Six New Polyacetylenic Alcohols from the Marine Sponges Petrosia sp. and Halichondria sp.

Article abstract of DOI:10.1248/cpb.c15-00203

Six new polyacetylenic alcohols, termed strongylotriols A and B; pellynols J, K, and L; and isopellynol A, together with three known polyacetylenic alcohols, pellynols A, B, and C were isolated from the marine sponges Petrosia sp., and Halichondria sp. collected in Okinawa, Japan. Their planer structures were determined based on 2D-NMR and mass spectrometric analysis of the degraded products by RuCl₃ oxidation. The absolute stereochemistry of isolates was examined by their Mosher's esters. The strongylotriols were found to be optically pure compounds, whereas the pellynols are diastereomeric mixtures at the C-6 position. Proliferation experiments using the HeLa and K562 cell lines suggested that the essential structural units for activity are the "hexa-2,4-diyn-1,6-diol" and "pent-1-en-4-yn-3-ol" on the termini.

Full text of DOI:10.1248/cpb.c15-00203

Vol. 63, No. 6
Chem. Pharm. Bull. 63, 469–475 (2015)
469
Regular Article
Six New Polyacetylenic Alcohols from the Marine Sponges Petrosia sp.
and Halichondria sp.
Adeyemi Francis Gabriel, Zhen Li, Ryouhei Kusuda, Chiaki Tanaka, and Tomofumi Miyamoto*
Department of Natural Products Chemistry, Graduate School of Pharmaceutical Sciences, Kyushu University; 3–1–1
Maidashi, Higashi-ku, Fukuoka 812–8582, Japan.
Received March 5, 2015; accepted March 18, 2015; advance publication released online April 9, 2015
Six new polyacetylenic alcohols, termed strongylotriols A and B; pellynols J, K, and L; and isopellynol
A, together with three known polyacetylenic alcohols, pellynols A, B, and C were isolated from the marine
sponges Petrosia sp., and Halichondria sp. collected in Okinawa, Japan. Their planer structures were deter-
mined based on 2D-NMR and mass spectrometric analysis of the degraded products by RuCl oxidation. The
3
absolute stereochemistry of isolates was examined by their Mosher’s esters. The strongylotriols were found
to be optically pure compounds, whereas the pellynols are diastereomeric mixtures at the C-6 position. Pro-
liferation experiments using the HeLa and K562 cell lines suggested that the essential structural units for
activity are the “hexa-2,4-diyn-1,6-diol” and “pent-1-en-4-yn-3-ol” on the termini.
Key words polyacetylenic alcohol; marine sponge; Petrosia sp.; Halichondria sp.; strongylotriol; pellynol
Polyacetylenic compounds are diverse groups of second- sp. was fractionated using a Sephadex LH-20 column chloro-
ary metabolites that have been isolated from various sources, form (CHCl ), and reverse-phase HPLC (5C , 91% methanol
3
22
including plants, marine organisms, nudibranchs, hard cor- (MeOH)/H O), yielding strongylotriols A (1) and B (2); and
2
8)
als and red algae. These compounds have been shown to pellynols J (4), A (7), B (8) and C (9). The Et O soluble frac-
2
possess various pharmacological activities, which including tion of the EtOH extract of Halichondria sp. was fractionated
anti-tumor, anti-viral, anti-microbial and anti-inflammatory using a silica-gel column, followed by reverse-phase HPLC
1
–5)
activities.
In our continuing research on biologically active (5C , 93% MeOH/H O or 5C , 95% MeOH/H O), yielding
30 2 18 2
6
)
metabolites in marine invertebrates, we have isolated six new isopellynol A (3); pellynols J (4), K (5), L (6), A (7), B (8) and
polyacetylenic alcohols from the marine sponges, Petrosia C (9) (Fig. 1).
sp., and Halichondria sp. collected in Okinawa. This paper
7
)
Strongylotriol A (1) was found to be a white, amorphous
describes the isolation, structure and biological activity of solid, with electrospray ionization-time of flight (ESI-TOF)-
1
acetylenic alcohols.
MS showing a molecular formula of C H O . H- and
25 40 3
13
C-NMR and heteronuclear single quantum coherence
(HSQC) spectroscopic data indicated the presence of one pri-
Results and Discussion
Ethanol (EtOH) extracts prepared from 93 specimens of mary methyl, one oxygenated methylene, two oxygenated me-
marine invertebrates collected at Iriomote, Okinawa, were thines, three di-substituted alkynes, and 15 aliphatic methy-
screened for anti-proliferative activity. Among them, an ex- lenes, suggesting that 1 was an acyclic polyacetylenic alcohol.
tract of the Petrosia sp. and an extract of the Halichondria Its heteronuclear multiple bond correlation (HMBC) spectrum
sp. were able to inhibit HeLa cell proliferation. These extracts showed partial structures, including the terminal “hexa-2,4-
were subjected to bioassay-guided separation. The diethyl diyn-1,6-diol” and the internal “prop-2-yn-1-ol” (Fig. 2a).
ether (Et O) soluble fraction of the EtOH extract of Petrosia The location of the “prop-2-yn-1-ol” unit was determined by
2
Fig. 1. Structures of Compounds 1–9
*
To whom correspondence should be addressed. e-mail: miyamoto@phar.kyushu-u.ac.jp
©
2015 The Pharmaceutical Society of Japan

4
70
Chem. Pharm. Bull.
