(-)-Duryne and its homologues, cytotoxic acetylenes from a marine sponge Petrosia sp.

Article abstract of DOI:10.1021/np200271n

Six linear acetylenes, (-)-duryne (1) and (-)-durynes B-F (2-6), were isolated from the marine sponge Petrosia sp. Their structures were elucidated by NMR and tandem FABMS analyses. The positions of the olefinic bonds were confirmed by ozonolysis experiments, and the absolute configurations were determined by the modified Mosher's method. Compound 1 was found to be the enantiomer of duryne, a previously reported sponge metabolite. Compounds 1-6 show cytotoxicity against HeLa cells with IC₅₀ values between 0.08 and 0.50 μM. (Chemical Equation Presented).

Full text of DOI:10.1021/np200271n

ARTICLE
pubs.acs.org/jnp
(
ꢀ)-Duryne and Its Homologues, Cytotoxic Acetylenes from a Marine
Sponge Petrosia sp.
Yuki Hitora, Kentaro Takada, Shigeru Okada, Yuji Ise, and Shigeki Matsunaga*
†
†
†
‡
,†
†
Laboratory of Aquatic Natural Products Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo,
Bunkyo-ku, Tokyo, 113-8657, Japan
‡
Misaki Marine Biological Station, Graduate School of Science, The University of Tokyo, Miura, Kanagawa, 238-0225, Japan
S Supporting Information
b
ABSTRACT: Six linear acetylenes, (ꢀ)-duryne (1) and (ꢀ)-durynes
BꢀF (2ꢀ6), were isolated from the marine sponge Petrosia sp. Their
structures were elucidated by NMR and tandem FABMS analyses. The
positions of the olefinic bonds were confirmed by ozonolysis experi-
ments, and the absolute configurations were determined by the
modified Mosher’s method. Compound 1 was found to be the
enantiomer of duryne, a previously reported sponge metabolite.
Compounds 1ꢀ6 show cytotoxicity against HeLa cells with IC
50
values between 0.08 and 0.50 μM.
inear acetylenes are prominent constituents of marine
sponges of the order Haplosclerida. A classification of this
1
L
class of metabolites from a chemical perspective has been
2
reported. There are two distinct groups of acetylenes with both
termini functionalized. One group contains compounds related
to the petroformynes, which possess chains of C or longer with
4
6
3
ꢀ7
a carbinol-interrupted diyne in the middle of the chain.
Compounds in the other group are shorter, with chains of
around C , and do not contain the carbinol-interrupted diyne
3
0
8
ꢀ10
moiety.
Even though their modes of action have not been
elucidated, compounds in these two groups are reported to
exhibit potent cytotoxic activity. In the course of our screening
for cytotoxic activity against HeLa cells, the extract of a marine
sponge Petrosia sp. exhibited significant activity. Bioassay-guided
fractionation of the extract afforded six related compounds,
(
ꢀ)-duryne (1) and its homologues (ꢀ)-durynes BꢀF (2ꢀ6).
This is the first isolation of the (ꢀ)-duryne as a natural product.
Duryne, the (þ)-enantiomer, was isolated from the marine
Compound 1, which analyzed for C30H O by HRESIMS,
48 2
8
1
sponge Cribrochalina dura, the taxonomy of which was later
exhibited H NMR signals characteristic of the terminal structure
of sponge-derived acetylenes [δ 2.57 (d, J = 2.1 Hz; H-1, H-30),
4.84 (brd, J = 6.4 Hz; H-3, H-28), 5.61 (dd, J = 15.1, 6.4 Hz; H-4,
H-27), and 5.92 (dt, J = 15.1, 6.9 Hz; H-5, H-26)]. A literature
2
suggested to be Petrosia, and its absolute configuration was
determined by synthesis of both enantiomers.
11
1
13
search showed that the H and C NMR data of 1 (Table 1)
’
RESULTS AND DISCUSSION
The sponge Petrosia sp. was collected by a remotely operated
were identical with those of duryne. However, an opposite sign
with a similar magnitude for the specific rotation ([R] = ꢀ19)
D
vehicle at Miyako sea-knoll. The sponge specimen was extracted
with EtOH and MeOH/CHCl (1:1), and the combined extracts
were concentrated and partitioned between CHCl and H O.
