Isolation and Characterization of Polybrominated Diphenyl Ethers as Inhibitors of Microtubule Assembly from the Marine Sponge Phyllospongia dendyi Collected at Palau

Article abstract of DOI:10.1021/np0304621

Two new polybrominated diphenyl ethers (1 and 2) were isolated by bioassay-guided separations together with nine known compounds (3-11) from the marine sponge Phyllospongia dendyi collected from Palau. The structures were assigned on the basis of their sp

Full text of DOI:10.1021/np0304621

472
J . Nat. Prod. 2004, 67, 472-474
Isola tion a n d Ch a r a cter iza tion of P olybr om in a ted Dip h en yl Eth er s a s
In h ibitor s of Micr otu bu le Assem bly fr om th e Ma r in e Sp on ge P h yllospon gia
d en d yi Collected a t P a la u
Hongwei Liu,†,‡ Michio Namikoshi,*,† Shiori Meguro,† Hiroshi Nagai,† Hisayoshi Kobayashi,§ and Xinsheng Yao‡
Department of Ocean Sciences, Tokyo University of Marine Science and Technology, Minato-ku, Tokyo 108-8477, J apan,
Department of Natural Products, Shenyang Pharmaceutical University, Wenhua Road 103,
Shenyang 110016, People’s Republic of China, and Institute of Molecular and Cellular Biosciences, The University of Tokyo,
Bunkyo-ku, Tokyo 113-0032, J apan
Received October 22, 2003
Two new polybrominated diphenyl ethers (1 and 2) were isolated by bioassay-guided separations together
with nine known compounds (3-11) from the marine sponge Phyllospongia dendyi collected from Palau.
The structures were assigned on the basis of their spectral data. Compounds 3, 7, and 10 showed inhibitory
activities to the assembly of microtubule proteins (IC50: 29.6, 33.5, and 20.9 µM, respectively) and to the
meiotic maturation of starfish oocytes (IC50: 3.6, 4.2, and 4.2 µM, respectively), while 1, 2, 4-6, 8, 9, and
11 were not active at 100 µM. Two phenolic hydroxyls are required for their bioactivities.
The microtubule system of eukaryotic cells is an impor-
tant target for the development of anticancer and antifun-
gal agents.1,2 A variety of compounds, such as vinca
alkaloids, colchicine, podophyllotoxin, paclitaxel, and
epothilone, act on microtubules to disrupt the cellular
microtubule structure and mitotic cell division.3,4 We have
recently reported a screening method for antimitotic sub-
stances, which detects the inhibition and stimulation of the
assembly and inhibition of the disassembly of microtubules
in the sequence utilizing purified porcine brain microtubule
proteins.5
During our continuous study on biologically active
metabolites from the sea, we reported the isolation of seven
new meroditerpenoids from the marine sponge Strongylo-
phora strongylata by a screening bioassay using starfish
oocytes.6 This screening method detects the inhibitory
activity on maturation of oocytes of Asterina pectinifera
stimulated by 1-methyladenine (1-MeAde). Immature oo-
Compound 1 was obtained as a viscous oil. ESIMS of 1
cytes are arrested at G2/M phase, and 1-MeAde, the
showed the presence of six Br atoms, and the molecular
maturation-inducing hormone, induces the germinal vesicle
formula C13H6Br6O3 was determined from the FABMS and
breakdown in activating the maturation-promoting factor
NMR spectral data. The 1H NMR spectrum of 1 showed
(MPF). We found that the ethanol extract of the marine
signals due to meta-coupled aromatic protons at δ 6.54 and
sponge Phyllospongia dendyi inhibited the maturation of
7.37, a methoxy methyl at δ 3.80, and a hydroxyl proton
oocytes in this bioassay. The extract also showed inhibitory
1
at δ 5.91. H and 13C NMR data for 1 (Table 1) resembled
activity to the assembly of microtubule proteins. Two
those for 3, except a methoxy methyl signal of 1. Com-
bioassays were conducted to find cell-cycle inhibitors.
pounds 1 and 3 gave the same dimethoxy compound 12.
