New sesquiterpene hydroquinones from a Taiwanese marine sponge Polyfibrospongia australis

Article abstract of DOI:10.1021/np9605302

Two new sesquiterpene hydroquinones, polyfibrospongols A (1) and B (2), were isolated from the Taiwanese marine sponge Polyfibrospongia australis Lendenfeld (Spongiidae) in addition to the known metabolites, dictyoceratin A (3), ilimaquinone (4), and 5-epi-ilimaquinone (9). The structures of these compounds have been determined mainly on the basis of spectral and chemical methods. Biological testing revealed that compounds 1 and 2 exhibited significant cytotoxicity against human KB-16, A-549, and murine P-388 tumor cell lines.

Full text of DOI:10.1021/np9605302

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J . Nat. Prod. 1997, 60, 93-97
93
New Sesqu iter p en e Hyd r oqu in on es fr om a Ta iw a n ese Ma r in e Sp on ge
P olyfibr ospon gia a u str a lis
Ya-Ching Shen* and Pei-Wen Hsieh
Institute of Marine Resources, National Sun Yat-sen University, 70 Lien-Hai Rd.,
Kaohsiung, Taiwan 80424, Republic of China
Received J une 24, 1996X
Two new sesquiterpene hydroquinones, polyfibrospongols A (1) and B (2), were isolated from
the Taiwanese marine sponge Polyfibrospongia australis Lendenfeld (Spongiidae) in addition
to the known metabolites, dictyoceratin A (3), ilimaquinone (4), and 5-epi-ilimaquinone (9).
The structures of these compounds have been determined mainly on the basis of spectral and
chemical methods. Biological testing revealed that compounds 1 and 2 exhibited significant
cytotoxicity against human KB-16, A-549, and murine P-388 tumor cell lines.
Sesquiterpene quinones and hydroquinones represent
a group of still expanding C15-C6 metabolites with
notable medicinal applications such as antitumor, an-
tibacterial, and anti-HIV activities.1-4 Of particular
interest are two antimitotic agents called avarol and
avarone, which were recently shown to inhibit HTLV-
III replication in human H9 cells as measured by
determination of reverse transcriptase.5 This result
suggested that sesquiterpene quinones and hydroquino-
nes may be useful in the treatment of AIDS. Due to
their potential antitumor and anti-HIV activities as well
as the novelty of their structures, the exploration of
natural sesquiterpene quinones has continued to grow
in the past decade. Several bioactive metabolites such
as ilimaquinone (4), peyssonols, and hyatellaquinone
from various species of sponges and algae have been
recently isolated and characterized.6-10 A comprehen-
sive review dealing with the chemistry and biological
properties of sesquiterpene-shikimate derivatives has
been published.11
In a program searching for bioactive substances from
organisms of coral reefs, we have focused on Taiwanese
marine sponges.12,13 An EtOH extract of the sponge
Polyfibrospongia australis Lendenfeld (Spongiidae), col-
lected at Nan-wan (at a depth of 25 m), exhibited
significant inhibition against human KB-16 (5.2 µg/mL)
and murine P-388 (2.3 µg/mL) tumor cells. After
extensive fractionation by chromatography, a series of
sesquiterpene quinones, including ilimaquinone (4),
5-epi-ilimaquinone (9), and dictyoceratin A (3), and two
new compounds, which we have named polyfibrospon-
gols A (1) and B (2), were isolated. In this paper we
report the isolation, structure elucidation, and anti-
tumor cytotoxicity of these new sesquiterpene hydro-
quinones.
pounds 4 and 9 (95 mg), dictyoceratin A (3, 6 mg), and
polyfibrospongol B (2, 5 mg, 0.002% wet wt). Chro-
matographic fractionation of an EtOH extract from an
unidentified sponge (SP 137) furnished ilimaquinone (4).