The CHCl layer was further partitioned between 90% MeOH
and n-hexane. Bioassay-guided fractionation of the 90% MeOH
fraction by ODS column chromatography, silica gel column
chromatography, and reversed-phase HPLC afforded six cyto-
toxic compounds (1ꢀ6).
showed that 1 was the enantiomer of the previously reported
3
8,11
duryne ([R] = þ29). The modified Mosher’s analysis of 1
D
3
2
12
supported this assignment (Figure 1). Therefore, 1 was firmly
3
13
established to be (ꢀ)-duryne.
Received: March 30, 2011
Published: May 02, 2011
Copyright r 2011 American Chemical Society and
American Society of Pharmacognosy
1262
dx.doi.org/10.1021/np200271n |J. Nat. Prod. 2011, 74, 1262–1267

Journal of Natural Products
ARTICLE
1
13
Table 1. H and C NMR Data (CDCl ) for (ꢀ)-Duryne (1), (ꢀ)-Duryne B (2), and (ꢀ)-Duryne F (6)
3
(
ꢀ)-duryne (1)
(ꢀ)-duryne B (2)
(ꢀ)-duryne F (6)
a
C
b
C
b
C
position
, 30
δ
δ
H
(J in Hz)
position
δ
δ
H
(J in Hz)
position
δ
H
δ (J in Hz)
1
2
3
4
5
6
7
8
1
1
1
74.1
83.5
2.57, d (2.1)
1
73.9
83.4
2.57, d (2.1)
1
74.1
83.4
2.57, d (2.1)
, 29
2
2
, 28
63.0
4.84, d (6.4)
5.61, dd (15.1, 6.4)
5.92, dt (15.1, 6.9)
2.07, m
3
62.8
4.84, brd (6.2)
5.61, dd (15.3, 6.2)
5.92, dt (15.3, 6.9)
2.07, m
3
62.8
4.84, brd (6.2)
5.62, dd (15.1, 6.2)
5.92, dt (15.1, 6.9)
2.07, m
, 27
128.3
134.8
32.1
4
128.3
134.5
31.9
4
128.1
134.4
31.8
, 26
5
5
, 25
6
6
c
, 24
30.0
1.40, m
7
28.7
1.40, m
7
28.9
1.40, m
c
ꢀ12, 19ꢀ23
3, 18
4, 17
5, 16
29.4ꢀ29.8
29.0
1.27ꢀ1.30, m
1.32, m
8ꢀ13
14
15
16
28.7ꢀ29.7
27.4
1.27ꢀ1.36
2.02, m
8ꢀ13
14
15
16
17
18ꢀ26
27R
27β
28
29
30
31
32
28.7ꢀ30.0
27.3
1.27ꢀ1.34
2.01, m
c
27.4
2.02, m
129.9
129.9
27.4
5.35, m
129.9
5.36, m
129.9
5.35, m
5.35, m
129.9
5.36, m
1
1
2
2
2
2
3
3
3
7
2.02, m
27.3
2.01, m
8ꢀ25
28.7ꢀ29.7
28.7
1.27ꢀ1.36
1.40, m
28.7ꢀ30.0
36.3
1.27ꢀ1.34
1.63, m
6
7
8
9
0
1
2
31.9
2.07, m
1.52, m
134.5
128.3
62.8
5.92, dt (15.3, 6.9)
5.61, dd (15.3, 6.2)
4.84, brd (6.2)
70.0
147.5
108.7
82.4
4.68, m
6.00, dd (11.0, 8.9)
5.54, dd (11.0, 2.5)
83.4
73.9
2.57, d (2.1)
79.5
3.14, d (2.5)
a 13
b
c
C NMR spectrum was measured at 150 MHz. Chemical shifts were assigned from the HSQC data. Assignments with the same superscript in the
same column may be interchanged.
1
1,17
13,17
were assigned by tandem FABMS to be Δ
and Δ
for 3, Δ
for 4,
1
5,21
for 5 (Figure 2). These assignments were confirmed
by the ozonolysis experiments (Scheme 1). Ozonolysis of 3, 4,
and 5 followed by oxidative workup afforded common FABMS
ion peaks at m/z 145 and 215, which suggested the formation of
adipic acid and undecanedioic acid, respectively. Additional
ions were observed at m/z 159 and 187 in the ozonolysis
products of 3 and 4, respectively, consistent with the formation
of heptanedioic acid and nonanedioic acid. The absolute
configurations of the secondary alcohols in the three com-
pounds were all determined as R by the modified Mosher’s
Figure 1. Modified Mosher's analysis for 1. Δδ(δ
S
ꢀ δ
R
) values are shown.
