Polybrominated diphenyl ethers from the Indonesian sponge Lamellodysidea herbacea

Article abstract of DOI:10.1021/np0605081

Four new (1-4) and 10 known polybrominated diphenyl ethers (5-14) have been isolated from the title sponge. The structures of the new entities were elucidated by interpretation of spectroscopic data and chemical transformations. These metabolites showed p

Full text of DOI:10.1021/np0605081

432
J. Nat. Prod. 2007, 70, 432-435
Polybrominated Diphenyl Ethers from the Indonesian Sponge Lamellodysidea herbacea
Novriyandi Hanif,† Junichi Tanaka,*,† Andi Setiawan,‡ Agus Trianto,§ Nicole J. de Voogd,# Anggia Murni,† Chiaki Tanaka,† and
Tatsuo Higa†
Department of Chemistry, Biology, and Marine Science, UniVersity of the Ryukyus, Nishihara, Okinawa 903-0213, Japan, Department of
Chemistry, Lampung UniVersity, Bandar Lampung 35145, Indonesia, Department of Marine Science, Diponegoro UniVersity,
Semarang 50234, Indonesia, and Naturalis, National Museum of Natural History, P.O. Box 9517, 2300 RA Leiden, The Netherlands
ReceiVed October 15, 2006
Four new (1-4) and 10 known polybrominated diphenyl ethers (5-14) have been isolated from the title sponge. The
structures of the new entities were elucidated by interpretation of spectroscopic data and chemical transformations.
These metabolites showed potent antimicrobial activity against Bacillus subtilis and moderate/weak cytotoxicity against
NBT-T2 rat bladder epithelial cells. The major constituent 14 was treated under debromination conditions to give eight
derivatives, which were subjected to a structure-activity relationship study. The results indicated that the presence of
two phenolic hydroxyl groups and bromines at C-2 and/or C-5, as in 2, is important for the exhibition of antibacterial
activity.
Sponges of the family Dysideidae have been the subject of
numerous chemical investigations and have yielded a number of
unique, bioactive substances such as arenastatin,1 dysidiolide,2
dysiherbaine,3 and dysidazirine,4 to name a few. Among the species
of the genus Lamellodysidea (formerly known as Dysidea), L.
herbacea is the one studied most extensively. The majority of its
metabolites can be grouped into three chemical classes: small
peptides with a characteristic trichloromethyl group, sesquiterpe-
noids, and polybrominated diphenyl ethers (PBDEs). The genus
Lamellodysidea is biologically characterized by the symbiotic
presence of the filamentous cyanobacterium Oscillatoria sponge-
liae.5,6 Faulkner and co-workers have reported that the PBDEs are
produced by the associated cyanobacteria.7 PBDEs have been found
to exhibit a variety of bioactivities: antibacterial and antifungal
properties,8-11 brine shrimp toxicity,10 antimicroalgal activity,12
antiinflammatory activity,13 and inhibition of a range of enzymes
implicated in tumor development such as inosine monophosphate
dehydrogenase, guanosine monophosphate synthetase, and 15-
lipoxygenase.14 More recently, PBDEs have been reported to inhibit
the assembly of microtubule protein, the maturation of starfish
oocytes,15 and also Tie2 kinase.16 In this collaborative project on
the studies of Indonesian marine organisms,17 we have examined
the constituents of L. herbacea collected at Sangiang Island,
Indonesia, and have isolated four new PBDEs (1-4) along with
known congeners (5-14). We also prepared 13 synthetic derivatives
of these compounds for the study of their structure-activity
relationships (SAR) against B. subtilis and NBT-T2 cells. We report
herein the isolation and structure elucidation of the new compounds
and the results of this SAR study.
HPLC to give the new compound 3 along with 6, 13, and 14. The
structures of the known compounds (5-14) were identified on the
basis of the interpretation of their spectroscopic data and by
comparison with literature values.14,15,18-22Mass spectrometry of
compound 1 established its molecular formula as C13H7Br5O3. The
1H NMR data showed the presence of meta-coupled protons (δ
6.81 and 7.48) and a methoxy group (δ 4.01) on ring B, as in 5.
