t), 32.5 (t), 33.5 (q), 37.6 (s), 61.9 (d), 123.3 (d),
25.7 (s), 127.2 (d), 130.6 (s), 133.6 (s), 137.9 (s), 138.9
3
2
and analyzed by RP-HPLC. 13: analytical RP-HPLC;
tR=24.8, 29.6 and 30.4 min (linear gradient, 20–80%
acetonitrile in 0.1% TFA in 60 min, 0.8 mL/min);mp
(
1
d), 139.5 (d), 144.1 (s), 148.0 (d), 148.0 (s), 156.4 (s),
70.8 (s), 184.1 (s) and 184.9 (s);selected NOESY
ꢃ
25
correlations H-3/H-4a, H-3/H-5a, H-4b/H-20, H-5b/
H-20, H-4a/H-5a, H-4b/H-5b, H-5b/H-18, and H-18/
H-20;HRMS (ESI-TOF) calcd for C 20H O (M+H)
3
2
4
140–144 C, [a] D ꢀ21 (c 0.91, CHCl –MeOH 1:1);UV
3
(MeOH) lmax 216 (e 21,200), 255 (18,400), 289 (13,500)
and 336 nm (7000);IR (KBr) nmax 1702, 1675, 1635,
1459, 1440, 1321, 1241 and 1137 cm
15
5
ꢀ
1
1
35.0914, found 335.0923. Anal. calcd for C H O
5
;
H NMR
2
0
14
/3H O: C, 69.36;H, 4.45%;found: C, 69.30;H,
2
.17%.
(600 MHz, CDCl –CD OD 4:1) d 1.36 (2H, m), 1.43
3
3
(2H, m), 1.52 (2H, m), 1.56/1.68 (1.5H each, s, H-20),
.62 (2H, m), 1.61–1.74 (4H, m), 1.94 (0.5H, dt, J=4.1
1
3
,4-Dehydroxestoquinone (11). 3,4-Dehydroxestoquinol
and 13.6 Hz, H-5), 2.12 (0.5H, brt, J=12.6 Hz, H-5),
2.20 (2H, m), 2.34 (2H, m), 2.38-2.55 (6H, m), 2.55-2.73
(5.5H, m), 2.74 (1H, d, J=12.9 Hz), 2.85 (0.5H, m), 2.93
dimethyl ether (9, 31.2 mg) was oxidized with CAN (IV),
as described above, to give quinone 11 (20.6 mg, 72%) as

3082
M. Nakamura et al. / Bioorg. Med. Chem. 11 (2003) 3077–3082
(
1H, dd, J=12.9, 5.0 Hz), 2.98 (0.5H, m), 3.08 (0.5H,
m), 3.13–3.30 (5H, m), 3.45 (2H, m), 3.56 (2H, m), 3.62–
.72 (2H, m), 3.80 (0.5H, m), 3.88 (0.5H, m), 4.32 (1H,
independent measurements were performed for each set of
conditions.
3
dd, J=7.8 and 4.5 Hz), 4.52 (1H, dd, J=7.8 and 4.9
Hz), 5.92 (0.5H, t, J=8.1 Hz, H-3a), 5.99 (0.5H, d,
J=4.9 Hz, H-3b), 7.11 (2H, s, H-14 and 15), 7.90 (0.5H,
d, J=2.9 Hz, H-1), 7.93 (0.5H, d, J=2.9 Hz, H-1), 8.28/
Acknowledgements
The authors wish to thank Drs Yasushi Ohizumi and
Tohru Yamakuni of Tohoku University for their help-
ful comments.
8
1
.29 (0.5H each, s, H-18) and 9.02/9.03 (0.5H each, s, H-
1
3
1); C NMR (150 MHz, CDCl –CD OD 4:1) d 24.9
3
3
(
(
(
(
t), 25.8 (t), 26.1 (t, C-4), 26.6/26.9 (0.5C each, t), 26.8
t), 28.0/32.6 (0.5C each, t, C-5), 28.3 (t), 28.6 (t), 29.1
t), 32.2/33.2 (0.5C each, q, C-20) 33.2 (t), 34.6 (t), 36.0
3C, t), 37.4/37.5 (0.5C each, s, C-6), 39.3 (t), 39.5 (d),
References and Notes
4
(
0.5 (t), 41.8 (t), 45.8 (t), 52.9 (t), 53.1 (t), 54.7 (t), 55.8
d), 60.4 (d), 62.2 (d), 62.8 (0.5C, d, C-3 of a-OCOR
isomer), 64.7 (0.5C, d, C-3 of b-OCOR isomer), 121.6/
1
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1
1
1
1
22.0 (0.5C each, s, C-2), 123.3/123.7 (0.5C each, d, C-
8), 127.2/127.3 (0.5C each, d, C-11), 130.8 (s, C-12),
33.8 (s, C-17), 137.2 (s, C-8), 137.7/137.8 (s, C-10), 139.1/
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6
+
7
+
2+
971.43 (M+H) (51), 497.22 (M+H+Na) (18), 486.22
2+
(M+2H)
C H N O S (M+2H) 486.1875, found 486.1884
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(100);HRMS (ESI-TOF) calcd for
1
49
60
6 11 2
2+
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2
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ꢃ
1
1
5
1
991, 32, 4761.
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ꢃ
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630 nm was measured within 5 min using a microplate
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