(
ꢀ)-Duryne B (2) had the molecular formula C H O , as
32 52 2
1
13
assigned by HRESIMS. The H and C NMR spectra of 2 were
identical with those of 1 except for the integration of the
methylene region (Table 1), suggesting that they differ only in
the length of the aliphatic chains. The position of the Z-olefin in
the middle of the chain was assigned by tandem FABMS to be
between C-15 and C-16 (Figure 2). The geometry of the
1
2
method.
HRESIMS showed that (ꢀ)-duryne F (6) was an isomer of 2.
1
5
The structure of one end was identical with those in the other
Δ -double bond was assigned by the chemical shifts of
1
13
14
compounds, as judged from the H and C NMR data (Table 1),
whereas at the other end, an acetylenic proton (δ 3.14, J = 2.5 Hz,
H-32) was coupled to a Z-olefinic proton (δ 5.54, J = 11.0, 2.5 Hz;
H-30), which was then coupled to another olefinic proton (δ 6.00,
J = 11.0, 8.9 Hz; H-29). This latter proton, H-29, was further
coupled to an oxymethine proton (δ 4.68, H-28), indicating a
rearranged terminus relative to the commonly observed motif.
There was a degenerate olefinic signal integrating for 2H, which
was assumed as a Z-olefin in the middle of the chain. The
Z-geometry was again assigned by the chemical shifts of the allylic
2
7.4 ppm for C-14 and C-17. The positions of the double
bonds were confirmed by ozonolysis with oxidative workup,
ꢀ
which gave FABMS ions at m/z 215 and 243 [M ꢀ H] ,
consistent with the formation of undecanedioic acid and tride-
canedioic acid, respectively (Scheme 1). The configurations of
the two secondary alcohols were both determined to be R by the
12
modified Mosher’s method.
ꢀ)-Durynes CꢀE (3ꢀ5) had molecular formulas of
C H O , C H O , and C H O , respectively, as assigned
(
3
2
50
2
34 54
2
36 58
2
1
by HRESIMS. The H NMR data of 3ꢀ5 were identical with
those of 1, except for doubled olefinic and allylic proton peaks
representing the central double bonds and an increased methy-
lene peak at δ 1.27ꢀ1.34, suggesting that 3ꢀ5 have longer
aliphatic chains and one additional double bond in their chains
14
carbons. Tandem FABMS data were not sufficient to locate the
double bond, because it was not possible to distinguish the two
termini by the MS data. Again we resorted an ozonolysis experiment
that afforded a reaction mixture with FABMS ion peaks at m/z 215
and 273, consistent with the formation of undecanoic acid and
2-hydroxytetradecanoic acid, respectively, demonstrating the loca-
tion of the double bond between C-15 and C-16 (Scheme 1). The
(
Table 2). The geometries of the olefins in the middle of the
chain in 3ꢀ5 were all assigned as Z judging from the chemical
1
4
shifts of the allylic carbons. The locations of the two Z-olefins
1
263
dx.doi.org/10.1021/np200271n |J. Nat. Prod. 2011, 74, 1262–1267

Journal of Natural Products
ARTICLE
Figure 2. Key tandem FABMS fragmentations for 1ꢀ6 by using the [M þ Li]þ ion as the precursor.
absolute configuration was determined to be 3R, 28S by the
600 NMR spectrometer at 300 K. Chemical shifts were referenced to
solvent peaks: δ 7.27 and δ 77.2 for CDCl . ESI mass spectra were
12
modified Mosher’s method.
ꢀ)-Duryne (1) and (ꢀ)-durynes BꢀF (2ꢀ6) show cytotoxic
activity against HeLa cells with IC values of 0.50, 0.26, 0.26, 0.10,
H
C
3
(
measured on a JEOL JMS-T100LC. FABMS and FABMSMS were
measured on a JEOL JMS-700T. HPLC was carried out on a Shimadzu
LC 20AT with a SCL-10Avp controller and a SPD-10Avp detector.