An additional aromatic singlet at δ 7.65 suggested that 1 is a
debromo analogue of 5. The presence of a phenolic hydroxyl group
was inferred from the low-field signal at δ 9.96 (brs) and the IR
absorption band at 3350 cm-1. HMBC correlations gave confirma-
tion of the position of the methoxyl at C-2′ and the substitution
pattern on ring A by the correlations H-4/C-2,3,5,6. Methylation
of 1 furnished dimethyl ether 15, which showed identical data with
those reported.21 Therefore, compound 1 was elucidated as 2,3,5-
tribromo-6-(3′,5′-dibromo-2′-methoxyphenoxy)phenol. Comparison
of the 13C NMR data for the ring A portion of 1 with those of the
demethyl analogue 13 showed good agreement (∆δ 0.0-0.3),
except for C-6 (∆δ 7.5), which is probably influenced by additional
hydrogen bonding in 13.
Compound 2 analyzed for C12H6Br4O3, indicating it to be a
tetrabromodiphenyl ether without a methyl ether function. The 1H
NMR spectrum exhibited a pair of meta-coupled signals at δ 6.64
and 7.39, as in two other members of this compound series, and
ortho-coupled resonances at δ 7.17 and 7.45. The presence of two
phenolic hydroxyls was inferred by the IR spectrum (3444 cm-1
)
and confirmed by methylation, giving the dimethyl ether 16. The
HMBC correlations H-6′/C-1′,2′,4′,5′ and H-4′/C-2′,6′ established
the same substitution pattern on ring B as in 5, while the correlations
H-3/C-1,2,5 and H-4/C-2,5,6 indicated the ring A moiety to be 2,5-
dibromo-6-phenoxyphenol. Compound 2 was elucidated as 2,5-
dibromo-6-(3′,5′-dibromo-2′-hydroxyphenoxy)phenol.
The EtOAc-soluble portion of a crude extract from the sponge
L. herbacea was partitioned between hexane and aqueous MeOH,
and the latter layer was then extracted with CH2Cl2. The hexane
extract was fractionated by silica gel flash chromatography followed
by HPLC separation and recrystallization to give the new com-
pounds 1, 2, and 4, in addition to nine known substances (5, 7-14).
The major constituent 14 was also obtained from the CH2Cl2 extract
by crystallization. The mother liquor portion was separated by
The molecular formula of 3, C12H6Br4O3, suggested that it is
isomeric with 2, but the substitution pattern is different. Ring B
was found to contain one bromine atom as shown by 1,2,4-
trisubstitution signals [δ 6.53 (d, J ) 2.5 Hz), 6.80 (d, J ) 8.5
Hz), 6.97 (dd, J ) 8.5, 2.5 Hz)], as in 6. Ring A was concluded to
contain three bromine atoms (δ 7.74 s) and was elucidated as a
1-hydroxy-2,4,5-tribromo-6-phenoxyl moiety by HMBC correla-
tions (H-3/C-1,2,4,5) and by comparing its 13C NMR data with those
reported for 17 and 18.10,23 Methylation of 3 gave 19, having two
methoxy groups. Therefore, 3 was deduced as 2,4,5-tribromo-6-
(5′-bromo-2′-hydroxyphenoxy)phenol.
Dedicated to the late Dr. Kenneth L. Rinehart of the University of
Illinois at Urbana-Champaign for his pioneering work on bioactive natural
products.
* Corresponding author. Tel: +81-98-895-8560. Fax: +81-98-895-8565.
E-mail: jtanaka@sci.u-ryukyu.ac.jp.
† University of the Ryukyus.
‡ Lampung University.
§ Diponegoro University.