Bioassay-guided separation yielded two new (1 and 2) and
Therefore, 1 was assigned as a monomethyl ether deriva-
nine known (3-11) polybrominated diphenyl ethers. Com-
tive of 3. The 13C signals due to C-1-C-6 were assigned by
pound 9 and four other polybrominated diphenyl ethers
HMQC and HMBC experiments. A correlation was detected
have been reported from the same sponge species.7 We
from OMe (δH 3.80) to the 13C signal at δC 151.0 in the
describe here the isolation, structure characterization, and
HMBC spectrum of 1, which was ascribed to the C-2′
bioactivities of these compounds.
position by comparing the chemical shift with those of 3
P. dendyi was extracted with EtOH, and the extract was
and 12. Consequently, the structure of 1 was assigned as
partitioned between water and EtOAc. The EtOAc extract
2-(3′,4′,5′,6′-tetrabromo-2′-methoxyphenoxy)-4,6-dibromophe-
was subjected to bioassay-guided separation using starfish
nol.
oocytes to give 11 polybrominated diphenyl ethers. The
Compound 2 was obtained as a viscous oil. The presence
structures of known compounds 3-11 were determined on
of five Br atoms was suggested by the ESIMS of 2. The
the basis of their spectral data, which were also compared
molecular formula C13H7Br5O3 was deduced from the
with those of the reported values.7-10
FABMS and NMR spectral data. The 1H NMR spectrum
of 2 showed an aromatic proton singlet at δ 7.28, two meta-
coupled aromatic proton doublets at δ 6.51 and 7.34, a
methoxy methyl singlet at δ 3.78, and a hydroxyl proton
singnal at δ 6.06. 1H and 13C NMR data for 2 (Table 1)
* To whom correspondence should be addressed. Tel: +81-3-5463-0451.
Fax: +81-3-5463-0398. E-mail: namikosh@s.kaiyodai.ac.jp.
† Tokyo University of Marine Science and Technology.
‡ Shenyang Pharmaceutical University.
§ The University of Tokyo.
10.1021/np0304621 CCC: $27.50
© 2004 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/21/2004

Notes
J ournal of Natural Products, 2004, Vol. 67, No. 3 473
Ta ble 1. 13C (125 MHz) and 1H (500 MHz) NMR Data for
Compounds 1 and 2 in CDCl3
1.16 The sponge was identified by Professor P. Bergquist
(University of Auckland, New Zealand).16
a
Isola tion of Com p ou n d s 1-11. The sponge (284 g) was
thawed, cut into small pieces, and extracted with ethanol
(2 × 1000 mL). The ethanol extract was evaporated, and the
residue was partitioned between water and EtOAc. The
organic extract (5.0 g) was chromatographed on SiO2 with
CHCl3-MeOH (gradient elution), and the bioactivity was
detected in CHCl3 (300 mg) and 1% MeOH/CHCl3 (1.0 g)
fractions. The CHCl3 fraction (300 mg) was further separated
by an ODS column with MeOH to afford 200 mg of bioactive
fraction, which was subjected to HPLC (SiO2, n-hexane-ethyl
acetate) to afford 1 (1.6 mg), 2 (5.7 mg), 4 (18.0 mg), 5 (3.2
mg), 6 (1.0 mg), 8 (13.0 mg), 9 (102.0 mg), 11 (5.6 mg), and a
mixture of 3 and 7. The 1% MeOH/CHCl3 fraction (0.5 g) was
separated by HPLC (ODS) with 87% MeOH-H2O (0.1% TFA)
to yield 3 (118.7 mg), 7 (33.3 mg), and 10 (1.7 mg).