Animal Material. The sponge Petrosia sp. was collected at Miyako
5
0
0
.08, and 0.34 μM, respectively. They are equipotent to duryne,
whose reported IC values are 0.16ꢀ0.23 μM against P388, HCT-8,
50
8
0
0
sea-knoll, southwestern Japan, 25°25.64 N, 125°42.48 E, at a depth of
15 m during a cruise of R/V Natsushima by using the remotely operated
A549, and MCF7 cell lines. The configuration of the allylic
propargylic alcohol is variable among acetylenes from Petrosia. Only
4
9,15
16
vehicle “Hyper-Dolphin” in October 2009. Sponge description: sub-
globular with an optically smooth surface; a single large oscule with a
peripheral sleeve opened on top of the body; color beige; consistency
hard and crumbly. The ectosomal skeleton is different from the
choanosomal skeleton, consisting of a tangential unispicular network.
The choanosomal skeleton consists of an isotropic network. Megascleres
strongyles appeared in two size classes, with few oxeas. Microscleres
were absent. The comparable species are P. spheroida Tanita, 1967 and P.
solusstrongyla Hoshino, 1981 on the basis of its external morphology. P.
spheroida is distinguished from the present species by its bean-shaped
microstrongyles. P. solusstrongyla has strongyles only, and the form and
size of strongyles are variable. The habitat of the present species is also
different from the two species. P. spheroida is reported from the shallow
water of the southwestern region of the Sea of Japan, and P. solusstrongyla
is reported from the subtidal zone to a depth of 15 m in Kagoshima. The
specimen used for the identification (NSMT-Po1954) was deposited at
National Museum of Nature and Science, Tokyo.
the S-isomers were reported for the petrosynols, adociacetylenes,
18,19
8
17
7
duryne, petroformynes, neopetroformynes, petrocortynes,
20
and the petroformynic acids. They invariably exhibit a positive sign
of optical rotation. However, the R-configuration was assigned for
the corresponding carbinol carbons in the triangulynes, all of which
showed a negative sign of optical rotation. Curiously scalemic
C -acetylenes were isolated from a Korean Petrosia sp.
21
22,23
It is
30
interesting to note that (ꢀ)-duryne was equipotent to its enantiomer,
suggesting that the cellular targets of these metabolites do not
recognize the configuration of the two secondary alcohols. The more
potent activity exhibited by the longer molecules is noticeable.
’
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were mea-
sured on a JASCO DIP-1000 digital polarimeter. UV spectra were measured
on a Shimadzu Biospec 1600. IR spectra were measured with a JASCO FT-
IR-230 spectrophotometer. NMR spectra were recorded on a JEOL alpha
Extraction and Isolation. The sponge was frozen after collection
and kept frozen until extraction. The sponge (130 g, wet weight) was
1
264
dx.doi.org/10.1021/np200271n |J. Nat. Prod. 2011, 74, 1262–1267

Journal of Natural Products
ARTICLE
1
13
Table 2. H and C NMR Data (CDCl ) for (ꢀ)-Duryne C (3), (ꢀ)-Duryne D (4), and (ꢀ)-Duryne E (5)
3
(ꢀ)-duryne C (3)
(ꢀ)-duryne D (4)
(ꢀ)-duryne E (5)
b
a
b
position
δ
C
δ
H
(J in Hz)
position
δ
C
δ
H
(J in Hz)
position
1, 36
δ
C
H
δ (J in Hz)
1
2
3
4
5
6
7
8
9
73.9
83.3
2.57, d (2.1)
1
74.1