Vol. 63, No. 6 (2015)
Fig. 2a. HMBC Correlations and Partial Structure of 1
Fig. 2b. RuCl Oxidation and Its Degradation Product of 1
Fig. 2c. Δδ Values of (R)- and (S)-MTPA Esters of 1
3
1
Table 1. H-NMR (600MHz) Data of Compounds 1–9 in CDCl3
Proton
1
2
3
4
5
6
7 (8)
9
1
6
7
4.32 (2H, s)
4.41 (t, 6.6)
1.70 (2H, m)
4.32 (2H, s)
4.41 (t, 6.6)
1.70 (2H, m)
1.64 (2H, m)
4.32 (brs)
4.33 (2H, s)
4.41 (t, 6.6)
1.70 (2H, m)
1.99 (2H, brd)
4.32 (2H, s)
4.41 (t, 6.6)
1.70 (2H, m)
1.99 (2H, q)
4.32 (2H, s)
4.41 (t, 6.6)
1.70 (2H, m)
1.99 (2H, q)
4.31 (2H, s)
2.26 (t, 6.6)
4.32 (2H, s)
4.41 (t, 6.6)
1.70 (2H, m)
1.99 (2H, q)
4.32 (2H, s)
4.41 (t, 6.6)
1.70 (2H, m)
2.26 (2H, q)
Internal 1.64 (2H, m)
2.26 (2H, q)
4
2
.32 (brs)
5.33 (2H, t-like) 5.33 (2H, t-like) 5.33 (2H, t-like) 5.80 (dt, 7.2, 10.8) 5.33 (2H, t-like) 5.79 (dt, 7.2, 10.8)
.18 (2H, t, 6.6) 2.18 (2H, t, 6.6) 1.95 (2H, brd)
1.99 (2H, q)
1.99 (2H, q)
5.43 (d, 10.8)
.33 (t, 6.6)
2.06 (q, 7.2)
1.99 (2H, q)
5.41 (d, 10.8)
2
Terminal
1.55 (2H, brd)
2.05 (q, 7.2)
2.05 (q, 7.2)
2.05 (q, 7.2)
2.05 (q, 7.2)
4
.66 (2H, q, 7.2) 5.90 (dt, 6.0, 15.6) 5.90 (dt, 6.0, 15.6) 5.91 (dt, 6.0, 15.6) 5.90 (dt, 6.0, 15.6) 5.90 (dt, 6.0, 15.6)
.97 (dd, 8.4, 10.8) 5.59 (dd, 6.0, 15.6) 5.59 (dd, 6.0, 15.6) 5.59 (dd, 6.0, 15.6) 5.59 (dd, 6.0, 15.6) 5.59 (dd, 6.0, 15.6)
5
1
.28 (2H, m)
1.49 (1H, m)
5.51 (brd, 10.8)
4.82 (brd, 6.0)
2.54 (d, 1.8)
4.82 (brd, 6.0)
2.54 (d, 1.8)
4.83 (brd, 6.0)
2.54 (d, 1.8)
4.82 (brd, 6.0)
2.54 (d, 1.8)
4.82 (brd, 6.0)
2.55 (d, 1.8)
0
.85 (3H, t, 7.8) 0.85 (6H, d, 7.2) 3.12 (d, 1.8)
1
analysis of the degradation product of 1 resulting from RuCl3 showed that its molecular formula was C H O . H- and
33
52
3
13
oxidation. Because the fragment ion peak observed at m/z
C-NMR and HSQC spectroscopic data indicated the pres-
01.1039 (C H O , Δmmu−9.3) was estimated to be a dec- ence of one oxygenated methylene, two oxygenated methines,
−
2
10
17
4
anedeoic acid, the “prop-2-yn-1-ol” unit was located at C-15 of two di-substituted alkenes, two di-substituted alkynes, one
, yielding the deduced planar structure shown in Fig. 2b. The terminal acetylene and 20 aliphatic methylenes. The HMBC
absolute stereochemistry of 1 was determined using a modi- spectrum of 3 showed three partial structures, a terminal
1
9)
fied Mosher’s method. The proton chemical shifts of (R)- and “hexa-2,4-diyn-1,6-diol,” a “pent-2-en-4-yn-1-ol” and an inter-
S)-α-methoxy(trifluoromethyl)phenyl acetic acid (MTPA) nal “Z-olefin” (Fig. 3a). The olefin geometry was determined
esters of 1 were assigned by correlated spectroscopy (COSY) to be Z both from its coupling constant (J=10.8Hz) and the
(
13
10)
and total correlation specroscopy (TOCSY) spectral data. The
C chemical shift of the allylic methylenes (δC 27.2). The
distribution of Δδ (δ –δ ) values indicated that the absolute location of the internal “Z-olefin” was determined by analysis
S
R
stereochemistry at C-6 and C-15 was 6R and 15S, respectively of the degradation products of 3 resulting from RuCl oxida-
3
(
Fig. 2c).
tion. The fragment ion peaks derived from tridecanedioic acid
Strongylotriol B (2) was found to be a white, amorphous (C H O ) and undecanedioic acid (C H O ) were observed
1
3
24
4
11 20
4
−
−
solid, with ESI-TOF-MS showing a molecular formula of at m/z 215.1305 (M−H) and m/z 243.1564 (M−H) , respec-
C H O . H- and C-NMR spectra were similar to those of tively, resulting in the deduced planar structure shown in Fig.
1
13
2
6
42
3
1
except for the signals due to the terminal isopropyl moiety 3b. The absolute stereochemistry of 3 was examined using
[
δ 0.85 (6H, d), δ 22.6 (q), 27.2 (d)] (Tables 1, 2). Mass spec- its Mosher’s esters, and the distribution of Δδ (δ –δ ) values
H
C
S
R
trometric analysis of the degradation product of 2 resulting closed to C-29 indicates that absolute stereochemistry at C-29
1
from RuCl oxidation showed that its “prop-2-yn-1-ol” unit was in the S configuration. However, the H-NMR spectra of
3
was located in the same position as in 1. The absolute stereo- (R)- and (S)-MTPA esters showed diastereomeric proton sig-
chemistry of 2 was also examined using a modified Mosher’s nals for H-6 in a ratio of 3/4.
method, and the absolute configurations at C-6 and C-15 were
Pellynol J (4) was obtained as a white, amorphous solid
determined to be 6R and 15S respectively, as in 1.
from the Et O extractives of both Petrosia sp. and Hali-
2
Isopellynol A (3) was isolated as a white, amorphous solid chondria sp. ESI-TOF-MS showed a molecular formula of
1
13
from the Et O extractive of Halichondria sp. ESI-TOF-MS C H O . H- and C-NMR and HSQC spectroscopic data
2
31 48
3

Vol. 63, No. 6 (2015)
Chem. Pharm. Bull.