compound 1
compound 2
C#
13C
1H
HMBC
13C
1H
HMBC
1
2
3
142.8
144.5
142.7
144.7
116.8 6.54 1H, d, 1, 2, 4,
116.3 6.51 1H, d, 1, 2, 4,
J ) 2.1 Hz
5
J ) 2.1 Hz
5
4
5
111.9
111.5
129.3 7.34 1H, d, 1, 3, 4,
J ) 2.1 Hz
129.5 7.37 1H, d, 1, 3, 4,
J ) 2.1 Hz
6
6
6
110.7
145.4
151.0
110.1
140.8
152.0
1′
2′
3′
116.6 7.28 1H, s 1′, 2′, 4′,
5′
119.1
121.3
4′
5′
6′
122.3
124.5
126.8
}
122.8
122.4
}
Com p ou n d 1: UV (EtOH) λmax (ꢀ) 232 (22 600), 292 (5000)
nm; IR (KBr) νmax 3434, 2924, 2854, 1584, 1477, 1445, 1406,
1390, 1344, 1264, 1206, 997, 963, 880, 835 cm-1; 1H (500 MHz,
CDCl3) and 13C (125 MHz, CDCl3) NMR data, Table 1; ESIMS
m/z 684, 686, 688, 690, 692, 694, and 696 (1:2:4:8:4:2:1, [M]+);
HRFABMS m/z 685.5380 (calcd for C13H679Br581BrO3, 685.5397),
687.5327 (calcd for C13H679Br481Br2O3, 687.5377), 689.5277
(calcd for C13H679Br381Br3O3, 689.5357), 691.5228 (calcd for
1-OH
2′-OMe
5.91 1H, s
61.6 3.80 3H, s 2′
6.06 1H, s
56.7 3.78 3H, s 2′
Signals were assigned by 1H-1H COSY, HMQC, and HMBC
a
experiments.
were very similar to those for 4. All 13C signals, except C-4′
and C-5′, were definitely assigned by HMQC and HMBC
experiments. A correlation from OMe (δH 3.78) to C-2′ (δC
152.0) was observed in the HMBC spectrum of 2. Moreover,
an NOE was detected between OMe and H-3′ (δ 7.28) in
the NOESY spectrum of 2. Thus, the structure of 2 was
deduced as 2-(4′,5′,6′-tribromo-2′-methoxyphenoxy)-4,6-di-
bromophenol.
Compounds 1-11 were tested for their inhibitory activi-
ties to the assembly of purified porcine brain microtubule
proteins5 and to the meiotic maturation of starfish oocytes
stimulated by 1-MeAde.6 Compounds 3, 7, and 10 inhibited
the assembly of microtubule proteins at 29.6, 33.5, and 20.9
µM (IC50), respectively (colchicine ) 10.0 µM). These
compounds also inhibited the maturation of oocytes at 3.6,
4.2, and 4.2 µM (IC50), respectively. Compounds 1, 2, 4-6,
8, 9, and 11 did not show inhibitory activity in either
bioassay at 100 µM. Two phenolic hydroxyls are, therefore,
necessary for their bioactivities. Substitution patterns of
Br may less affect the activity.
A few bioactivities have been reported for polybromi-
nated diphenyl ethers, such as antibacterial and antifungal
activities,11-13 antimicroalgal activity against Prorocentrum
micans and Brachiomonas submaria,7 toxicity to brine
shrimp,14 antiinflammatory activity,15 and inhibitory activ-
ity to several enzymes including inosine monophosphate
dehydrogenase, guanosine monophosphate synthetase, and
15-lipoxygenase.10 This is, therefore, the first to report
inhibitory activities of polybrominated diphenyl ethers to
the assembly of microtubule proteins and to the maturation
of starfish oocytes.
C
13H679Br281Br4O3, 691.5337), 693.5294 (calcd for C13H679Br-
81Br5O3, 693.5318).
Com p ou n d 2: UV (EtOH) λmax (ꢀ) 232 (25 000), 294 (6100)
nm; IR (KBr) νmax 3503, 3085, 2924, 1596, 1573, 1476, 1429,
1405, 1350, 1287, 1263, 1246, 1033, 942, 890, 835 cm-1 1H
;
(500 MHz, CDCl3) and 13C (125 MHz, CDCl3) NMR data, Table
1; ESIMS m/z 606, 608, 610, 612, 614, and 616 (1:4:8:8:4:1,
[M]+); HRFABMS m/z 607.6285 (calcd for C13H779Br481BrO3,
607.6292), 609.6259 (calcd for C13H779Br381Br2O3, 609.6272),
611.6224 (calcd for C13H779Br281Br3O3, 611.6252), 613.6219
(calcd for C13H779Br81Br4O3, 613.6233).