83.6
2.57, d (2.1)
74.1
83.2
2.57, d (2.2)
2
2, 35
62.8
4.84, brd (6.2)
5.61, dd (15.1, 6.2)
5.92, dt (15.1, 6.9)
2.07, m
3
62.9
4.84, brd (6.2)
5.61, dd (15.1, 6.2)
5.92, dt (15.1, 6.9)
2.07, m
3, 34
62.8
4.84, brd (6.2)
5.61, dd (15.1, 6.2)
5.93, dt (15.1,6.9)
2.07, m
128.4
134.5
31.9
4
128.6
134.7
32.1
4, 33
128.2
134.5
31.9
5
5, 32
6
6, 31
28.7
1.40, m
7
29.0
1.40, m
7, 30
28.7
1.40, m
29.1ꢀ29.4
29.4
1.28ꢀ1.33
1.36,m
8ꢀ10
11
12
13
14
15
16
17
18
19
20
21
22
23ꢀ27
28
29
30
31
32
33
34
29.3ꢀ29.9
29.5
1.28ꢀ1.32
1.36, m
8ꢀ12, 25ꢀ29
13, 24
14, 23
15, 22
16, 21
17, 20
18, 19
28.7ꢀ29.7
29.4
1.27ꢀ1.34
1.36, m
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
3
3
0
27.1
2.02, m
27.1
2.03, m
27.1
2.03, m
1
130.0
130.0
27.1
5.36, m
129.9
5.35, m
129.7
129.7
27.1
5.36, m
d
2
5.36, m
130.2
5.35, m
5.36, m
3
2.02, m
27.1
29.5
29.5
27.1
2.03, m
2.03, m
4
29.4
1.36, m
1.36, m
29.4
1.36, m
5
29.4
1.36, m
1.36, m
6
27.1
2.02, m
2.03, m
d
7
130.0
130.0
27.1
5.36, m
130.1
5.35, m
8
5.36, m
129.9
27.1
5.35, m
9
2.02, m
2.03, m
0
29.4
1.36, m
29.5
1.36, m
1ꢀ25
29.1ꢀ29.4
28.7
1.28ꢀ1.33
1.40, m
29.3ꢀ29.9
29.0
1.28ꢀ1.32
1.40, m
6
7
8
9
0
1
2
31.9
2.07, m
32.1
2.07, m
134.5
128.4
62.8
5.92, dt (15.1, 6.9)
5.61, dd (15.1, 6.2)
4.84, brd (6.2)
134.7
128.6
62.9
5.92, dt (15.1, 6.9)
5.61, dd (15.1, 6.2)
4.84, brd (6.2)
83.3
83.6
3
a 13
73.9
2.57, d (2.1)
74.1
2.57, d (2.1)
b
d
C NMR spectrum was measured at 150 MHz. Chemical shifts were assigned from the HSQC data. Assignments with the same superscript in the
same column may be interchanged.
Scheme 1. Ozonolysis of 2ꢀ6
subjected to ODS column chromatography, eluting with 50% MeOH,
0% MeOH (IC50 10 μg/mL), 90% MeOH (IC50 10 μg/mL), MeOH
IC50 0.4 μg/mL), and CHCl /MeOH/H O (6:4:1, IC 22 μg/mL).
7
(
3
2
50
The MeOH eluate was concentrated and subjected to silica gel column
chromatography eluting with CHCl (IC 0.4 μg/mL), CHCl /MeOH
3
50
3
(
98:2, IC50 4.5 μg/mL), CHCl
CHCl /MeOH (9:1), CHCl /MeOH/H
MeOH/H O (7:3:0.5). The CHCl eluate was subjected to silica gel
3
/MeOH (95:5, IC50 4.5 μg/mL),
3
3
2
O (8:2:0.1), and CHCl
3
/
2
3
column chromatography, eluting with n-hexane/EtOAc (9:1, IC50 4.5
μg/mL), n-hexane/EtOAc (8:2, IC50 0.4 μg/mL), and n-hexane/EtOAc
(1:1, IC50 4.5 μg/mL). The n-hexane/EtOAc (8:2) eluate was concen-
trated and separated by ODS HPLC (Cosmosil AR-II; 10 ꢁ 250 mm)
with 90ꢀ100% MeOH to afford 14 fractions. The second fraction was
purified by ODS HPLC (Cosmosil MS-II; 10 ꢁ 250 mm) with 85%
MeCN to afford 1 (6.3 mg). The third fraction was purified by ODS
HPLC with 86% MeCN to afford 3 (2.0 mg). The sixth fraction was
purified by ODS HPLC with 90% MeCN to afford 6 (1.8 mg). The
seventh fraction was purified by ODS HPLC with 85% MeCN to afford
2 (15.6 mg). The eighth fraction was purified by ODS HPLC with 90%
MeCN to afford 4 (16.0 mg). The 13th fraction was purified by ODS
HPLC with 93% MeCN to afford 5 (1.2 mg).
homogenized and extracted with EtOH (350 mL ꢁ 3) and CHCl
3
/
MeOH (1:1) (350 mL). The combined extracts were concentrated and
partitioned between H
2
O (200 mL) and CHCl
3
(200 mL ꢁ 3). The
CHCl layer (IC value against HeLa cells 0.4 μg/mL) was further
3
50
1
7
partitioned between n-hexane (100 mL) and 90% MeOH (100 mL).