471
13
Table 2.
C-NMR (150MHz) Data of Compounds 1–9 in CDCl3
Carbon
1
2
3
4
5
6
7 (8)
9
1
2
51.4 (t)
77.6 (s)
69.8 (s)
68.9 (s)
80.5 (s)
62.8 (d)
37.4 (t)
38.2 (t)
51.5 (t)
77.5 (s)
69.8 (s)
69.8 (s)
80.6 (s)
62.8 (d)
37.4 (t)
38.2 (t)
62.8 (d)
85.4 (s)
81.1 (s)
18.7 (t)
51.5 (t)
77.5 (s)
69.8 (s)
68.8 (s)
80.6 (s)
62.8 (d)
37.5 (t)
27.2 (t)
129.9 (d)
129.9 (d)
27.2 (t)
51.5 (t)
77.5 (s)
69.8 (s)
68.8 (s)
80.6 (s)
62.8 (d)
37.5 (t)
27.2 (t)
129.9 (d)
129.9 (d)
27.2 (t)
51.5 (t)
77.5 (s)
69.8 (s)
68.8 (s)
80.6 (s)
62.8 (d)
37.5 (t)
27.2 (t)
129.9 (d)
129.9 (d)
27.2 (t)
51.6 (t)
75.2 (s)
72.0 (s)
65.2 (s)
84.0 (s)
32.0 (t)
51.4 (t)
77.5 (s)
69.8 (s)
68.8 (s)
80.6 (s)
62.8 (d)
37.5 (t)
27.2 (t)
129.9 (d)
129.9 (d)
27.2 (t)
51.5 (t)
77.5 (s)
69.8 (s)
68.8 (s)
80.6 (s)
62.8 (d)
37.5 (t)
25.0 (t)
142.5 (d)
109.4 (d)
94.5 (s)
77.5 (s)
31.9 (t)
134.6 (d)
128.4 (d)
62.8 (d)
83.3 (s)
74.0 (d)
3
4
5
6
7
Int.
25.0 (t)
142.5 (d)
109.4 (d)
94.5 (s)
77.5 (s)
31.9 (t)
6
2.8 (d)
8
8
5.6 (s)
1.2 (s)
1
8.7 (t)
ω6
ω5
ω4
ω3
ω2
ω1
36.5 (t)
70.1 (d)
147.4 (d)
108.9 (d)
82.8 (s)
79.5 (d)
31.9 (t)
134.6 (d)
128.4 (d)
62.9 (d)
83.4 (s)
74.0 (d)
31.9 (t)
134.6 (d)
128.4 (d)
62.9 (d)
83.4 (s)
74.0 (d)
31.9 (t)
134.6 (d)
128.4 (d)
62.9 (d)
83.4 (s)
74.0 (d)
134.6 (d)
128.4 (d)
62.8 (d)
83.3 (s)
73.9 (d)
27.2 (d)
22.6 (q)
22.6 (q)
14.1 (q)
Fig. 3a. HMBC and COSY Correlations and Partial Structure of 3
Fig. 3b. RuCl Oxidation and Its Degradation Products of 3
3
“Z-olefin” unit (Tables 1, 2). The position of “Z-olefin” at C16
was determined by the ESI-TOF-MS analysis of 4 with RuCl3
oxidation. The absolute stereochemistry of C-29 was deter-
mined to be R configuration, however the stereochemistry at
C-6 was a diastereomeric mixture, as in isopellynol A (3).
Pellynol K (5) was obtained as a white, amorphous solid
from the Et O extractive of Halichondria sp. ESI-TOF-MS
2
1
1
13
Fig. 3c. H-NMR Chemical Shifts at H-6 and H -7, and Δδ Values of gave a molecular formula of C H O . H- and C-NMR and
2
34 54
3
(
R)- and (S)-MTPA Esters of 3
HSQC spectroscopic data were similar to those of pellynols
A (7), B (8), and J (4), suggesting that pellynol K was a C34
indicated the presence of one oxygenated methylene, two polyacetylenic alcohol possessing terminal “hexa-2,4-diyn-1,6-
oxygenated methines, two di-substituted alkenes, two di- diol” and “pent-1-en-4-yn-3-ol” units and an internal “Z-
substituted alkynes, one terminal acetylene and 18 aliphatic olefin” unit. The position of “Z-olefin” at C-19 was determined
methylenes. Because these spectral features corresponded to by the ESITOF-MS analysis of 5 with RuCl oxidation. A
3
those of the known polyacetylenic alcohols, pellynols A (7; comparison of the specific rotation of 5 with those of 4, 7, and
C H O ) and B (8; C H O ), pellynol J was regarded as 8 indicated that the stereochemistry of 5 was 32R.
33
52
3
32 50
3
a C31 polyacetylenic alcohol possessing terminal “hexa-2,4-
Pellynol L (6) was obtained as a white, amorphous solid
diyn-1,6-diol” and “pent-1-en-4-yn-3-ol” units and an internal from the Et O extractive of Halichondria sp. ESI-TOF-MS
2

4
72
Chem. Pharm. Bull.
Vol. 63, No. 6 (2015)
Fig. 4a. HMBC and COSY Correlations and Partial Structure of 6
Fig. 4b. RuCl Oxidation and Its Degradation Products of 6
3
more, the activities of isopellynol A (3) and pellynol L (6),
with terminal “pent-2-en-4-yn-1-ol” and “pent-2,4-diyn-1-ol”
units, respectively, were decreased slightly. These results were
6
,11)
consistent with earlier findings.
