Sesquiterpenes from the hainan sponge Dysidea septosa

Article abstract of DOI:10.1021/np8002035

Five new sesquiterpenes, named lingshuiolides A (1) and B (2), lingshuiperoxide (6), isodysetherin (10), and spirolingshuiolide (12), along with several known related analogues (7-9, 11, and 13-15), were isolated from the Hainan marine sponge Dysidea septosa. The structures of new compounds 1, 2, 6, 10, and 12 were determined by detailed analysis of 1D and 2D NMR spectra and by comparison with related model compounds. The absolute configuration of lingshuiolide B (2) was established by using the modified Mosher's method. Spirolingshuiolide (12) represents the first example of a sesquiterpene with a rearranged drimane skeleton. Compounds 7-9 and 15 exhibited significant inhibitory activity against human protein tyrosine phosphatase 1B (hPTP1B), an enzyme involved in the regulation of insulin signaling. In particular, 15 showed the most potent effect, with an IC₅₀ value of 1.9 μg/mL.

Full text of DOI:10.1021/np8002035

J. Nat. Prod. 2008, 71, 1399–1403
1399
Sesquiterpenes from the Hainan Sponge Dysidea septosa
Xiao-Chun Huang, Jia Li, Zhen-Yu Li, Lei Shi, and Yue-Wei Guo*
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203,
People’s Republic of China
ReceiVed April 1, 2008
Five new sesquiterpenes, named lingshuiolides A (1) and B (2), lingshuiperoxide (6), isodysetherin (10), and
spirolingshuiolide (12), along with several known related analogues (7-9, 11, and 13-15), were isolated from the
Hainan marine sponge Dysidea septosa. The structures of new compounds 1, 2, 6, 10, and 12 were determined by
detailed analysis of 1D and 2D NMR spectra and by comparison with related model compounds. The absolute
configuration of lingshuiolide B (2) was established by using the modified Mosher’s method. Spirolingshuiolide (12)
represents the first example of a sesquiterpene with a rearranged drimane skeleton. Compounds 7-9 and 15 exhibited
significant inhibitory activity against human protein tyrosine phosphatase 1B (hPTP1B), an enzyme involved in the
regulation of insulin signaling. In particular, 15 showed the most potent effect, with an IC50 value of 1.9 µg/mL.
Numerous investigations on the chemical composition of marine
sponges of the genus Dysidea have been carried out, and many
novel metabolites spanning a wide range of structure classes and
biosynthetic origins were discovered. In particular, many Dysidea
sponge metabolites exhibited various promising biological activities,
ranging from ichthyotoxic, to cytotoxic, to anti-inflammatory, which
attracted considerable attention from natural product chemists and
pharmacologists.1
Chart 1
As part of our ongoing research on the biologically active
substances from Chinese marine invertebrates,2–5 we made a
collection of the sponge Dysidea septosa off Lingshui Bay, Hainan
Province, China. Separation of the Et2O-soluble fraction of the
acetone extract of this sponge resulted in the isolation of five new
sesquiterpenes, namely, lingshuiolides A (1) and B (2), lingshuiper-
oxide (6), isodysetherin (10), and spirolingshuidolide (12), together
with several known analogues (7-9, 11, and 13-15). This paper
describes the isolation and structure elucidation of these new
compounds.
Results and Discussion
The acetone extract of the sponge D. septosa was divided into
Et2O- and BuOH-soluble portions. Then, the Et2O extract was
repeatedly chromatographed on silica gel columns and reversed-
phase HPLC to yield pure compounds 1, 2, 6-12, and 13-15,
respectively.
The structures of known compounds were readily identified as
hydroxybutenolide (7),6 microcionin-4 (8),7 dihydropallescensin-2
(9),8 dysetherin (11),9,10 furodysinin 3ꢀ,4ꢀ-epoxy lactone (13),11
furodysinin lactone (14),12 and nakafuran-8 (15),13 respectively,
by comparison of their NMR data with those reported in the
literature.
Among the five new sesquiterpenes, compounds 1, 2, and 6
showed NMR data characteristic for a γ-hydroxyl-bearing buteno-
lide moiety, like that of co-occurring 7, while the NMR spectra of
10 and 12 were reminiscent of those of co-occurring 13 and 14,
displaying a common epoxy-bearing butanolactone segment. The
structural elucidation of these new metabolites is described as
follows.