# National Museum of Natural History.
10.1021/np0605081 CCC: $37.00
© 2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/21/2007

Notes
Journal of Natural Products, 2007, Vol. 70, No. 3 433
Chart 1
Compound 4 was shown to have the same molecular formula,
Table 1. 13C NMR Spectroscopic Data (125 MHz, acetone-d6)
of 1-3
C13H7Br5O3, as 1. A pair of meta-coupled protons (δ 6.77, 7.43)
as in 1, 2, and 5 suggested that ring B is either 3′,5′-dibromo-2′-
hydroxyphenyl or 3′,5′-dibromo-2′-methoxyphenyl. The presence
of an aromatic singlet (δ 7.91) indicated that ring A of 4 contains
three bromine atoms. Of the four possible sites for the proton,
position 3 was considered more likely than 2, 4, and 5 because
C#
1
2
3
1
2
3
4
5
6
1′
2′
3′
4′
5′
6′
150.8
115.1
123.4
127.9
117.0
139.0
151.9
146.5
119.5
129.7
116.9
117.5
61.1
150.2
111.6
131.9
125.2
116.7
140.6
146.9
145.2
111.4
129.6
110.7
116.8
149.5
115.1
133.8
111.7
120.2
141.9
146.1
147.1
119.2
127.1
110.9
117.5
H-3 appears at a lower field as in 3 and related compounds.24
A
methoxy group (δ 3.86) can be placed at ring A by comparing
chemical shifts of methyl ethers in this work and refs 14, 15, 17,
and 22. In all compounds (1, 5, 8, 9, 12, 15, 16, 19, 20, and 22)
having a 3′,5′-dibromo-2′-methoxyphenoxy group (ring B), the
methoxy group is observed in the range δ 3.92-4.03, while a
methoxy at ring A (8-11, 15, 16, 19-22) appears higher than δ
3.87. Scarcity of the sample precluded the running of the 13C NMR
spectrum for a more rigorous assignment of the ring A substitution,
but the above evidence suggested that 4 is 2,4,5-tribromo-6-(3′,5′-
dibromo-2′-hydroxyphenoxy)anisole.
OMe-2′
In the cytotoxicity assay against NBT-T2 rat bladder epithelial
cells, IC50 values of compounds 12 and 27 were obtained as 2.8
and 8.5 µg/mL, while compounds 2, 3, 5, 6, 13, 14, 23-26, and
28-29 showed no significant activities (IC50 >15 µg/mL).
In order to determine the effect of the substituents in PBDEs on
antibacterial activity and cytotoxicity, methyl ethers 20-22 were
prepared from 5, 6, and 14. Furthermore, the major compound 14
was treated under debromination conditions using HBr and Na2-
SO3.25 Eight products (13, 23-29) were obtained with acetic acid
as the reaction solvent, while only 23 was produced when MeOH
was used instead of acetic acid. The structures of these compounds
Experimental Section
General Experimental Procedures. UV spectra were obtained on
a Hitachi U-2001 spectrophotometer and FTIR spectra on a JASCO
FTIR 300 spectrometer. NMR spectra were recorded on a JEOL R500
FT NMR spectrometer in acetone-d6, CDCl3, or CD3OD. Chemical
shifts were referenced to TMS or solvent signals (acetone-d6: δC 206.7;
CDCl3: δC 77.2; CD3OD: δC 49.2). Multiplicities of 13C NMR data
were determined by DEPT experiments. ESIMS were recorded on an
ESITOFMS QSTAR mass spectrometer (PE Biosystem), while EIMS
were measured on a Hitachi M-2500 instrument. HPLC separations
were carried out on a Tosoh CCPE pump equipped with a Tosoh UV-
8011 detector and a Shodex RI-101 refractive index detector or on a
Hitachi L-6000 pump outfitted with a Waters R403 RI monitor and a
Hitachi L-4000 UV detector. Columns used for HPLC were silica gel
(250 × 10 mm, Mightysil Si-60) or reversed-phase silica gel (250 ×
10 mm, Mightysil RP18 GP). Merck silica gel 60 (0.063-0.20 mm)
was used for initial column chromatography. Analytical TLC was
performed on commercial silica gel 60 F254 plates and visualized with
iodine vapor.
1
were characterized mainly by their H NMR and EIMS data and
by comparison with values reported in the literature.22 Six products
(23, 25-29) were new compounds, of which 23 is an unprecedented
hexabromodioxin. The structure of 23 was assigned by observing
meta-coupled proton signals as in 14, HMBC correlations (H-7/
C-5a,6,8,9, H-9/C-5a,7,8,9a), and molecular ions corresponding to
the loss of H2O from 14.