In summary, six new polyacetylenic alcohols, strongy-
lotriols A (1) and B (2); pellynols J (4), K (5), and L (6); and
isopellynol A (3), were isolated from the marine sponges
Petrosia sp. and Halichondria sp. Compounds 4, 7, and 8 were
Fig. 4c. Δδ Values of (R)- and (S)-MTPA Esters of 6
1
13
gave a molecular formula of C H O . H- and C-NMR and isolated from both sponges, despite the difference in appear-
35
52
2
HSQC spectral data indicated the presence of one oxygen- ance of these marine sponges. Furthermore, other abundant
ated methylene, one oxygenated methine, two di-substituted compounds differed in these species, in that meroditerpenoids
12)
alkenes, three di-substituted alkynes, one terminal acetylene such as strongylophorines were found only in Petrosia sp.,
and 21 aliphatic methylenes. The HMBC spectrum of 6 while polyacetylenic acids such as pellynic acids were found
8)
yielded partial structures, terminal “pent-2,4-diyn-1-ol” and only in Halichondria sp. (Supplementary material). The
pent-1-en-4-yn-3-ol” units and an internal “but-1-en-3-yn” isolated strongylotriols (1, 2) were optically pure, whereas the
“
unit (Fig. 4a). These data suggested that 6 is a 6-deoxy-type pellynols were diastereomeric mixtures at the C-6 position.
derivative of pellynol C (9; C H O ). The coupling constants The positions of the internal olefins differed in the pellynols,
33
48
3
of the olefins (J=10.8, 15.6Hz) indicated that their geometries but their locations from the end terminal were identical to
were Z and E, respectively. The location of the internal “but-1- each other. Some symbionts may therefore biosynthsize these
en-3-yn” unit was determined by the degradation products of polyacetylenic compounds linking to two types of acetylene
6
with RuCl oxidation followed by GC-MS analysis. Mass groups at this position.
3
spectral data identified two dimethyl ester derivatives, dimeth-
yl hexadecanedioate (C H O ) and dimethyl nonanedioate Experimental
18
34
4
(
C H O ) indicating that the “but-1-en-3-yn” unit was located
General Experimental Procedures Optical rotations
11
20
4
at C-20 (Fig. 4b). The absolute stereochemistry of 6 was also were measured with a Jasco Dip-370 digital polarimeter at
examined using a modified Mosher’s method, with C-33 deter- 24°C. The NMR spectra were recorded on a Varian INOVA
1
13
mined to have an absolute R configuration (Fig. 4c).
600 operating at 600MHz for H and 150MHz for C in
1
13
The absolute stereochemistry of the known compounds, CDCl . H- and C-NMR chemical shifts is reported in ppm
pellynols A (7) and C (9) was also assessed using the modified (δ) and referenced to tetramethylsilane (TMS) for H (δ =0)
Mosher’s method yielding results identical to those of isopel- and the solvent signal for C (CDCl :, δ =77.2). ESI-TOF-MS
3
1
H
13
3
C
lynol A (3) and pellynol J (4) with the secondary alcohol at were measured with a Bruker microTOF mass spectrometer.
C-6 being a diastereomeric mixture (Fig. 5).
GC-MS was performed on Gas Chromatography Mass Spec-
The ability of the isolated compounds to inhibit the pro- trometer-QP2010 using INERT Cap5MS/Sil, 30m×0.25mm
liferation of HeLa and K562 cells were examined. Pellynols i.d., GL Science); column temp. 100–280°C, injection temp.,
J (4), A (7), and B (8), all of which have the same structural 300°C, interface temp., 250°C. Chromatographic separations
units “hexa-2,4-diyn-1,6-diol” and “pent-1-en-4-yn-3-ol,” on were carried out using Sephadex LH-20 (GE Healthcare),
both ends, showed significant activity, with almost equivalent silica gel (Merck Silica gel 60), and reversed phase-HPLC
IC50 values (Table 3). In contrast, strongylotriols A (1) and B (Cosmosil 5C22AR-II or 5C18AR-II, 250×4.6mm i.d., Na-
(
2) with structural unit “pent-1-en-4-yn-3-ol” on one-half, had kalai Tesque, Develosil C30 UG-3, 250×4.6mm i.d., Nomura
nearly one-third the activity of pellynols J, A and B. Further- Chemicals) with a Jasco PU2089 gradient pump and PU2075

Vol. 63, No. 6 (2015)
Chem. Pharm. Bull.
473
1
Fig. 5. H-NMR Spectra of (R)-MTPA Esters of 7 and 1
Table 3. IC50 Values (µM) of Compounds 1–9 on the Proliferation of HeLa and K562 Cells
Compound
1
2
3
4
5
6
7
8
9
HeLa
K562
18.1
11.5
14.3
11.3
4.13
1.95
0.62
0.55
nt
nt
0.94
1.71
0.49
0.46
0.52
0.55
0.95
0.83
UV/Vis detector. Thin layer chromatography (TLC) analysis
were carried out using Merck precoated TLC plates Silica gel (ca. 1.0mg) in CCl (1.0mL) was added NaIO (10eq), RuCl
3
RuCl –NaIO4 Oxidation To a solution of each sample
3
4
4
6
0F254 and RP-18F254s
Animal Material Petrosia sp. and Halichondria sp. were was stirred vigorously for 1h, then 1M HCl (2.0mL) was
collected by hand at a depth of 10–15m off the shore of Irio- added and extracted with Et O. Et O layer was analyzed ESI-
.
(0.04mg), CH CN (1.0mL), and H O (1.0mL). The solution
3
2
2
2
mote Island, Okinawa Prefecture, Japan, in Nobember 2009. TOF-MS (negative ion) directly or methylated with TMS–
Voucher specimens were deposited at the Graduate School of CH N in 20% MeOH/benzene followed by GC-MS analysis.
2
2
Pharmaceutical Sciences, Kyushu University under reg. no.