The 90% MeOH layer (IC50 0.4 μg/mL) was concentrated and
(ꢀ)-Duryne (1): white solids; [R] ꢀ19 (c 0.27, CHCl ); IR (film)
D
3
ν
3400ꢀ3360 (br), 3282, 2917, 2852, 2118, 1654, 1467, 1088,
max
1
265
dx.doi.org/10.1021/np200271n |J. Nat. Prod. 2011, 74, 1262–1267

Journal of Natural Products
ARTICLE
ꢀ
1
1
13
1
9
(
62 cm
;
H NMR data (CDCl
3
), see Table 1; C NMR data
(S)-MTPA Ester of 4 (4b): H NMR (CDCl
3
) δ 6.08 (H-5, H-30),
þ
CDCl ), see Table 1; HRESIMS m/z 463.3524 [M þ Na] (calcd
6.02 (H-3, H-32), 5.61 (H-4, H-31), 5.35 (H-13, H-14, H-19, H-20),
2.60 (H-1, H-34), 2.09 (H-6, H-29), 2.02 (H-12, H-15, H-18, H-21),
1.26ꢀ1.39 (H-7ꢀH-11, H-16ꢀH-17, H-22ꢀH-28); ESIMS m/z 949
3
for C30
H
48
O
2
Na, 463.3552).
26
D
(
ꢀ)-Duryne B (2): white solids; [R]
ꢀ17 (c 0.34, CHCl
3
); UV
þ
(
2
MeOH) λ (log ε) 206 (3.57); IR (film) ν 3440ꢀ3400 (br), 2916,
[M þ Na] .
max
max
ꢀ
1
1
1
849, 2121, 1647, 1468, 1215 cm ; H NMR data (CDCl ), see
(R)-MTPA Ester of 5 (5a): H NMR (CDCl
) δ 6.04 (H-3, H-34),
3
3
13
Table 1; C NMR data (CDCl
3
), see Table 1; HRESIMS m/z 491.3857
6.02 (H-5, H-32), 5.50 (H-4, H-33), 5.35 (H-15, H-16, H-21, H-22),
2.64 (H-1, H-36), 2.05 (H-6, H-31), 2.02 (H-14, H-17, H-20, H-23),
1.26ꢀ1.37 (H-7ꢀH-13, H-18ꢀH-19, H-24ꢀH-30); ESIMS m/z 977
þ
[
M þ Na] (calcd for C32
52 2
H O Na, 491.3865).
25
(
ꢀ)-Duryne C (3): white solids; [R]
D
ꢀ61 (c 0.05, CHCl
3
); UV
þ
(
3
MeOH) λ (log ε) 207 (3.60); IR (film) ν 3400ꢀ3360 (br), 3307,
[M þ Na] .
max
max
ꢀ
1
1
1
005, 2925, 2853, 2118, 1655, 1459, 1012, 968 cm ; H NMR data
(S)-MTPA Ester of 5 (5b): H NMR (CDCl
3
) δ 6.08 (H-5, H-32),
13
(CDCl
3
), see Table 2; C NMR data (CDCl
3
), see Table 2; HRESIMS
Na, 489.3708).
ꢀ38 (c 0.22, CHCl ); UV
6.02 (H-3, H-34), 5.61 (H-4, H-33), 5.35 (H-15, H-16, H-21, H-22),
2.60 (H-1, H-36), 2.09 (H-6, H-31), 2.02 (H-14, H-17, H-20, H-23),
1.27ꢀ1.41 (H-7ꢀH-13, H-18ꢀH-19, H-24ꢀH-30) ESIMS m/z 977
þ
m/z 489.3706 [M þ Na] (calcd for C 23 2
H
50 2
O
6
(
ꢀ)-Duryne D (4): white solids; [R]
D
3
þ
(
2
(
MeOH) λmax (log ε) 206 (3.59); IR (film) νmax 3400ꢀ3340 (br), 3290,
[M þ Na] .