The results of the antibacterial assays carried out are shown in
Table 2. In the standard disk diffusion assay, all compounds, except
for 7-9, 11, 15, 16, 19, 20, and 22, were active against the Gram-
positive bacterium B. subtilis in the range of 1-10 µg/disk, while
compounds 1-4, 6, 10, 12, 14, 23, 24, 28, and 29 were still active
at the concentration of 0.1 µg/disk. Compound 2 was most active,
giving clear zones of inhibition of 20, 20, 13, and 7 mm at 10, 5,
1, and 0.1 µg/disk, respectively. Among the derivatives, 24 showed
inhibition zones of 7-16 mm at the concentrations of 0.1-10 µg/
disk. These results point out that the presence of two phenolic
hydroxyl groups as well as bromine atoms at C-2 and/or C-5 as in
2 is important for the resultant antibacterial activity.
Animal Material. A specimen of the sponge Lamellodysidea
herbacea was collected by hand using scuba in Sangiang Island, West
Java, Indonesia, in August 2004. Voucher specimens have been
deposited at the Departement of Chemistry, Biology, and Marine
Science, University of the Ryukyus (Code No. 04C35) and also at

434 Journal of Natural Products, 2007, Vol. 70, No. 3
Notes
Table 2. Antibacterial Activity against B. subtilis (inhibition
1; EIMS m/z 605.6 (10), 607.6 (51), 609.6 (100), 611.6 (97), 613.6
(48), 615.6 (10) [M]+; HRESIMS m/z 634.6116 [M + Na]+ (634.6149
calcd for C13H779Br281Br3O3Na).
zone in mm)
concentration (µg/disk)
Compound 2: white solid; UV (MeOH) λmax (log ꢀ) 208.5 (4.87)
compound
0.1
1
5
10
1
nm; IR (KBr) νmax 3444, 1574, 1474 cm-1; H NMR (acetone-d6) δ
1
2
3
4
5
6
7
8
6
7
7
6
0
7
0
0
0
6
0
6
0
6
0
0
0
0
0
0
7
7
0
0
0
6
6
11
13
8
14
20
16
10
10
16
0
0
0
9
0
10
8
12
0
0
0
0
0
0
14
20
17
13
13
18
0
0
0
9
0
10
10
12
0
0
0
0
0
0
6.64 (1H, d, J ) 2.0 Hz, H-6′), 7.17 (1H, d, J ) 9.0 Hz, H-4), 7.39
(1H, d, J ) 2.0 Hz, H-4′), 7.45 (1H, d, J ) 9.0 Hz, H-3); 13C NMR,
see Table 1; EIMS m/z 513.7 (16), 515.7 (70), 517.7 (100), 519.7 (66),
521.7 (18); HRESIMS m/z 540.7031 [M + Na]+ (540.7009 calcd for
C12H679Br281Br2O3Na).
7
10
13
0
0
0
9
0
9
7
7
0
0
0
0
0
0
Compound 3: white solid; UV (MeOH) λmax (log ꢀ) 211 (4.83) nm;
1
IR (KBr) νmax 3444, 1698, 1487 cm-1; H NMR (acetone-d6) δ 6.53
(1H, d, J ) 2.5 Hz, H-6′), 6.80 (1H, d, J ) 8.5 Hz, H-3′), 6.97 (1H,
dd, J ) 8.5, 2.5 Hz, H-4′), 7.74 (1H, s, H-3); 13C NMR, see Table 1;
EIMS m/z 513.7 (16), 515.7 (70), 517.7 (100), 519.7 (66), 521.7 (18)
[M]+; HREIMS m/z 519.6970 (519.6988 calcd for C12H679Br81Br3O3).
9
10
11
12
13
14
15
16
19
20
21
22
23
24
25
26
27
28
29
Compound 4: white solid; UV (MeOH) λmax (log ꢀ) 211.5 (4.90)
1
nm; IR (KBr) νmax 3368, 1540, 1477 cm-1; H NMR (acetone-d6) δ
3.86 (3H, s, OMe-1), 6.77 (1H, d, J ) 2.0 Hz, H-6′), 7.43 (1H, d, J )
2.0 Hz, H-4′), 7.91 (1H, s, H-3); EIMS m/z 605.6 (10), 607.6 (51),
609.6 (100), 611.6 (98), 613.6 (48), 615.6 (10) [M]+; HREIMS m/z
609.6274 (609.6271 calcd for C13H779Br381Br2O3).