Lingshuiolide A (1), a colorless oil, showed a molecular ion peak
at m/z 266 (M+) in the EIMS, and the molecular formula, C15H22O4,
was established by HREIMS (m/z 266.1532, calcd 266.1518, ∆ )
-1.4 mmu), indicating five degrees of unsaturation. The IR
spectrum showed absorption bands at 3376 and 1762 cm-1
assignable to a hydroxyl group and an ester carbonyl group,
respectively. The 1H NMR spectrum displayed a broad one-proton
singlet at δH 6.85 indicating an R,ꢀ-unsaturated γ-lactone, a broad
one-proton singlet at δH 6.06 suggesting a hydrogen atom on the
carbon bearing the lactone oxygen and hydroxyl oxygen atoms,
one methyl singlet and a one-proton doublet at δH 1.66 and 5.85
(d, J ) 4.6 Hz), respectively, ascribed to a Me-bearing trisubstituted
double bond, and a one-proton broad singlet at δH 4.06 indicative
of a hydrogen on a carbon bearing an allylic hydroxyl group. The
13C NMR spectrum (Table 1) exhibited 15 signals (3CH3, 3CH2,
5CH, 4C), whose chemical shift values and multiplicities (DEPT)
confirmed the presence of an R,γ-disubstituted butenolide moiety
[δC 97.2 (CH), 138.3 (qC), 143.7 (CH), and 172.3 (qC)], a Me-
bearing trisubstituted double bond [δC 19.1 (CH3), 145.1 (qC), 126.1
* Corresponding author. Tel: 86-21-50805813. Fax: 86-21-50805813.
E-mail: ywguo@mail.shcnc.ac.cn.
10.1021/np8002035 CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/16/2008

1400 Journal of Natural Products, 2008, Vol. 71, No. 8
Table 1. 13C NMR Data of Compounds 1, 2, 6, 10, and 12a
Huang et al.
carbon
1
2
6
10
12
1
2
3
4
5
6
145.1, qC
141.6, qC
81.0, qC
48.4, qC
58.6, qC
41.9, CH2 30.8, CH2
130.8, CH
127.9, CH
126.1, CH 129.5, CH 137.9, CH
64.5, CH 67.8, CH 130.1, CH
35.7, CH2 37.0, CH2 76.3, CH
27.8, CH
41.0, qC
38.5, CH
31.6, CH2 38.4, CH 121.8, CH
40.8, qC 40.7, qC 135.4, qC
31.2, CH
48.0, qC
34.5, CH2
7
8
33.5, CH2 33.1, CH2 35.1, CH2 30.4, CH2 31.1, CH2
20.4, CH2 20.2, CH2 20.9, CH2 20.9, CH2 58.4, qC
Figure 2. ∆δ values (δS - δR) (ppm) for the protons near C-3 of
(S)- and (R)-MTPA esters of 2.
9
138.3, qC
143.7, CH 143.1, CH 143.0, CH
97.2, CH
172.3, qC
19.1, CH3 18.7, CH3 16.1, CH3 77.9, CH
15.6, CH3 15.8, CH3 15.5, CH3 179.1, qC
19.5, CH3 20.5, CH3 18.4, CH3 23.6, CH3 24.4, CH3
138.5, qC
138.9, qC
51.0, CH
29.1, CH3 177.7, qC
22.4, CH3 78.0, CH
51.9, qC
10
11
12
13
14
15
96.8, CH
171.7, qC
96.7, CH
171.4, qC
corresponding proton in model compound 4. Moreover, no ROESY
cross-peak between H-3 and H-5 gave further support to this
assignment.
59.2, CH
59.4, CH
15.1, CH3
15.8, CH3
Lingshuiolide B (2) has a molecular formula C15H22O4, as
deduced from its HREIMS spectrum, the same as 1. Detailed
analysis of 1D and 2D NMR spectra revealed that the planar
structure of 2 was identical to that of 1. As shown in Table 1, the
13C NMR data from C-6 to C-12 and C-13 to C-15 of 2 are almost
the same as those of 1, whereas the differences between them
mainly happened at the cyclohexene ring. The multiplicity of
carbinolic signal H-3 (δH 4.06, br s, in 1 and δH 4.20, m, in 2)
indicated that compound 2 differs from 1 only in the relative
stereochemistry of the hydroxyl group at C-3. Since the configu-
ration of 3-OH of 1 was already assigned as ꢀ, the hydroxyl group
at C-3 of 2 was determined adopting the R-configuration. The
diagnostic ROESY correlation between H-3 and H-5 further
confirmed this assignment. The different chemical shift values from
C-1 to C-5 between 1 and 2, due to different orientations of the
hydroxyl group, were in good agreement with what was observed
in the model compounds 4 and 5,14 a pair of C-3 epimers. Thus,
lingshuiolide B was determined as the C-3 epimer of 1.
a Bruker DRX-400 spectrometer. The chemical shift values are given
in ppm and referenced to CDCl3 (77.0 ppm).