Ilimaquinone (4), 5-epi-ilimaquinone (9), and dictyo-
ceratin A (3) have been previously isolated from Hip-
pospongia sp. and other sponges.6,8 The structures of
compounds 3, 4, and 9 were identified by comparison
1
of their EIMS, IR, [R], H-, and 13C-NMR spectral data
with those of the previously reported compounds.6 The
structure of compound 3 was also confirmed by prepara-
tion of its diacetate (7), which had spectroscopic data
identical to those of dictyoceratin A diacetate.6
Polyfibrospongol A (1), [R] +5.3° (CHCl3), was isolated
as an amorphous solid. The molecular formula C24H34O4
was deduced from its molecular ion at m/ z 386.2447 in
1
its HREIMS and was confirmed by H, 13C, and DEPT
NMR. Its UV and IR bands indicated the presence of
phenyl (227, 269 nm, 1602 cm-1), hydroxyl (3416 cm-1),
and ester (1714 cm-1) groups. This finding was also
supported by fragment ions at m/ z 355 [M - OMe]+
and m/ z 196 (ArCH2•+) in the EIMS spectrum of 1. The
presence of a rearranged drimane skeleton was inferred
from the observations of a significant fragment ion at
• +
m/ z 191 (M - ArCH2 ) and characteristic resonances,
such as two methyl singlets (δ 1.06, 0.88), a methyl
doublet (δ 1.03), and a pair of exocyclic methylene
signals (δ 4.41, 4.36) as well as corresponding carbon
signals (Table 1).6 A carbomethoxy moiety was also
observed at δ 3.87, 51.9, and 167.2. The assignment of
each aliphatic proton in the drimane moiety of 1 was
determined by a COSY experiment. In addition, a
correlation via meta-coupling was observed between
aromatic protons at δ 7.46 and 7.38, indicating the
presence of a 1,2,3,5-tetrasubstituted benzene moiety.
A comparison with literature data indicated that poly-
fibrospongol A (1) exhibited a molecular weight 14 mass
Resu lts a n d Discu ssion
The CHCl3-soluble fraction from the alcoholic extract
of the sponge P. australis was chromatographed on a
Si gel column using eluents of increasing polarity of
CHCl3 and MeOH to yield a mixture of ilimaquinone
and 5-epi-ilimaquinone (4 and 9, 125 mg) and polyfi-
brospongol A (1, 7 mg, 0.003% wet wt). Further
separation by LC and preparative TLC on Si gel (CHCl3/
Me2CO, 10:1) yielded an additional mixture of com-
1
units greater than that of dictyoceratin A (3).6 The H-
and 13C-NMR spectra of 1 were similar to those of
dictyoceratin A except that an additional methoxyl
singlet was observed at δ 3.93 in 1. On acetylation
compound 1 yielded a monoacetate (5), which showed a
molecular ion at m/ z 428 and characteristic fragment
ions at m/ z 191 (M - ArCH•)+ and m/ z 238 (ArCH2•+).
1
Besides, an aromatic acetyl singlet (δ 2.36) in the H-
X Abstract published in Advance ACS Abstracts, J anuary 15, 1997.
NMR spectrum of 5 suggested that the hydroxy might
S0163-3864(96)00530-7 CCC: $14.00
© 1997 American Chemical Society and American Society of Pharmacognogy

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94 J ournal of Natural Products, 1997, Vol. 60, No. 2
Ta ble 1. 1H- and 13C-NMR Data for Polyfibrospongol A (1)
Shen et al.
Ta ble 2. 1H- and 13C-NMR Data for Polyfibrospongol B (2)
position
1Ha
COSY
13Cb
position
1Ha
COSY
13Cb
1a
1b
2a
2b
3a
3b
4
5
6
7
8
2.10 m
1.60 m
1.92 m
1.40 m
2.34 (dd, 14.4, 5.4)
2.10 m
H-2a, 1b, 2b, 10
H-2a, 1a, 2b, 10
H-3a, 2b
H-3b, 2a, 1a
H-3b, 2a, 2b, 11
H-3a, 2a
23.1 (t)
1a
1b
2a
2b
3a
3b
4
5
6
7
8
2.10 m
H-1b, 2b
H-1a, 2b, 10
H-3a, 2b
H-3a, 2a, 1a
H-3b, 2a, 2b,
H-3a
24.1 (t)
1.40 m
1.88 m
1.30 m
2.30 m
2.08 m
27.9 (t)
33.1 (t)
28.3 (t)
33.1 (t)
160.3 (s)
40.2 (s)
36.6 (t)
27.7 (t)
36.4 (d)
42.1 (s)
48.0 (d)
102.6 (t)
159.8 (s)
40.1 (s)
36.9 (t)
28.0 (t)
36.7 (d)
46.6 (s)
49.5 (d)
103.2 (t)
1.20-1.50 m
1.20-1.50 m
1.30 m
H-7
H-6, 8
H-13
1.20-1.50 m
1.30-1.50 m
1.38 m
H-7
H-6, 8
H-13, 7
9
10
11
9
10
11
0.95 (dd, 1.8, 11.7)
4.41 (s)
H-1a, 1b
H-3a
1.10 m
4.44 (s)
H-1b
4.36 (s)
4.40 (s)
12
13
14
15
16
17
18
19
20
21
OMe
COOMe
1.06 (s)
1.03 (d, 6.3)
0.88 (s)
20.6 (q)
17.6 (q)
17.7 (q)
36.8 (t)
124.4 (s)
149.2 (s)
145.8 (s)
109.0 (d)
120.4 (s)
127.5 (d)
56.1 (q)
167.2 (s)
51.9 (q)
12
13
1.06 (s)
20.9 (q)
19.0 (q)
64.5 (q)
H-8
1.11 (d, 6.3)
3.81 (d, 11.6)
3.92 (d, 11.6)
3.09 (d, 14.4)
2.85 (d, 14.4)
H-8
14a
14b
15a
15b
16
17
18
19
20
H-14b
H-14a
H-15b
H-15a
2.68 (s)
37.3 (t)
124.3 (s)
149.2 (s)
146.0 (s)
109.3 (d)
120.6 (s)
127.8 (d)
56.1 (q)
7.38 (d, 2.1)
H-21
H-19
7.46 (d, 2.1)
3.93 (s)
3.87 (s)
7.40 (d, 2.1)
H-21
H-19
21
OMe
COOMe
7.50 (d, 2.1)
3.94 (s)
3.88 (s)
167.1 (s)
51.2 (q)
a
Multiplicities and coupling constants in Hz are in parentheses.