Methylation of 5. To a solution of 5 (1.2 mg) in MeOH (1.1 mL)
was added dropwise 10% TMSCHN2 in hexane. The solution was
allowed to stand at room temperature (15 min) and concentrated to
dryness under a stream of nitrogen to yield the methyl derivative 20:
12
10
0
0
0
13
13
8
16
7
14
16
13
18
13
14
11
1
white solid; H NMR (CDCl3) δ 3.86 (3H, s), 4.00 (3H, s), 6.97 (1H,
d, J ) 2.5 Hz), 7.50 (1H, d, J ) 2.5 Hz).
8
6
10
8
Partial Methylation of 14. Compound 14 (3.1 mg) was treated with
diluted TMSCHN2 solution, and the resulting mixture was separated
by HPLC (silica, hexane-CH2Cl2, 1:2) to give 5 (0.2 mg, 6%), 20
(0.4 mg, 12%), 21 (1.0 mg, 31%), and recovery of 14 (1.3 mg, 42%).
Compound 21: 1H NMR (acetone-d6) δ 3.84 (3H, s), 6.84 (1H, d, J )
2.0 Hz), 7.40 (1H, d, J ) 2.0 Hz).
Naturalis, National Museum of Natural History, The Netherlands (No.
RMNH Por 2653).
Extraction and Isolation. An air-dried sample of the sponge (1.8
kg) was extracted at room temperature with MeOH (4 × 2.5 L). The
MeOH extract was concentrated, and the residue was partitioned
between water and EtOAc. The organic extract (47.71 g) was further
partitioned between hexane and aqueous MeOH (50%) to afford a
hexane-soluble fraction (3.89 g). The aqueous MeOH layer was
extracted with CH2Cl2 to give a CH2Cl2 fraction (35.21 g). Both the
hexane and CH2Cl2 fractions showed strong activity against the Gram-
positive bacterium B. subtilis and weak toxicity against NBT-T2 cells.
Bioassay-guided fractionation of the hexane-soluble portion was carried
out by flash column chromatography over Si gel 60 using stepwise
gradient elution with hexane-EtOAc-MeOH to yield nine fractions.
The first fraction (0.75 g) was purified by silica HPLC (hexane-EtOAc)
to give 16 subfractions. Compound 7 (24.8 mg) was isolated from the
second subfraction by silica HPLC (hexane-CH2Cl2). The fifth
subfraction was similarly purified to give compound 8 (69.7 mg). The
last subfraction gave compound 9 (7.3 mg). Compound 1 (111.1 mg)
was obtained from the third fraction (0.46 g) by fractional crystallization
from hexane-acetone. The fourth fraction (0.39 g) was washed with
CH2Cl2 and then recrystallized with the same solvent to afford
compound 10 (5.2 mg). The fifth fraction (0.43 g), showing strong
activity against B. subtilis, was separated by reversed-phase HPLC
(RP18, MeOH) to give 10 subfractions. The second subfraction (4.5
mg) was recrystallized from hexane-CHCl3 to give compound 2 (4.2
mg) as a white solid. Repeated recrystallization of the fourth, fifth,
sixth, and seventh subfractions using the same solvent system afforded
compounds 4 (0.2 mg), 5 (17.0 mg), 11 (5.3 mg), and 12 (50.6 mg),
respectively. The mother liquor of the sixth fraction (0.62 g) was washed
with CH2Cl2, and the residue of the CH2Cl2 solution was recrystallized
from hexane-acetone to give compound 13 (10.7 mg). The eighth
fraction (0.34 g) was similarly recrystallized to afford 14 (69.4 mg).
The residue of the initial CH2Cl2 fraction (35.21 g) was also washed
with a small amount of CH2Cl2, and the residue was recrystallized from
hexane-acetone to afford 14 (12.57 g). Separation of the CH2Cl2-
soluble portion using ODS VFC gave three fractions. The fraction eluted
with 60% aqueous MeOH was purified by recrystallization and HPLC
(Si60, CH2Cl2) to afford compounds 3 (2.2 mg) and 6 (0.7 mg).