1
Figure 1. Key H-1H COSY and HMBC correlations of com-
pound 1.
(CH)], and a hydroxyl-bearing carbon [δC 64.5 (CH)]. From these
spectral data compound 1 was deduced to be a bicyclic structure
with two olefins and one carbonyl group. Analysis of the 2D NMR
spectra of 1 revealed that compound 1 was composed of two parts
(Figure 1): partial structures X (from C-1 to C-8 including C-13 to
C-15) and Y (from C-9 to C-12).
Since lingshuiolide B contains a secondary hydroxyl group at
C-3, the absolute configuration of 2 was determined by applying
the modified Mosher’s method.16 Compound 2 was esterified
separately with (R)- and (S)-MTPA chloride in dry pyridine at room
temperature to yield the corresponding MTPA esters 2S and 2R,
respectively. Assignment of the 1H NMR signals of the esters was
1
achieved by carefully analyzing the H-1H COSY spectra. The
For the partial structure X, the presence of two spin-spin systems
(H-2 to H-5 and H3-14; H2-7 to H2-8) were evident by analysis of
the 1H-1H COSY spectrum. Further, HMBC correlations from H3-
15 to C-1, C-5, C-6, and C-7, from H3-14 to C-4, C-5, and C-6,
and from H3-13 to C-1, C-2, and C-6 suggested that the two above-
mentioned spin systems were connected through the quaternary
carbon C-6. Moreover, the 1H and 13C NMR chemical shifts of the
moiety X were found almost identical to those of pelseneeriol-1
(4),14 a furanosesquiterpene recently isolated from the Portuguese
nudibranch Doriopsilla pelseneeri, securing the assignment for the
partial structure X. The identification of partial structure Y was
∆δ (δS-ester - δR-ester) values of the protons near the chiral carbon
(C-3) were summarized as shown in Figure 2. Negative ∆δ values
were recorded for the protons of H2-4, H-5, and Me-14 and positive
∆δ values were observed for the protons of H-2 and Me-13.
According to the Mosher’s determination rule, the absolute stere-
ochemistry of the hydroxyl group at C-3 was established as S.
Consequently, the absolute stereochemistry at C-5 and C-6 was
determined as both R, respectively. The configuration of C-11
remains undefined. Although the hydroxyl group on C-11 could
also be esterified by (R)- and (S)-MTPA chloride, the ∆δ (δS -
δR) values of H-10 and H-11 in both 11-MTPA esters were nearly
zero. It should be pointed out that these results were in good
agreement with the phenomenon observed when treating cacospon-
gionolide F,17 which contains a similar γ-hydroxybutenolide moiety,
with (R)- and (S)-MTPA chloride.
1
straightforward. Direct comparison of the H and 13C NMR data
of C-9 to C-12 of 1 with those of 2-oxomicrocionin-2-lactone (3)15
and co-occurring 7 readily identified an R,γ-disubstituted butenolide
moiety. Finally, subunits X and Y were linked through the bond
1
C-8 to C-9 by the observation of H-13C long-range correlations
Lingshuiperoxide (6) was obtained as an optically active, white,
amorphous powder. The EIMS of 6 showed a molecular ion peak
at m/z 280, and its molecular formula was inferred as C15H20O5
from the HREIMS (m/z 280.1307, calcd 280.1311). It was im-
from H2-8 to C-9, C-10, and C-12 and from H-10 to C-8 (Figure
1). Thus, the planar gross structure of 1 was determined.
There are three chiral centers (C-3, C-5, and C-6) around the
cyclohexene ring of 1. The cis relationship between H3-14 and H3-
15 was determined by analogy with model compound 4.14As
described in ref 14, the 13C NMR chemical shift of CH3-14 has a
similar value (15.7-16.0 ppm) in both cis and trans isomers,
whereas the carbon value of CH3-15 is smaller in the cis (20.9-21.1
ppm) than in the trans (26.3-26.6 ppm) isomer due to the greater
γ-type interaction between the two methyl groups in cis compounds.