ꢀ
1
1
1
997, 2920, 2849, 2120, 1654, 1459, 1215, 964 cm ; H NMR data
(R)-MTPA Ester of 6 (6a): H NMR(CDCl
) δ6.04 (H-3), 6.02 (H-5),
3
13
CDCl ), see Table 2; C NMR data (CDCl ), see Table 2; HRESIMS
5.95 (H-28), 5.83 (H-29), 5.64 (H-30), 5.36 (H-15, H-16), 5.50 (H-4),
3.25 (H-32), 2.64 (H-1), 2.05 (H-6), 2.02 (H-14, H-17), 1.81 (H-27R),
1.68 (H-27β), 1.26ꢀ1.36 (H-7ꢀH-13, H-18ꢀH-26); ESIMS m/z 923
3
3
þ
m/z 517.3986 [M þ Na] (calcd for C H O Na, 517.4021).
3
4 54 2
2
6
(
ꢀ)-Duryne E (5): white solids; [R]
D
ꢀ120 (c 0.02, MeOH); UV
1
þ
(
MeOH) λmax (log ε) 210 (3.73); H NMR data (CDCl ), see Table 2;
3
[M þ Na] .
1
3
1
C NMR data (CDCl ), see Table 2; HRESIMS m/z 545.4351
(S)-MTPA Ester of 6 (6b): H NMR (CDCl
3
) δ 6.08 (H-5), 6.02 (H-
3
þ
[
M þ Na] (calcd for C36
H
58
O
2
Na, 545.4334).
3), 6.00 (H-29), 5.96 (H-28), 5.67 (H-30), 5.61 (H-4), 5.36 (H-15,
H-16), 3.26 (H-32), 2.60 (H-1), 2.09 (H-6), 2.02 (H-14, H-17), 1.74
(H-27R), 1.23ꢀ1.61 (H-7ꢀH-13, H-18ꢀH-26, H-27β); ESIMS m/z
26
D
(
ꢀ)-Duryne F (6): white solids; [R]
ꢀ32 (c 0.04, CHCl
3
); UV
(
2
MeOH) λmax (log ε) 225 (3.76); IR (film) νmax 3400ꢀ3360 (br), 3308,
ꢀ
1 1
þ
922, 2851, 2120, 1655, 1459, 1014, 968 cm ; H NMR data (CDCl ),
923 [M þ Na] .
3
13
see Table 1; C NMR data (CDCl
3
), see Table 1; HRESIMS m/z
Na, 491.3865).
Ozonolysis. Separately, solutions of acetylenes 2ꢀ6 (0.5 mg each)
4
91.3846 [M þ Na]þ (calcd for C32
H
52
O
2
in CH
2
Cl
2
(1 mL) were treated with O
3
at ꢀ78 °C for 15 min. After
excess O was removed by a stream of N , the reaction mixtures were
treated with 90% HCOOH/35% H O (2:1, 1 mL) at room tempera-
ture for 15 min. The products were concentrated and subjected to
FABMS without further separation.
Preparation of MTPA Esters. To solutions of samples 1ꢀ6 (0.1
3
2
mg each) in dry pyridine (10 μL) was added (S)-MTPACl (2 μL). The
2 2
solutions were left at room temperature for 15 h, and the reaction mixtures
were diluted with H
were purified by reversed-phase HPLC to afford the (R)-MTPA esters.
S)-MTPA esters were prepared in a similar way using (R)-MTPACl.
R)-MTPA Ester of 1 (1a): H NMR (CDCl
.02 (H-5, H-26), 5.50 (H-4, H-27), 5.35 (H-15, H-16), 2.64 (H-1,
2 3
O and extracted with CHCl . The organic layers
Cytotoxicity Assay. The cytotoxicities of 1ꢀ6 against HeLa cells
2
4
were evaluated by an MTT assay. HeLa human cervical cancer cells
were cultured in Dulbecco’s modified Eagle’s medium containing 10%
fetal bovine serum, 2 μg/mL gentamycin, and 2 μg/mL antibio-
(
1
(
3
) δ 6.04 (H-3, H-28),
6
ticꢀantimicotic (Gibco) at 37 °C under an atmosphere of 5% CO
2
.
H-30), 2.05 (H-6, H-25), 2.02 (H-14, H-17), 1.26ꢀ1.36 (H-7ꢀH-13,
þ
To each well of a 96-well microplate containing 200 μL of tumor cell
H-18ꢀH-24): ESIMS m/z 895 [M þ Na] .