Compound 1: white solid; UV (MeOH) λmax (log ꢀ) 214 (4.94) nm;
Methylation of 1, 2, 3, and 6. Each of these samples was similarly
treated with TMSCHN2 as for 5 to give compounds 15, 16, 19, and
22, respectively. Compound 15: 1H NMR (CDCl3) δ 3.82 (3H, s), 3.99
(3H, s), 6.50 (1H, d, J ) 2.5 Hz), 7.40 (1H, d, J ) 2.5 Hz), 7.76 (1H,
s). Compound 16: 1H NMR (acetone-d6) δ 3.82 (3H, s), 4.01 (3H, s),
6.69 (1H, d, J ) 2.0 Hz), 7.49 (1H, d, J ) 2.0 Hz), 7.50 (1H, d, J )
9.0 Hz), 7.56 (1H, d, J ) 9.0 Hz). Compound 19: 1H NMR (acetone-
d6) δ 3.82 (3H, s), 3.92 (3H, s), 6.68 (1H, d, J ) 2.5 Hz), 6.96 (1H, d,
J ) 8.5 Hz), 7.09 (1H, dd, J ) 8.5, 2.5 Hz,), 7.94 (1H, s). Compound
22: 1H NMR (acetone-d6) δ 3.81 (3H, s), 3.93 (3H, s), 6.46 (1H, d, J
) 2.5 Hz), 6.85 (1H, d, J ) 8.5 Hz), 6.98 (1H, d, J ) 8.5, 2.5 Hz),
7.07 (1H, d, J ) 8.5 Hz), 7.40 (1H, d, J ) 8.5 Hz).
Treatment of 14 with HBr and Na2SO3 in MeOH.25 Hydrobromic
acid (47%, 3.0 mL) was added to a stirred solution of 14 (50.1 mg)
and sodium sulfite (93 mg, 10 equiv) in MeOH (10 mL). After the
solution was stirred under reflux for 1 h, it was then basified with
aqueous KOH to pH 10-11. The resulting mixture was extracted with
EtOAc. The organic layer was dried (Na2SO4) and concentrated to give
a residue. The residue was purified by HPLC (RP18, MeOH-H2O,
10:1) to give 23 (19.9 mg, 40%) and 14 (1.2 mg, 2%). Compound 23:
1H NMR (CD3OD) δ 7.28 (1H, d, J ) 2.0 Hz), 6.94 (1H, d, J ) 2.0
Hz); 13C NMR (CD3OD) δ 159.2, 148.8, 147.6, 145.0, 130.1, 124.9,
120.7, 120.4, 119.5, 113.1, 110.5, 108.4; EIMS m/z 651.5 (6), 653.5
(23), 655.5 (100), 657.5 (87), 659.5 (63), 661.5 (32), 663.5 (4) [M]+.
Debromination of 14 in AcOH. Hydrobromic acid (47%, 2.5 mL)
was added to a stirred solution of 14 (51 mg) and sodium sulfite (95
mg, 10 equiv) in AcOH (16 mL). The mixture was stirred under reflux
for 1 h, neutralized with aqueous KOH, and partitioned between EtOAc
and water. The organic layer was taken, and the product was purified
by HPLC (RP18, MeOH) to afford 13 (4.5 mg, 10%), 23 (12.5 mg,
25%), and 24 (0.6 mg, 1%). Compound 13: 1H NMR (acetone-d6) δ
6.73 (1H, d, J ) 2.0 Hz), 7.39 (1H, d, J ) 2.0 Hz), 7.62 (1H, s); 13C
NMR (acetone-d6) δ 151.0, 146.5, 144.8, 139.4, 129.7, 127.8, 123.4,
116.9, 116.5, 114.9, 111.5, 111.1; HMBC H-4/C-3,5,6, H-4′/2′,3′,5′,6′,
H-6′/C-1′,2′,4′,5′; EIMS m/z 591.6 (9), 593.6 (49), 595.6 (99), 597.6
(100), 599.6 (51), 601.6 (12) [M]+. Compound 24:12,15 1H NMR
(acetone-d6) δ 6.65 (1H, d, J ) 2.5 Hz), 7.24 (1H, d, J ) 2.5 Hz), 7.38
(1H, s).