b s ) C, d ) CH, t ) CH2, q ) CH3. Multiplicities and assignments
were made by DEPT.
a
Multiplicities and coupling constants in Hz are in parentheses.
b s ) C, d ) CH, t ) CH2, q ) CH3. Multiplicities and assignments
are made by DEPT.
The presence of enhancements among C12-Me, C13-Me,
and C14-Me agreed with a cis relationship among these
methyl protons. The absence of a NOE between H-10
and the methyl protons of C-12 and C-14 suggested that
H-10 was trans to C-12 and C-14. The intensity of H-10
and H-21 was increased (2.2%, 2.7%) as the C-15
methylene singlet (δ 2.56, 5) was irradiated, confirming
a cis-disposition for H-10 and H-15, and a close relation-
ship for H-15 and H-21 in space. A proposed model
consistent with the results of NOE experiments for 1 is
shown in Figure 1.
F igu r e 1. NOE studies of polyfibrospongol A (1, RdH) and
polyfibrospongol A monoacetate (5, RdAc).
Polyfibrospongol B (2), [R] +1.8° (CHCl3), had the
composition C24H34O5 as derived from a molecular ion
at m/ z 402.2408 (calcd 402.2406) in the HREIMS of 2,
and also from FABMS (m/ z 425, [M + Na]+, m/ z 403
[M + H]+), and DEPT NMR. Its UV and IR spectra
indicated the presence of hydroxyl (3420 cm-1), ester
be located at the C-17 or C-18 position. Detailed
comparison of the resonances of H-19 and H-21 in the
1H-NMR spectrum of 1 and 5 with those of 3 and 7
suggested that the location of the hydroxyl group should
be at C-17. This is because the chemical shifts of H-19
and H-21 in 5 remain unchanged after acetylation of 1.
The position of the acetyl group in 5 and the corre-
sponding hydroxyl group in 1 should be located at the
meta-direction of H-19 and H-21 of 5 and 1, respectively.
This was confirmed by an NOE study of 1. Irradiation
of the methoxy singlet at δ 3.93 causes enhancement of
the signal of H-19 (2.9%), and vice versa (3.4%), con-
firming the hydroxy at C-17 in 1. The stereochemistry
of 1 was assigned by observation of NOE experiments
and detailed comparison of the observed chemical shifts,
coupling constants, and specific rotation with those of
dictyoceratin A (3).
(1710 cm-1), and phenolic (227, 270 nm, 1645 cm-1
)
groups. This was also supported by fragment ions at
• +
m/ z 371 [M - OMe]+, m/ z 207 (M - ArCH2 ) , and a
base peak at m/ z 196 (ArCH2•+) in the EIMS spectrum.
The presence of a carbomethoxy and two meta-coupling
aromatic protons was verified by the observation of 1H-
and 13C-NMR spectral data (Table 2). As in 1, the
characteristic resonances of a methyl singlet at δ 1.06
(12-Me), a methyl doublet at δ 1.11 (13-Me), and a group
of aliphatic protons between δ 1.20 and 2.30 indicated
that compound 2 is an analogue of 1. A pair of broad
singlets at δ 4.44 and 4.40 accounted for the C-11
exocyclic methylene protons of the drimane skeleton.6
Although compounds 1 and 2 showed similar 1H- and
13C-NMR spectra, the major difference between them
was in the chemical shifts and coupling pattern of the
C-15 methylene protons. In compound 2 they appeared
as an AB spin system at δ 3.09 (J ) 14.4 Hz) and δ
Assuming that 1 has the same absolute configuration
at C-10 as other natural sesquiterpene hydroquinones,11
selective NOE difference studies were performed on 1
and its monoacetate (5) (Figure 1) to determine the
relative stereochemistry around other chiral centers.