Bioa ssa y. The microtubule assembly assay was performed
as reported.5,17 In brief, fresh porcine brains were homogenized
at 0 °C in a buffer solution (100 mM 4-morphorineethansul-
fonic acid, 1 mM EGTA, 0.5 mM MgCl2, 1 mM GTP, and 1
mM 2-mercaptoethanol, pH 6.5) and centrifuged at 50000g at
4 °C. A glycerol buffer (8 M glycerol in the above buffer
solution, pH 6.5) was added to the supernatant, and the
mixture was incubated at 37 °C for 30 min and centrifuged at
100000g to afford the precipitate (microtubule). The depolym-
erization and polymerization procedure was further performed
twice to purify the microtubule proteins. The concentration of
proteins was quantified using the Coomassie Protein Assay
Kit (Pierce). Polymerization was observed by the turbidity at
400 nm in a glass UV cell at 37 °C with a Shimadzu model
U-3000 spectrophotometer equipped with an electronic tem-
perature controller.5 Each sample was dissolved in DMSO and
added to the suspension of microtubule proteins in the buffer
solution (1.3 mg in 1 mL). The final concentration of DMSO
was less than 2%.
Effects of compounds on meiotic maturation of starfish
oocytes were measured according to the previous paper6 and
earlier report.18 Starfish, Asterina pectinifera, were collected
from the coastal waters off J apan during the breeding season.
Immature oocytes were obtained from the starfish ovaries and
adjusted to 100 cells/mL. This oocyte suspension (1 mL) and
10 µL of each test sample were added into a 24-well plate and
placed on ice for 10 min. After addition of 10 µL of 1-MeAde
(1.25 µM), the plate was incubated at 20 °C for 60 min, and
the states of germinal vesicles were observed under an inverted
microscope. DMSO was used to dissolve the samples. The final
concentration of DMSO was adjusted to less than 1%.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. UV and IR spectra
were recorded on a Hitachi U-3000 and on a J ASCO A-102,
respectively. NMR spectra were measured on either a J EOL
J NM A-500 NMR spectrometer or a Varian Unity Inova-500
spectrometer. Mass spectra were obtained by either a J EOL
HX-110 mass spectrometer (FAB mode, m-nitrobenzyl alcohol
as matrix) or a Finnigan TSQ 700 triple quadrupole mass
spectrometer (ESI mode).
Ma r in e Sp on ge. Phyllospongia dendyi was collected by
scuba diving in J uly 2000 at Palau and kept in a freezer at
-30 °C until extraction. The voucher specimen is deposited
at the Department of Ocean Sciences, Tokyo University of
Marine Science and Technology, as TUF number 00-07-11)2-
P r ep a r a tion of Meth yl Eth er s. A solution of compound
(0.01 mmol), MeI (1 mL), and anhydrous K2CO3 (500 mg) in
dry acetone (10 mL) was refluxed for 12 h. The residue, after
evaporation, was partitioned between water (10 mL) and
chloroform (3 × 10 mL), and the organic extract was dried (Na2-
SO4) and evaporated to give the corresponding methyl ether.

474 J ournal of Natural Products, 2004, Vol. 67, No. 3
Notes
(9) Norton, R. S.; Croft, K. D.; Wells, R. J . Tetrahedron 1981, 37, 2341-
2349.
(10) 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.
(11) Sharma, G. M.; Vig, B. Tetrahedron Lett. 1972, 17, 1715-1718.
(12) Salva, J .; Faulkner, D. J . J . Nat. Prod. 1990, 53, 757-760.
(13) Kurata, K.; Amiya, T. Phytochemisty 1980, 19, 141-142.
(14) 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.
(15) Wiemer, D. F.; Idler, D. D.; Fencial, W. Experientia 1991, 47, 851-
853.
(16) The voucher specimen of the same sponge is also kept in the Marine
Biotechnology Institute, J apan, as 00P-010, which was identified by
Professor P. Bergquist as Phyllospongia dendyi.
(17) Takahashi, M.; Iwasaki, S.; Kobayashi, H.; Okuda, S.; Murai, T.; Sato,
Y.; Haraguchi-Hiraoka, T.; Nagano, H. J . Antibiot. 1987, 40, 66-72.
(18) Toraya, T.; Maoka, T.; Tsuji, H.; Kobayashi, M. Appl. Environ.
Microbiol. 1995, 61, 1799-1804.
Ack n ow led gm en t. This work was supported in part by a
grant from the Naito Foundation. We thank Varian Technolo-
gies J apan, Ltd. for the measurement of a few NMR spectra
and M. Endo and Y. Shizuri of the Marine Biotechnology
Institute, J apan, for the sample collection at Palau and for
the information on the identification of the sponge.
Refer en ces a n d Notes
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NP0304621