1
IR (KBr) νmax 3350, 1620, 1475 cm-1; H NMR (acetone-d6) δ 4.01
(3H, s, OMe-2′), 6.81 (1H, d, J ) 2.5 Hz, H-6′), 7.48 (1H, d, J ) 2.5
Hz, H-4′), 7.65 (1H, s, H-4), 9.96 (1H, brs, OH-1); 13C NMR, see Table

Notes
Similar treatment of 14 (55 mg) with the above procedure using a
Journal of Natural Products, 2007, Vol. 70, No. 3 435
(2) Gunasekera, S. P.; McCarthy, P. J.; Kelly-Borges, M.; Lobkovsky,
E.; Clardy, J. J. Am. Chem. Soc. 1996, 118, 8759-8760.
(3) Sakai, R.; Kamiya, H.; Murata, M.; Shimamoto, K. J. Am. Chem.
Soc. 1997, 119, 4112-4116.
(4) Molinski, T. F.; Ireland, C. M. J. Org. Chem. 1988, 53, 2103-2105.
(5) Faulkner, D. J.; Unson, M. D.; Bewley, C. A. Pure Appl. Chem.
1994, 66, 1983-1990.
(6) Unson, M. D.; Faulkner, D. J. Experientia 1993, 49, 349-353.
(7) Unson, M. D.; Holland, N. D.; Faulkner, D. J. Mar. Biol. 1994, 119,
1-11.
(8) Sharma, G. M.; Vig, B. Tetrahedron Lett. 1972, 17, 1715-1718.
(9) Salva, J.; Faulkner, D. J. J. Nat. Prod. 1990, 53, 757-760.
(10) Handayani, D.; Edrada, R. A.; Proksch, P.; Wray, V.; Witte, L.; van
Soest, R. W. M.; Kunzmann, A.; Soedarsono. J. Nat. Prod. 1997,
60, 1313-1316.
(11) Sionov, E.; Roth, D.; Losica, H. S.; Kashman, Y.; Rudi, A.; Chill,
L.; Berdicevsky, I.; Segal, E. J. J. Infect. 2005, 50, 453-460.
(12) Hattori, T.; Konno, A.; Adachi, K.; Shizuri, Y. Fish. Sci. 2001, 67,
899-903.
(13) Kuniyoshi, M.; Yamada, K.; Higa, T. Experientia 1985, 41, 523-
524.
(14) Fu, X.; Schmitz, F. J.; Govindan, M.; Abbas, S. A.; Hanson, K. M.;
Horton, P. A.; Crews, P.; Laney, M.; Schatzman, R. C. J. Nat. Prod.
1995, 58, 1384-1391.
(15) Liu, H.; Namikoshi, M.; Meguro, S.; Nagai, H.; Kobayashi, H.; Yao,
X. J. Nat. Prod. 2004, 67, 472-474.
larger amount of sodium sulfite (15 equiv) and extended reflux (6 h)
resulted in the formation of 13 (10.5 mg, 21%), 25 (0.1 mg, 0.2%),
and 26 (1.0 mg, 2%) and recovery of unreacted 14 (1.2 mg, 2%).
Compound 25: 1H NMR (acetone-d6) δ 6.45 (1H, t, J ) 8.0 Hz), 6.74
(1H, s), 7.18 (1H, dd, J ) 8.0, 2.0 Hz), 7.66 (1H, dd, J ) 8.0, 2.0 Hz);
EIMS m/z 513.7 (18), 515.7 (64), 517.7 (100), 519.7 (68), 521.7 (16)
[M]+. 26: 1H NMR (acetone-d6) δ 6.64 (1H, d, J ) 2.5 Hz), 7.24 (1H,
d, J ) 2.5 Hz), 7.37 (1H, d, J ) 2.5 Hz), 7.38 (1H, d, J ) 2.5 Hz);
EIMS m/z 513.7 (17), 515.7 (69), 517.7 (100), 519.7 (66), 521.7 (16)
[M]+.