Preparation of the (R)- or (S)-MTPA Esters To a solu-
0
91120-36 for Petrosia sp., and no. 091120-35 for Halichon- tion of each 6 (1.0mg, 2µmol) in dry CH Cl (0.2mL) stirring
2
2
dria sp.
at room temperature was added (R)- or (S)-MTPA (7.4mg,
Extraction and Isolation Petrosia sp. (wet weight 32.0 µmol), N,N′-dicyclohexyl carbodiimide (DCC) (6.5mg,
05.89g) was homogenized and extracted with EtOH 32.0 µmol) and a catalytic amount of 4-dimethylaminopyridine
4
(
3×0.5L) and filtrated. The extract was evaporated in vacuo, (DMAP). The solution was stirred at room temperature for 8h
and the resulting aqueous suspension was diluted H O (0.5L) and filtered. The filtrate was dried with N and subjected to
2
2
and extracted with Et O (3×0.2L) and n-BuOH (3×0.2L). The silica gel column with n-hexane–EtOAc (9:1) to afford (R)-
2
organic layers were evaporated to give an Et O extract (8.71g) MTPA ester (1.9mg) and (S)-MTPA ester (2.3mg).
2
and n-BuOH extract (0.99g). The part of Et O extract (4.99g)
Strongylotriol A (1) Amorphous solid; [α] −8.3 (c=0.012,
2
D
+
was subjected to Sephadex LH-20 column chromatography CHCl ); ESI-TOF-MS m/z: 411.2900 [M+Na] (Calcd for
3
1
13
with CHCl to give twelve fractions, and fraction 8 (33.8mg) C H NaO , 411.2870); H- and C-NMR see Tables 1 and 2.
3
25 40
3
−
was applied to reversed phase HPLC (Cosmosil 5C22 AR-II) Oxidative product: decanedeoic acid: m/z: 201.1039 [M−H]
with 91% MeOH/H O to give compounds 1 (2.0mg), 2 (Calcd for C H O , 201.1132).
2
10 17
4
+
(1.0mg), 4 (2.4mg), 7 (6.7mg), and 8 (2.5mg), respectively.
(R)-MTPA ester of 1, ESI-TOF-MS m/z: 1059.4119 [M+Na]
1
Halichondria sp. (wet weight 100.0g) was homogenized and (Calcd for C H F NaO , 1059.4064); H-NMR (CDCl ) δ:
55
61
9
9
3
extracted with EtOH (3×1.0L) and filtrated. The extract was 0.878 (3H, t, H-25), 1.260 (2H, m, H-12), 1.280 (2H, m, H-8),
evaporated in vacuo, to give EtOH extract (5.0g). The EtOH 1.315 (2H, m, H-20), 1.390 (2H, m, H-13), 1.405 (2H, m, H-19),
extract was partitioned between Et O, n-BuOH and H O to 1.770 (2H, t, H-7), 1.775 (2H, m, H-14), 2.145 (2H, t, H-18),
2
2
give an Et O extract (2.73g) and n-BuOH extract (1.06g). The 4.891 (1H, d, H-1), 4.981 (1H, d, H-1′), 5.484 (1H, t, H-15),
2
part of Et O extract (1.43g) was subjected to silica gel column 5.563 (1H, t, H-6), 3.544 (3H, s, OMe), 3.568 (6H, s, OMe),
2
chromatography using n-hexane–EtOAc (1:9–0:1) to give 7.3–7.7 (15H, phenyl); (S)-MTPA ester of 1, ESI-TOF-MS m/z:
+
seven fractions. Fraction 3 (92.9mg) was subjected to silica 1059.4158 [M+Na] (Calcd for C H F NaO , 1059.4064);
55
61
9
9
1
gel column followed by reversed phase HPLC (Cosmosil 5C18
H-NMR (CDCl ) δ: 0.878 (3H, t, H-25), 1.210 (2H, m, H-12),
3
AR-II) with 95% MeOH/H O to give compound 6 (5.2mg), 1.400 (2H, m, H-8), 1.340 (2H, m, H-20), 1.290 (2H, m, H-13),
2
and part of fraction 5 (165.8mg) was applied to reversed phase 1.470 (2H, m, H-19), 1.830 (2H, t, H-7), 1.720 (2H, m, H-14),
HPLC (Develosil C30 UG-3) with 100% MeOH to give com- 2.175 (2H, t, H-18), 4.870 (1H, d, H-1), 4.987 (1H, d, H-1′),
pounds 3 (2.8mg), 4 (7.2mg), 7 (71.2mg), 8 (30.5mg), and 9 5.510 (1H, t, H-15), 5.528 (1H, t, H-6), 3.540 (3H, s, OMe),
(13.7mg), respectively.
3.568 (6H, s, OMe), 7.3–7.7 (15H, phenyl).

4
74
Chem. Pharm. Bull.
Vol. 63, No. 6 (2015)
Strongylotriol B (2) Amorphous solid; [α] −8.3 (c=0.013, (0.5H, t, H-6), 5.582 (0.5H, t, H-6), 5.600 (1H, dd, H-28),
D
+
CHCl ); ESI-TOF-MS m/z: 425.3009 [M+Na] (Calcd for 6.009 (1H, d, H-29), 6.067 (1H, m, H-27), 3.541 (3H, s, OMe),
3
1
13
C H NaO , 425.3026); H- and C-NMR see Tables 1 and 2. 3.547 (3H, s, OMe), 3.568 (3H, s, OMe), 7.398–7.519 (15H,
2
6
42
3
−
Oxidative product: decanedeoic acid: m/z: 201.1035 [M−H]
Calcd for C H O , 201.1132).
phenyl).
Pellynol K (5) Amorphous solid; [α]D −6.3 (c=0.027,
(
10 17 4
+
(
R)-MTPA ester of 2, ESI-TOF-MS m/z: 1073.4332 CHCl ); ESI-TOF-MS m/z: 533.3950 [M+Na] (Calcd for
3
1 1 13
+
[
(
(
(
(
(
(
M+Na] (Calcd for C H F NaO , 1073.4221); H-NMR C H NaO , 533.3971); H- and C-NMR see Tables 1 and 2.