4
1
suspension (1 ꢁ 10 cells/mL) was added a sample after 24 h
(
S)-MTPA Ester of 1 (1b): H NMR (CDCl
3
) δ 6.08 (H-5, H-26),
preincubation, and the plate was incubated for 72 h. After addition of
6
.02 (H-3, H-28), 5.61 (H-4, H-27), 5.35 (H-15, H-16), 2.60 (H-1,
3
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
saline solution (1 mg/mL, 50 μL) to each well, the plate was incubated
for 3 h. After the incubation, the supernatant was discarded and DMSO
150 μL) was added. The absorbance was measured to determine
IC50 values. In this assay, adriamycin was used as a positive control
IC50 1.64 μM).
H-30), 2.09 (H-6, H-25), 2.02 (H-14, H-17), 1.27ꢀ1.39 (H-7ꢀH-13,
þ
H-18ꢀH-24): ESIMS m/z 895 [M þ Na] .
1
(
R)-MTPA Ester of 2 (2a): H NMR (CDCl
3
) δ 6.04 (H-3, H-30),
(
6
.02 (H-5, H-28), 5.50 (H-4, H-29), 5.35 (H-15, H-16), 2.64 (H-1,
H-32), 2.05 (H-6, H-27), 2.02 (H-14, H-17), 1.26ꢀ1.37 (H-7ꢀH-13,
þ
(
H-18ꢀH-26); ESIMS m/z 923 [M þ Na] .
1
(
S)-MTPA Ester of 2 (2b): H NMR (CDCl
3
) δ 6.08 (H-5, H-28),
6
.02 (H-3, H-30), 5.61 (H-4, H-29), 5.35 (H-15, H-16), 2.60 (H-1,
’ ASSOCIATED CONTENT
Supporting Information. NMR data and tandem FABMS
H-32), 2.09 (H-6, H-27), 2.02 (H-14, H-17), 1.27ꢀ1.40 (H-7ꢀH-13,
þ
S
H-18ꢀH-26); ESIMS m/z 923 [M þ Na] .
b
1
(
R)-MTPA Ester of 3 (3a): H NMR (CDCl
3
) δ 6.04 (H-3, H-30),
data for 1ꢀ6and photograph of the sponge. This material is available
6
2
1
.02 (H-5, H-28), 5.50 (H-4, H-29), 5.35 (H-11, H-12, H-17, H-18),
.64 (H-1, H-32), 2.05 (H-6, H-27), 2.02 (H-10, H-13, H-16, H-19),
.27ꢀ1.38 (H-7ꢀH-9, H-14ꢀH-15, H-20ꢀH-26); ESIMS m/z 921
free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
þ
[M þ Na] .
1
Corresponding Author
*
(
S)-MTPA Ester of 3 (3b): H NMR (CDCl ) δ 6.08 (H-5, H-28),
3
Tel: 81-3-5841-5297. Fax: 81-3-5841-8166. E-mail: assmats@
6
2
1
.02 (H-3, H-30), 5.61 (H-4, H-29), 5.35 (H-11, H-12, H-17, H-18),
.60 (H-1, H-32), 2.09 (H-6, H-27), 2.02 (H-10, H-13, H-16, H-19),
.27ꢀ1.40 (H-7ꢀH-9, H-14ꢀH-15, H-20ꢀH-26); ESIMS m/z 921 [M
mail.ecc.u-tokyo.ac.jp.
þ
þ Na] .
1
’ ACKNOWLEDGMENT
(
3
R)-MTPA Ester of 4 (4a): H NMR (CDCl ) δ 6.04 (H-3, H-32),
6
2
1
.02 (H-5, H-30), 5.50 (H-4, H-31), 5.35 (H-13, H-14, H-19, H-20),
.64 (H-1, H-34), 2.05 (H-6, H-29), 2.02 (H-12, H-15, H-18, H-21),
The sponge was collected during the NT-09-17 cruise of
Natsushima, JAMSTEC Japan Agency for Marine-Earth Science
and Technology. We thank Dr. Y. Ito, JEOL, for valuable advice
about the measurement of tandem FABMS.
.26ꢀ1.36 (H-7ꢀH-11, H-16ꢀH-17, H-22ꢀH-28); ESIMS m/z 949
þ
[M þ Na] .
1
266
dx.doi.org/10.1021/np200271n |J. Nat. Prod. 2011, 74, 1262–1267

Journal of Natural Products
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