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Sesquiterpene Hydroquinones from Sponge
J ournal of Natural Products, 1997, Vol. 60, No. 2 95
Ta ble 3. Cytotoxicities (IC50, µg/mL) of Compounds 1-8 and
10
compd/tumor cells
P-388
KB-16
A-549
1
2
0.7
1.0
1.4
2.0
0.6
1.0
3
1.4
1.4
0.7
4
0.2
0.7
0.4
5
3.4
2.1
3.1
6
7
>50
>50
>50
1.9
1.8
1.5
8
0.7
1.9
2.7
10
0.6
2.1
1.2
a
The concentration of compound inhibiting 50% (IC50) of the
growth of murine and human tumor cell lines, P-388 (murine
leukemia), KB-16 (human nasopharyngeal carcinoma), and A-549
(human lung adenocarcinoma), after 3, 3, and 6 days of drug
exposure according to a published method.17
2.85 (J ) 14.4 Hz) but were at δ 2.68 ppm in compound
1. Moreover, The C-14 methyl singlet (δ 0.88) in 1 was
missing in 2, and an oxygenated methylene AB quartet
was observed instead (δ 3.92, 3.81, J ) 11.6 Hz). These
findings clearly indicated that polyfibrospongol B (2) has
an additional free hydroxyl group at C-14. The assigned
structure was also supported by the 13C-NMR spectrum
of 2, which exhibited a primary hydroxylated carbon at
δ 64.5. Upon acetylation compound 2 provided a diac-
etate (6), which showed the molecular ion at m/ z 486
in the mass spectrum and two acetyl singlets at δ 2.09
and 2.34 in the 1H-NMR spectrum. In addition, the
chemical shift of the C-14 oxygenated methylene was
shifted downfield to 4.24 ppm. The fragment ions at
F igu r e 2. Selective NOESY studies of compounds 8 and 10.
similar and promising activity against three tumor cell
lines. However, their acetates (compounds 5-7) were
less active. The most active of the compounds isolated
was ilimaquinone (4). In order to investigate structural
effects on its activity, its 4(11) epoxide (8) and the
stereoisomeric epoxide (10) were prepared from 4 and
a mixture of 4 and 9, respectively. The structures of
both 8 and 10 were determined by spectral analysis and
confirmed by the NOESY experiments. Oxygenation of
the 4(11) double bonds of 4 and 9 occurred primarily
from the less-hindered Re face in preference to the
hindered Si face.15 The proposed configurations of 8 and
10 were consistent with the results of NOESY studies
as shown in Figure 2. The biological activities of 8 and
10 were slightly less than the activity of 4 in each cell
line tested, indicating that the 4(11) double bond does
not play a major role in the bioactivity of compound 4.
• +
m/ z 249 (M - ArCH2 ) and m/ z 238 (ArCH2•+) in the
EIMS spectrum of 6 were also consistent with the
assigned structure of 2. Because the observed chemical
shifts, coupling pattern, and specific rotation of 2 are
similar to those of 1, the stereochemistry of 2 is
presumed to be identical to that of 1.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Optical rota-
tions were recorded on a J ASCO DIP-1000 polarimeter.
IR and UV spectra were measured on Hitachi T-2001
and Hitachi V-3210 spectrophotometers, respectively.
EI, FAB, and HREIMS spectra were recorded on a VG
Quattro 5022 and J EOL J MS-HX 110 mass spectrom-
eters. The 1H-, 13C-NMR, COSY and NOE, and NOESY
spectra were recorded on a Varian FT-300, a Varian FT-
400, or a Bruker AMX 400 spectrometer. The chemical
shifts are given in δ (ppm) and coupling constants in
Hz. Sephadex LH-20 (Pharmacia) and Si gel 60 (Merck)
were used for column chromatography, and precoated
Si gel plates (Merck, Kieselgel 60 F254, 1 mm) were used
for preparative TLC.