5
6
63
9
9
34 54
3
−
CDCl ) δ: 0.853 (6H, d, H-25, 26), 1.260 (2H, m, H-12), 1.280 Oxidative product: undecanedioic acid: m/z: 215.1281 [M−H]
3
2H, m, H-8), 1.315 (2H, m, H-20), 1.390 (2H, m, H-13), 1.405 (Calcd for C H O , 215.1289), tetradecanedioic acid: m/z:
2H, m, H-19), 1.770 (2H, t, H-7), 1.775 (2H, m, H-14), 2.145 257.1782 [M−H] (Calcd for C H O , 257.1758) .
2H, t, H-18), 4.891 (1H, d, H-1), 4.981 (1H, d, H-1′), 5.484
1H, t, H-15), 5.563 (1H, t, H-6), 3.544 (3H, s, OMe), 3.568 CHCl ); ESI-TOF-MS m/z: 527.3964 [M+Na] (Calcd for
6H, s, OMe), 7.3–7.7 (15H, phenyl); (S)-MTPA ester of 2, ESI- C H NaO , 527.3865); H- and C-NMR see Tables 1 and
TOF-MS m/z: 1073.4302 [M+Na] (Calcd for C H F NaO , 2. Oxidative product: dimethyl nonanedioate (C H O ),
073.4221); H-NMR (CDCl ) δ: 0.850 (3H, t, H-25), 1.210 t =10.04min, m/z: 185 (M−31) , 152, 111, 83, 55 (base peak),
11 19 4
−
14 25 4
Pellynol L (6) Amorphous solid; [α]D −2.6 (c=0.019,
+
3
1
13
35 52 2
+
5
6
63
9
9
11 20
4
1
+
1
(
(
(
(
(
3
R
2H, m, H-12), 1.400 (2H, m, H-8), 1.335 (2H, m, H-20), 1.280 dimethyl hexadecanedioate (C H O ), t =17.66min, m/z: 283
18 34 4 R
+
2H, m, H-13), 1.465 (2H, m, H-19), 1.830 (2H, t, H-7), 1.730 (M−31) , 241, 112, 98 (base peak).
2H, m, H-14), 2.180 (2H, t, H-18), 4.880 (1H, d, H-1), 4.982 (R)-MTPA ester of 6, ESI-TOF-MS m/z: 959.4668 [M+Na]
1H, d, H-1′), 5.517 (1H, t, H-15), 5.527 (1H, t, H-6), 3.536 (Calcd for C H F NaO , 959.4656); H-NMR (CDCl ) δ:
+
1
55
66
9
6
3
3H, s, OMe), 3.565 (3H, s, OMe), 3.586 (3H, s, OMe) 7.3–7.7 1.798 (2H, m, H-30), 2.276 (2H, q, H-19), 2.343 (2H, t, H-24),
(15H, phenyl). 2.556 (1H, s, H-35), 4.886 (2H, d, H-1), 5.433 (1H, d, H-21),
Isopellynol A (3) Amorphous solid; [α]D −15.0 (c=0.022, 5.497 (1H, dd, H-32), 5.796 (1H, q, H-20), 6.015 (1H, m, H-31),
+
CHCl ); ESI-TOF-MS m/z: 519.3854 [M+Na] (Calcd for 6.036 (1H, d, H-33), 3.552 (3H, s, OMe), 3.569 (3H, s, OMe),
3
1
13
C H NaO , 519.3817); H- and C-NMR see Tables 1 and 2. 3.587 (3H, s, OMe), 7.394–7.516 (15H, phenyl); (S)-MTPA
Oxidative product: undecanedioic acid: m/z: 215.1305 [M−H]
Calcd for C H O , 215.1289), tridecanedioic acid: m/z: C H F NaO , 959.4656)); H-NMR (CDCl ) δ: 1.778 (2H, m,
43.1564 [M−H] (Calcd for C H O , 243.1602).
R)-MTPA ester of 3, ESI-TOF-MS m/z: 1167.5015 [M+Na]
Calcd for C H F NaO , 1167.5003); H-NMR (CDCl3) H-32), 5.816 (1H, q, H-20), 6.065 (1H, m, H-31), 6.006 (1H, d,
3
3
52
3
−
+
ester of 6, ESI-TOF-MS m/z: 959.4727 [M+Na] (Calcd for
1
(
2
1
1
19
4
55 66
9
6
3
−
H-30), 2.306 (2H, q, H-19), 2.373 (2H, t, H-24), 2.516 (1H, s,
H-35), 4.906 (2H, d, H-1), 5.453 (1H, d, H-21), 5.597 (1H, dd,
13
23
4
+
(
1
(
63 73 9 9
δ: 1.765 (2H, m, H-28), 1.779 (1H, m, H-7), 1.839 (1H, m, H-33), 3.550 (3H, s, OMe), 3.569 (3H, s, OMe), 3.587 (3H, s,
H-7), 1.998–2.023 (4H, brd, H-17, 20), 3.220 (1H, s, H-33), OMe), 7.394–7.516 (15H, phenyl).
4
.872–4.983 (2H, ddd, H-1), 5.328 (2H, brdt, H-18, 19), 5.529
Pellynol A (7) Amorphous solid; [α]D −5.0 (c=0.024,
(
0.5H, t, H-6), 5.565 (0.5H, t, H-6), 5.611 (1H, d, H-31), 5.802 CHCl ), ref. 9 [α] −8.5 (c=1.0, CHCl ); ESI-TOF-MS m/z:
3
D
+
3
1
(1H, dd, H-30), 5.925 (1H, q, H-29), 3.524 (3H, s, OMe), 3.532 519.3864 [M+Na] (Calcd for C H NaO , 519.3817); H- and
33 52 3
13
(3H, s, OMe), 3.553 (3H, s, OMe), 7.380–7.500 (15H, phenyl);
C-NMR see Tables 1 and 2. Oxidative product: undec-
anedioic acid: m/z: 215.1281 [M−H]− (Calcd for C H O ,
+
(
S)-MTPA ester of 3, ESI-TOF-MS m/z: 1167.5007 [M+Na]
1
1
19
4
1
−
(Calcd for C H F NaO , 1167.5003); H-NMR (CDCl ) δ: 215.1289), tridecanedioic acid: m/z: 243.1585 [M−H] (Calcd
63 73 9 9 3
1
.721 (2H, m, H-28), 1.778 (1H, m, H-7), 1.839 (1H, m, H-7), for C H O , 243.1602).