Further treatment of 14 (60 mg) using the above procedure with
larger amounts of hydrobromic acid (10 mL) and sodium sulfite (20
equiv) gave compounds 27 (2.4 mg, 6%), 28 (0.2 mg, 0.6%), and 29
(2.2 mg, 6%). Compound 27: 1H NMR (acetone-d6) δ 6.67 (1H, d, J
) 2.0 Hz), 6.75 (1H, d, J ) 8.5 Hz), 6.93 (1H, dd, J ) 2.0, 8.5 Hz),
7.03 (1H, d, J ) 2.0 Hz), 7.12 (1H, d, J ) 2.0 Hz); EIMS m/z 435.8
(34), 437.8 (100), 439.8 (100), 441.8 (34) [M]+. 28: 1H NMR (CD3-
OD) δ 6.48 (1H, dd, J ) 8.0, 2.0 Hz), 6.62 (1H, td, J ) 8.0, 2.0 Hz),
6.87 (2H, m), 7.05 (1H, d, J ) 2.5 Hz), 7.22 (1H, d, J ) 2.5 Hz);
EIMS m/z 357.8 (51), 359.8 (100), 361.8 (51) [M]+. 29: 1H NMR
(CD3OD) δ 6.74 (1H, d, J ) 8.5 Hz), 6.79 (1H, d, J ) 2.0 Hz), 6.80
(1H, d, J ) 2.0 Hz), 6.97 (1H, dd, J ) 2.0, 8.5 Hz), 7.07 (1H, d, J )
2.0 Hz); EIMS m/z 435.8 (31), 437.8 (100), 439.8 (98), 441.8 (36)
[M]+.
Cytotoxicity Assay. Compounds 2, 3, 5, 6, 13, 14, and 23-29 were
evaluated for their cytotoxicity against NBT-T2 rat bladder epithelial
cells as described previously.26 IC50 values were obtained by using the
MTT method.
(16) Xu, Y.; Johnson, R. K.; Hecht, S. M. Bioorg. Med. Chem. 2005, 13,
657-659.
(17) Issa, H. H.; Tanaka, J.; Rachmat, R.; Higa, T. Tetrahedron Lett. 2003,
44, 1243-1245.
Agar-Plate Diffusion Assay. Paper disks were impregnated with
isolated compounds ranging from 0.1 to 10 µg/disk and placed on agar
plates inoculated with B. subtilis. The plates were checked for inhibition
zones after incubation at 37 °C for 24 h. Prior to and after the testing,
all materials were sterilized at 121 °C for 20 min. Acetone was used
to dissolve the compounds.
(18) Hect, S.; Deng, J.-Z.; Dedkova, L. U.S. Patent 2006235047.
(19) Segraves, E. N.; Shah, R. R.; Segraves, N. L.; Johnson, T. A.;
Whitman, S.; Sui, J. K.; Kenyon, V. A.; Cichewicz, R. H.; Crews,
P.; Holman, T. R. J. Med. Chem. 2004, 47, 4060-4065.
(20) Cameron, G. M.; Stapleton, B. L.; Simonsen, S. M.; Brecknell, D.
J.; Garson, M. J. Tetrahedron 2000, 56, 5247-5252.
(21) Carte, B.; Faulkner, D. J. Tetrahedron 1981, 37, 2335-2339.
(22) Norton, R. S.; Croft, K. D.; Wells, R. J. Tetrahedron 1981, 37, 2341-
2349.
(23) Agrawal, M. S.; Bowden, B. F. Mar. Drugs 2005, 3, 9-14.
(24) Higa, T. In Marine Natural Products: Chemical and Biological
PerspectiVes; Scheuer, P. J., Ed.; Academic Press: New York, 1981;
Vol. IV, p 119.
Acknowledgment. This work was supported by Grant-in-Aids
(15406005, 18032061) for Scientific Research from the Ministry of
Education, Culture, Sports, Science and Technology of Japan. We thank
Dr. E. R. O. Siwu and Prof. D. Uemura, Nagoya University, for
measuring the ESIMS and Dr. H. Naoki and Mr. T. Yoshida, Okinawa
Health Biotechnology Research Development Center, for using a
microplate reader.
(25) Choi, H. Y.; Chi, D. Y. J. Am. Chem. Soc. 2001, 123, 9202-9203.
(26) Tanaka, C.; Yamamoto, Y.; Otsuka, M.; Tanaka, J.; Ichiba, T.;
Marriott, G.; Rachmat, R.; Higa, T. J. Nat. Prod. 2004, 67,
1368-1373.
References and Notes
(1) Kobayashi, M.; Aoki, S.; Ohyabu, N.; Kurosu, M.; Wang, W.;
Kitagawa, I. Tetrahedron Lett. 1994, 35, 7969-7972.
NP0605081