An im a l Ma ter ia l. P. australis Lendenfeld (Spongi-
idae) was collected from Nan-Wan, Taiwan, in October
1993, and stored at -20 °C before extraction. The
sponge has an irregular shape and exhibited a brown
massive architecture. The soft thorny-looking tissue
was surrounded by numerous small ostia and exhalent
pores rising to the surface of the sponge. In order to
observe the spicules, the sponge was treated with a
mixture of HNO3 (12 N, 3 mL) and H2SO4 (18 N, 1 mL),
The cytotoxicities of the new sesquiterpene hydro-
quinones 1 and 2 and their derivatives were evaluated
in vitro against human and murine tumor cell lines. As
shown in Table 3, compounds 1-5, 7, 8, and 10
exhibited significant cytotoxicities against human lung
(A-549) and nasopharyngeal (KB-16) cancers and against
murine lymphocytic leukemia (P-388) cells. Polyfibro-
spongols A (1) and B (2) and dictyoceratin A (3) showed

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96 J ournal of Natural Products, 1997, Vol. 60, No. 2
Shen et al.
and heated for 2 h. Its needle-like spicules were
observed under a microscope.14 A voucher specimen (sp
125), identified by Dr. G. H. Lee, has been deposited in
the Department of Marine Resources, National Sun Yat-
sen University.
(Ac2O-Py, each 0.2 mL, room temperature) of 1 (3 mg)
gave after workup 5 (3.1 mg) as a solid: [R]25D + 6.5° (c
0.18, CHCl3); UV (MeOH) λ max (log ꢀ) 227 (4.55), 245
(4.49), 289 (4.11) nm; IR (neat) ν max 2932, 1768, 1724,
1636, 1596, 1464, 1438, 1422, 1344, 1302, 1244, 1168,
1080, 892, 770 cm-1; 1H NMR (CDCl3, 300 MHz) δ 0.9-
2.4 (12H, m, H-1-H-10), 4.43 (1H, s, H-11a), 4.37 (1H,
s, H-11b), 1.07 (3H, s, H-12), 1.00 (3H, d, J ) 6.3 Hz,
H-13), 0.86 (3H, s, H-14), 2.56 (2H, s, H-15), 7.49 (1H,
d, J ) 1.5 Hz, H-17), 7.47 (1H, d, J ) 1.8 Hz, H-19),
3.90 (3H, s, OMe), 3.86 (3H, COOCH3), 2.36 (3H, s, OAc);
EIMS (70 eV) m/ z [M]+ 428 (0.4), 369 (0.3), 238 (4.5),
196 (20.9), 191 (42.4), 177 (17.6), 135 (19.8), 121 (27.0),
109 (29.2), 95 (100).
Extr a ction a n d Isola tion . The fresh sponge (230
g, wet wt) was crushed in a blender in 95% EtOH and
extracted with the same solvent (2 L × 3). The
combined filtrate was concentrated under vacuum to
give a suspension, which was adjusted to 400 mL by
the addition of H2O. The suspension was extracted
exhaustively with CHCl3 (400 mL). The CHCl3 layer
was concentrated to give a residue (0.4 g), which was
applied to a Si gel (20 g) column eluted with CHCl3 and
CHCl3-MeOH (10:1) to afford fractions A-D. Collec-
tion of fraction A (CHCl3, 500 mL) yielded polyfibro-
spongol A (1, 7 mg). A mixture (125 mg) of ilimaquinone
(4) and 5-epi-ilimaquinone (9) was obtained from frac-
tion B (CHCl3-MeOH,10:1, 500 mL). Fraction D (130
mg) was chromatographed on a Si gel column eluted
with CHCl3-Me2CO (10:1, 440 mL) to furnish an
additional mixture (95 mg) of compounds 4 and 9,
compound 3 (6 mg), and an unknown fraction, which
was purified by a preparative TLC plate (Si gel, CHCl3-
Me2CO, 10:1) to yield polyfibrospongol B (2, 5 mg).