13 23 4
+
1.994 (4H, brd, H-17, 20), 3.227 (1H, s, H-33), 4.873–4.985
(R)-MTPA ester of 7, ESI-TOF-MS m/z:1167.4934 [M+Na]
1
(2H, ddd, H-1), 5.330 (2H, brdt, H-18,19), 5.529 (0.5H, t, H-6), (Calcd for C H F NaO , 1167.5003); H-NMR (CDCl3)
63
73
9
9
5.566 (0.5H, t, H-6), 5.642 (1H, d, H-31), 5.944 (1H, dd, H-30), δ: 1.727 (1H, m, H-7), 1.787 (1H, m, H-7), 1.925 (2H, m,
5.971 (1H, q, H-29), 3.524 (3H, s, OMe), 3.533 (3H, s, OMe), H-28), 1.966–1.978 (4H, brd, H-17,20), 2.550 (1H, s, H-33),
3.553 (3H, s, OMe), 7.380–7.500 (15H, phenyl).
4.814–4.944 (2H, ddd, H-1), 5.276 (2H, brdt, H-18,19), 5.427
Pellynol J (4) Amorphous solid; [α]D −9.6 (c=0.016, (1H, dd, H-30), 5.478 (0.5H, t, H-6), 5.514 (0.5H, t, H-6),
+
CHCl ); ESI-TOF-MS m/z: 491.3541 [M+Na] (Calcd for 5.938 (1H, m, H-29), 5.957 (1H, d, H-31), 3.472 (3H, s, OMe),
3
1
13
C H NaO , 491.3503); H- and C-NMR see Tables 1 and 2. 3.500 (3H, s, OMe), 3.519 (3H, s, OMe), 7.322–7.335 (15H,
Oxidative product: undecanedioic acid: m/z: 215.1305 [M−H]
Calcd for C H O , 215.1289).
R)-MTPA ester of 4, ESI-TOF-MS m/z: 1139.4659 [M+Na]
Calcd for C H F NaO , 1139.4690); H-NMR (CDCl3) m, H-28), 1.966–1.978 (4H, brd, H-17,20), 2.512 (1H, s, H-33),
31
48
3
−
phenyl); (S)-MTPA ester of 2, ESI-TOF-MS m/z: 1167.4978
1
(
[M+Na]+ (Calcd for C H F NaO , 1167.5003); H-NMR
11
19
4
63 73
9
9
+
(
(CDCl ) δ: 1.726 (1H, m, H-7), 1.787 (1H, m, H-7), 1.966 (2H,
3
1
(
61 69 9 9
δ: 1.798 (2H, m, H-26), 1.805 (1H, m, H-7), 1.857 (1H, m, 4.894–4.944 (2H, ddd, H-1), 5.277 (2H, brdt, H-18,19), 5.528
H-7), 2.012–2.034 (4H, brd, H-15, 18), 2.620 (1H, s, H-31), (1H, dd, H-30), 5.476 (0.5H, t, H-6), 5.514 (0.5H, t, H-6), 5.999
4
(
6
3
.884–4.999 (2H, ddd, H-1), 5.343 (2H, brdt, H-16, 17), 5.495 (1H, m, H-29), 5.767 (1H, d, H-31), 3.473 (3H, s, OMe), 3.502
1H, dd, H-28), 5.546 (0.5H, t, H-6), 5.583 (0.5H, t, H-6), (3H, s, OMe), 3.519 (3H, s, OMe), 7.322–7.335 (15H, phenyl).
.006 (1H, m, H-27), 6.030 (1H, d, H-29), 3.540 (3H, s, OMe), Pellynol B (8) Amorphous solid; [α]D −8.8 (c=0.019,
.570 (3H, s, OMe), 3.587 (3H, s, OMe), 7.392–7.516 (15H, CHCl ), ref. 9 [α]D −7.6 (c=0.28, CHCl ) ; ESI-TOF-MS
3
3
+
phenyl); (S)-MTPA ester of 4, ESI-TOF-MS m/z: 1139.4697 m/z: 505.3771 [M+Na] (Calcd for C H NaO , 505.3660);
3
2
50
3
+
1
1
13
[
M+Na] (Calcd for C H F NaO , 1139.4690); H-NMR
H- and C-NMR see Tables 1 and 2. Oxidative product: un-
61
69
9
9
−
(CDCl ) δ: 1.793 (1H, m, H-7), 1.856 (1H, m, H-7), 1.843 (2H, decanedioic acid: m/z: 215.1281 [M−H] (Calcd for C H O ,
3
1
1
1
9
4
−
m, H-26), 2.007–2.074 (4H, brd, H-15,18), 2.579 (1H, s, H-31), 215.1289), dodecanedioic acid: m/z: 229.1393 [M−H] (Calcd
.884–4.998 (2H, ddd, H-1), 5.343 (2H, brdt, H-16, 17), 5.546 for C H O , 229.1445).
4
12
21
4

Vol. 63, No. 6 (2015)
Chem. Pharm. Bull.
475
Pellynol C (9) Amorphous solid; [α]D −8.7 (c=0.031,
Acknowledgments We wish to thank the Ministry of
CHCl ), ref. 9 [α] −11.2 (c=2.38, CHCl ); ESI-TOF-MS Education, Culture, Sports, Science and Technology (MEXT)
3
D
3
+
m/z: 515.3496 [M+Na] (Calcd for C H NaO , 515.3503); of Japan for the financial support to Mr. Gabriel Adeyemi
H- and C-NMR see Tables 1 and 2. Oxidative product: Francis. This work was supported by JSPS KAKENHI Grant
33
48
3
1
13
dimethyl nonanedioate (C H O ), t =10.08min, m/z: 185 Number 24590139.