P olyfibr osp on gol B Dia ceta te (6). Acetylation
(Ac2O-Py, each 0.1 mL, room temperature) of 2 (1.5 mg)
gave after workup 6 (1.8 mg) as a solid: [R]25 +1.5° (c
D
0.18, CHCl3); UV (MeOH) λ max (log ꢀ) 228 (4.72), 278
(4.04) nm; IR (neat) ν max 2940, 1775, 1730, 1650, 1465,
1
1345, 1305, 1240, 1200, 1170, 910, 810, 760 cm-1; H
NMR (CDCl3, 300 MHz) δ 0.7-2.4 (12H, m, H-1-H-10),
4.44 (1H, s, H-11a), 4.39 (1H, s, H-11b), 1.07 (3H, s,
H-12), 1.09 (3H, d, J ) 6.3 Hz, H-13), 4.24 (2H, s, H-14),
2.73 (1H, d, J ) 14.4 Hz, H-15a), 2.90 (1H, d, J ) 14.4
Hz, H-15b), 7.58 (1H, d, J ) 1.8 Hz, H-17), 7.50 (1H, d,
J )1.8 Hz, H-19), 3.90 (3H, s, OMe), 3.87 (3H, COOCH3),
2.34 (3H, s, OAc), 2.09 (3H, s, OAc); EIMS (70 eV) m/ z
[M]+ 486 (0.7), 445 (0.9), 444 (2.9), 413 (1.3), 377 (2.0),
362 (2.4), 249 (62.2), 238 (11.6), 196 (38.3), 189 (100),
161 (12.8), 147 (20.7), 133 (34.3), 119 (25), 107 (32.2),
95 (67.1), 81 (53.1), 69 (38.0), 55 (36.3), 43 (78.1);
FABMS m/ z [M + Na]+ 509 (2.0), [M + H]+ 487 (3.4).
In another batch of unidentified sponge (sp-137), the
fresh body (100 g) was crushed with EtOH (200 mL ×
2). After removal of solvent in vacuo, the residue (7 g)
was partitioned between equal volumes of CHCl3 and
H2O (each, 300 mL). The CHCl3-soluble fraction (180
mg) was subjected to a Sephadex LH-20 column (30 g)
and eluted with MeOH to yield a residue. Purification
of the residue by a Si gel column (15 g) using CHCl3 as
solvent gave compound 4 (15 mg).
Com p ou n d 7. Acetylation (Ac2O-Py, each 0.1 mL,
room temperature) of 3 (1.5 mg) gave after workup 7
P olyfibr osp on gol A (1): amorphous solid; [R]25
+
(1.8 mg) as a solid: [R]25 +0.1° (c 0.04, CHCl3); IR
D
D
5.3° (c 0.3, CHCl3); UV (MeOH) λ max (log ꢀ) 227 (4.51),
269 (4.22), 297 (3.88) nm; IR (neat) ν max 3416 (OH),
1714 (O-CdO, conjugated ester), 1602, 1496, 1464,
(neat) ν max 2932, 1778, 1728, 1438, 1374, 1328, 1308,
1234, 1200, 1164, 1032, 895, 756 cm-1; EIMS (70 eV)
m/ z [M]+ 456 (1.5), 425 (2.0), 413 (1.2), 383 (1.6), 224
(20), 191 (88), 177 (36), 135 (34), 121 (54), 109 (58), 95
1
1432, 1304, 1220, 1110, 1080, 948, 894, 766 cm-1; H-
and 13C-NMR data, see Table 1; EIMS (70 eV) m/ z [M]+
386 (0.9), 355 (1.9), 196 (83.1), 191 (61.4), 175 (22.1),
135 (22.1), 121 (35.8), 95 (100); HREIMS (70 eV) m/ z
386.2447, calcd for C24H34O4 386.2457.
1
(100), 81 (56). The H-NMR spectrum was consistent
with that reported for dictyoceratin A diacetate.6
Ilim a qu in on e Ep oxid e (8). Ilimaquinone (4, 10 mg)
was treated with m-CPBA (5 mg) at room temperature
for 15 min. The reaction mixture after evaporation
under vacuum was purified by a TLC plate (Si gel,
P olyfibr osp on gol B (2): amorphous solid; [R]25
D
+1.8° (c 0.18, CHCl3); UV (MeOH) λ max (log ꢀ) 227
(4.58), 270 (4.23), 301 (3.85) nm; IR (neat) ν max 3420,
2940, 1710, 1645, 1440, 1310, 1220, 1025, 900, 815, 760
CHCl3) to yield ilimaquinone epoxide (8) (3 mg): [R]25
D
-9.7 (c 0.1, CHCl3); 1H NMR (CDCl3, 300 MHz) δ 0.7-
2.2 (12H, m, H-1-H-10), 2.74 (1H, d, J ) 4.5 Hz, H-11a),
2.30 (1H, d, J ) 4.5 Hz, H-11b), 1.10 (3H, s, H-12), 0.96
(3H, d, J ) 6.3 Hz, H-13), 0.82 (3H, s, H-14), 2.60 (1H,
d, J ) 14.1 Hz, H-15a), 2.50 (1H, d, J ) 14.1 Hz, H-15b),
5.87 (1H, s, H-19), 3.87 (3H, s, OMe); 13C NMR (CDCl3,
75.4 MHz) δ 22.7 (t, C-1), 27,1 (t, C-2), 29.7 (t, C-3), 65.4
(s, C-4), 37.9 (t, C-5), 30.8 (t, C-6), 30.0 (t, C-7), 37.3 (d,
C-8), 42.9 (s, C-9), 46.8 (d, C-10), 49.9 (t, C-11), 19.7 (q,
C-12), 17.8 (q, C-13), 17.2 (q, C-14), 32.0 (t, C-15), 117.1
(s, C-16), 153.5 (s, C-17), 182.3 (s, C-18), 102.1 (d, C-19),
161.8 (s, C-20), 182.2 (s, C-21), 56.9 (q, OMe); EIMS (70
eV) m/ z [M]+ 374 (1.8), 207 (87), 189 (30), 168 (100),
147 (14), 133 (19), 119 (20), 109 (24), 95 (47), 81 (26),
69 (26), 55 (37).
1
cm-1; H- and 13C-NMR data, see Table 2; EIMS m/ z
[M]+ 402 (7.2), 371 (2.7), 207 (3.8), 197 (25.8), 196 (100),
195 (39.0), 189 (15), 164 (20.5), 149 (11.5), 137 (30.1),
119 (15.9), 107 (23.7), 95 (67.9), 81 (51.0), 69 (34.2),
55(36.8); FABMS m/ z [M + Na]+ 425 (2.0), [M + 1]+
403 (4.4), [M]+ 402 (12.4); HREIMS (70 eV) m/ z
402.2408, calcd for C24H34O5 402.2406.
Com p ou n d 3: isolated as an amorphous solid; [R]25
D
+4.9° (c 0.17, CHCl3); 13C NMR (CDCl3, 75.4 MHz) δ
23.2 (t, C-1), 27.9 (t, C-2), 33.0 (t, C-3), 160.1 (s, C-4),
40.2 (t, C-5), 36.6 (t, C-6), 27.7 (t, C-7), 36.3 (d, C-8),
42.1 (s, C-9), 48.0 (d, C-10), 102.7 (t, C-11), 20.6 (q, C-12),
17.6 (q, C-13), 17.7 (q, C-14), 36.9 (t, C-15), 125.1 (s,
C-16), 127.3 (s, C-17), 120.4 (s, C-18), 113.9 (d, C-19),
142.4 (s, C-20), 148.7 (s, C-21), 167.5 (s, COOCH3), 52.0
5-epi-Ilim a qu in on e Ep oxid e (10). A mixture (40
mg) of ilimaquinone (4) and 5-epi-ilimaquinone (9) was
treated with m-CPBA (20 mg) at room temperature for
30 min. The reaction mixture after evaporation under
1
(q, COOCH3); the UV, IR, H-NMR, and MS data of 3
were identical with those reported for dictyoceratin A.6
P olyfibr osp on gol A Mon oa ceta te (5). Acetylation

+
+
Sesquiterpene Hydroquinones from Sponge
J ournal of Natural Products, 1997, Vol. 60, No. 2 97
vacuum was chromatographed on a Sephadex LH-20
column eluted with MeOH-CHCl3-n-hexane (2:1:1) to
give ilimaquinone epoxide (8) and 5-epi-ilimaquinone
Yat-sen University, for carrying out cytotoxicity assays.
Dr. G. H. Lee, Institute of Oceanology, Academia Sinica,
is gratefully acknowledged for his identification of
Polyfibrospongia australis (Lendenfeld). We appreciate
Ms. Chao-lein Ho and Shiu-ching Yu of the NSC
Southern NMR and MS Instrument Center in National
Sun Yat-sen University, and Ms. Chyi-jia Wang, De-
partment of Chemistry, Kaohsiung Medical College, for
measurements of NMR and MS spectral data. This
research was supported by grants from the National
Science Council, Republic of China (NSC 85-2321-B110-
003 BH) and by the National Institute of Health,
Republic of China (DOH 85-HR-509).