1
1
20
4
R
+
(
M−31) , 152, 111, 83, 55 (base peak), dimethyl tridecanedio-
ate (C H O ), t =14.74min, m/z: 241 (M−31) , 199, 167, 112,
8 (base peak).
R)-MTPA ester of 9, ESI-TOF-MS m/z: 1163.5035 [M+Na]+
Calcd for C H F NaO , 1163.4690); H-NMR (CDCl ) δ:
+
Conflict of Interest The authors declare no conflict of
interest.
15
28
4
R
9
(
1
(
Supplementary Materials The online version of this ar-
63
69
9
9
3
1
2
4
.797 (2H, m, H-28), 1.806 (1H, m, H-7), 1.857 (1H, m, H-7), ticle contains supplementary materials.
.274 (2H, q, H-17), 2.342 (2H, t, H-22), 2.557 (1H, s, H-33),
.886–4.998 (2H, ddd, H-1), 5.432 (1H, d, H-19), 5.498 (1H, References
dd, H-30), 5.548 (0.5H, t, H-6), 5.573 (0.5H, t, H-6), 5.794
1H, q, H-18), 6.013 (1H, m, H-29), 6.033 (1H, d, H-31),
.542 (3H, s, OMe), 3.568 (3H, s, OMe), 3.577 (3H, s, OMe),
1) Jung H. J., Im S. K., Bae H. B., “Biomaterials from Aquatic and
Terrestial Organisms, Chap. 12, Bioactive Polyacetylenic Com-
pounds from Marine Sponges,” ed. by Fingerman M., Nagabhusha-
nam R., Science Publishers, New Hampshire, 2006.
(
3
7.392–7.516 (15H, phenyl); (S)-MTPA ester of 9, ESI-TOF-MS
+
2) Bohlmann F., Lam J., Breteler H., Arnason T., Hansen L., “Chem-
istry and Biology of Naturally Occurring Acetylenes and Related
Compounds,” Elsevier, Amsterdam, 1998.
m/z: 1163.4941 [M+Na] (Calcd for C H F NaO , 1163.4690);
63
69
9
9
1
H-NMR (CDCl ) δ: 1.837 (2H, m, H-28), 1.793 (1H, m, H-7),
3
1.857 (1H, m, H-7), 2.274 (2H, q, H-17), 2.343 (2H, t, H-22),
3)
Bohlmann F., Burkhardt T., Zdero C., “Naturally Occuring Acety-
lenes,” Academic Press, London and New York, 1973.
2
.517 (1H, s, H-33), 4.886–4.997 (2H, ddd, H-1), 5.432 (1H, d,
H-19), 5.597 (1H, dd, H-30), 5.545 (0.5H, t, H-6), 5.573 (0.5H,
t, H-6), 5.795 (1H, q, H-18), 6.064 (1H, m, H-29), 5.853 (1H, d,
H-31), 3.542 (3H, s, OMe), 3.568 (3H, s, OMe), 3.577 (3H, s,
OMe), 7.392–7.516 (15H, phenyl).
4) Perry N. B., Span E. M., Zidorn C., Tetrahedron Lett., 42, 4325–
4328 (2001).
5) Allen E. H., Thomas C. A., Phytochemistry, 10, 1579–1582 (1971).
6
)
Shirouzu T., Watari K., Ono M., Koizumi K., Saiki I., Tanaka C.,
van Soest R. W. M., Miyamoto T., J. Nat. Prod., 76, 1337–1342
Cell Culture and Cell Proliferation Assay Human
malignant epithelial cells (HeLa) were cultured in Eagle’s
minimum essential medium (EMEM) supplemented with 10%
fetal bovine serum (FBS) kept in an incubator at 37°C in a
(2013).
7
)
The genus of Petrosia was classified to Strongylophora and Ha-
lichondria was classified to Pellina until 2002. De Weerdt W. H.,
“
Family Chalinidae Gary. 1867 in Systema Porifera, a Guide to the
humidified air containing 5% CO . FBS was purchased from
2
Classification of Sponges,” ed. by Hooper J. N. A., Van Soest R. W.
M., Kluwer Academic/Plenum Publishers, New York, 2002.
Fu X., Abbas S. A., Schmitz F. J., Vidavsky I., Gross M. L., Laney
M., Schatzman R. C., Cabuslay R. D., Tetrahedron, 53, 799–814
(1997).
Nichirei Bioscience Inc. (Tokyo, Japan). Human immortal-
ized myelogenous leukemia cells (K562) were cultured in
RPMI-1640 medium supplemented with 10% FBS kept in an
8)
incubator at 37°C in a humidified air containing 5% CO . FBS
2
was purchased from Nichirei Bioscience Inc. Cell viability
was determined by a Cell-Titer 96 AQueous Non-Radioactive
Cell Proliferation (MTS) Assay (Promega, WI, U.S.A.) ac-
9) Ohtani I., Kusumi T., Kashman Y., Kakisawa H., J. Am. Chem.
Soc., 113, 4092–4096 (1991).
0) Fusetani N., Yasumuro K., Matsunaga S., Hirota H., Tetrahedron
Lett., 30, 6891–6894 (1989).
11) Zhou G.-X., Molinski T. F., Mar. Drugs, 1, 46–53 (2003).
1
cording to the manufacturer’s protocol. HeLa and K562 cells
4
(
3×10 cells/well) were seeded in 96 well plates and incubated
1
2) Braekman J. C., Daloze D., Hulot G., Tursch B., Declercq J. P.,
for 24h, subsequently grown with compounds for additional
8h, and then cell proliferation assay was performed.
Germain G., Van Meerssche M., Bull. Soc. Chim. Belg., 87, 917–926
4
(1978).