epoxide (10, 20 mg). Compound 10: [R]25 -34.6° (c
D
0.38, CHCl3); UV (MeOH) λ max (log ꢀ) 228 (4.46), 290
(4.57) nm; IR (neat) ν max 3324 (OH), 2936 (CH,
aliphatic), 1646, 1610, 1538, 1462, 1448, 1380, 1324,
1230, 1038, 916, 754 cm-1; 1H NMR (CDCl3, 300 MHz)
δ 0.7-2.4 (12H, m, H-1-H-10), 3.04 (1H, d, J ) 4.2 Hz,
H-11a), 2.58 (1H, d, J ) 4.2 Hz, H-11b), 1.10 (3H, s,
H-12), 0.96 (3H, d, J ) 6.3 Hz, H-13), 0.95 (3H, s, H-14),
2.66 (1H, d, J ) 13.8 Hz, H-15a), 2.55 (1H, d, J ) 13.8
Hz, H-15b), 5.88 (1H, s, H-19), 3.89 (3H, s, OMe); 13C
NMR (CDCl3, 75.4 MHz) δ 22.5 (t, C-1), 23.8 (t, C-2),
30.0 (t, C-3), 60.9 (s, C-4), 36.3 (t, C-5), 32.1 (t, C-6),
31.2 (t, C-7), 38.1 (d, C-8), 44.3 (s, C-9), 48.3 (d, C-10),
55.9 (t, C-11), 29.6 (q, C-12), 19.2 (q, C-13), 18.2 (q,
C-14), 32.6 (t, C-15), 117.4 (s, C-16), 153.4 (s, C-17),
182.4 (s, C-18), 102.0 (d, C-19), 161.8 (s, C-20), 182.0 (s,
C-21), 56.9 (q, OMe); EIMS (70 eV) m/ z [M]+ 374 (0.5),
207 (37), 168 (100), 149 (22), 139 (18), 119 (27), 109 (35),
95 (66), 81 (53), 69 (76), 55 (73).
Cytotoxicity Assa ys. The cytotoxicity of compounds
against P-388 (leukemia), KB-16 (nasopharynx), and
A-549 (lung) tumor cells was determined by the MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide] colorimetric assay with some modifications.16
Each cell line was plated at 1000 cells/well in 96-well
microtiter plates. Twofold serial dilutions of tested
compounds were added to the cells, and P-388, KB-16,
and A-549 cells were enumerated using MTT after 3, 3,
and 6 days, respectively. MTT (50 µL, 1 mg/mL) was
added to each well, and the plates were incubated at
37 °C for 5 h. Formazan crystals were redissolved in
DMSO (Merck) for 10 min with shaking, and plates were
then immediately read on a microtiter plate reader
(Dynatech) at 540 nm. The IC50 was defined as the
concentration of test compound resulting in a 50%
reduction of absorbance compared to untreated cells in
the MTT assay. Results are given in Table 3. As
described in a previous paper mithramycin was used
as a standard (IC50 0.06 µg/mL).17
Refer en ces a n d Notes
(1) Luibrand, R. T.; Erdman, T. R.; Vollmer, J . J .; Scheuer, P. J .;
Finer, J .; Clardy, J . Tetrahedron 1979, 35, 609-612.
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M.; Crews, P. Tetrahedron 1992, 32, 6667-6680.
(3) Talpir, R.; Rudi, A.; Kashman, Y.; Loya, Y.; Hizi, A. Tetrahedron
1994, 50, 4179-4184.
(4) Urban, S.; Capon, R. J . J . Nat. Prod. 1992, 55, 1638-1642.
(5) Sarin, P. S.; Sun, D.; Thornton, A.; Muller, W. E. G. J . Nat.
Cancer Inst. 1987, 78, 663-666.
(6) Nakamura, H.; Deng, S.; Kobayashi, J .; Ohizumi, Y.; Hirata, Y.
Tetrahedron 1986, 42, 4197-4201.
(7) Kondracki, M.-L.; Guyot, M. Tetrahedron 1989, 45, 1995-2004.
(8) Kushlan, D. M.; Faukner, D. J .; Parkanyi, L.; Clardy, J .
Tetrahedron 1989, 45, 3307-3312.
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(11) Capon, R. J . In Studies in Natural Products Chemistry, Structure
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(12) Shen, Y. C.; Tai, H. R.; Duh, C. Y. Chin. J . Pharm. 1996, 48,
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(14) This species was formerly misidentified as Hippospongia sp.
because the shape and the chemical constituents of this species
were similar to those of Hippospongia. The genera Polyfibro-
spongia and Hippospongia both belong to the family Spongiidae
(Dictyoceratida) and can be distinguished by the occurrence of
polyfiber and polyspicule architecture on the surface or in the
bodies of sponges.
(15) March, J . Advanced Organic Chemistry: Reactions, Mechanisms,
and Structure; McGraw-Hill: New York, 1973; p 578.
(16) Mosmann, T. J . Immunol. Methods 1983, 65, 55-63.
(17) Wang, S. K.; Duh, C. Y.; Wu, Y. C.; Cheng, M. C.; Wang, Y.;
Soong, K.; Fang, L. S. J . Nat. Prod. 1992, 55, 1430-1435.
Ack n ow led gm en t. The authors thank Dr. Chang-
yih Duh, Institute of Marine Resources, National Sun
NP9605302