The ꢀ-orientation of the hydroxyl group at C-3 was inferred by
analysis of the splitting pattern of H-3, which was the same as the
1
mediately apparent from the H and 13C NMR data that 6 differs
from 1 and 2 only in the ring A, where the methyl-bearing
trisubstituted double bond in 1 and 2 was replaced by a disubstituted
double bond [δH 6.39 (1H, br d, J ) 8.2 Hz), 6.59 (1H, dd, J )
8.2 and 6.1 Hz); δC 130.1 (CH), 137.9 (CH)]. In addition, careful
1
analysis of H-1H COSY and HMQC spectra revealed the clear
proton connectivities from H-2 to H-5 and H3-14. Thus, starting
from H-4 (δH 4.33, δC 76.3), a series of distinct correlations between
H-4 and H-3 (δH 6.59), between H-3 and H-2 (δH 6.39), between

Sesquiterpenes from the Sponge Dysidea septosa
Journal of Natural Products, 2008, Vol. 71, No. 8 1401
Figure 3. Key 2D NMR correlations of compound 6.
Figure 4. Key 2D NMR correlations of compound 12.
H-4 and H-5 (δH 2.29), and between H-5 and H3-14 (δH 0.82) were
observed (Figure 3). One butenolide moiety (ring B) and one
cyclohexene ring accounted for the five sites of unsaturation.
Consequently, the last two oxygen atoms unassigned had to be
ascribed to a peroxide bridge that linked at C-1 and C-4,
respectively, to complete the required unsaturation degrees of 6.
Finally, by analogy to compounds 1 and 2, the relative stereo-
chemistry of H3-14 and H3-15 was assigned as cis (both R), whereas
the configuration for the peroxide bridge was established by analysis
of the ROESY spectrum. The ROESY correlations between H-4
and H-5, between H-3 and H-4, between CH3-13 and H-7a, between
CH3-13 and H-2, and between H-5 and H-7b indicated that the
peroxide bridge is also R-oriented. The cis nature of the double
bond at ∆2(3) was assigned on the basis of the coupling constant
between H-2 and H-3 (J ) 8.2 Hz).
for three of the required six sites of unsaturation. Consequently,
spirolingshuiolide must possess three rings. Analysis of the 1H NMR
1
spectrum of 12, aided by H-1H COSY, HMQC, and TOCSY,
revealed the proton connectivities for three partial structures, a-c
(Figure 4).
For the partial structure a, the olefinic proton (δH 5.32, H-1)
exhibited clear correlation with the adjacent olefinic proton (δH 5.80,
H-2), which, in turn, was further correlated with the methylene
proton at δ 2.02 (H-3b); on the other hand, the sharp doublet signal
at δ 0.95 (H3-14) was linked to the methine at δ 1.94 (H-4), which,
in turn, was also coupled with H2-3. For the partial structure b, the
correlations between H2-6 (δ 1.76, 1.96) and H2-7 (δ 1.98, 2.18)
were observed. The clear cross-peaks between H-11 (δ 5.48) and
H-12 (δ 3.86) indicated the presence of the partial structure c. All
the subunits, bearing in mind two unassigned methyls [δH 0.79 (s),
1.03 (s); δC 15.1 (CH3), 24.4 (CH3)] and three quaternary carbons
(C-5, δ 48.0; C-8, δ 58.4, C-9, δ 51.9), were connected by extensive
interpretation of well-resolved HMBC spectra. Significant 1H-13C
long-range correlations, as shown in Figure 4, connected H3-13 to
C-5 and H3-15 to C-9 and located the spirocarbon at C-8. Thus,
the planar structure of 12 was unambiguously assigned.
Isodysetherin (10)18 was isolated as an optically active, colorless
oil with [R]2D0 +81 (c 0.26, CHCl3). The molecular formula of 10
was determined to be C15H20O3 by HREIMS {m/z 248.1415 [M]+,
∆ ) +1.1 mmu}. The carbon framework of 10 was readily
recognized to be the same as dysetherin (11)9,10 by comparing their
1H-1H COSY, HMQC, and HMBC spectra. In fact, the significant
1
difference between the H NMR spectra of 10 and 11 was the
signals assigned to the methine proton pairs (H-4 δ 2.85 in 10 and
2.96 in 11; H-9 δ 1.66 in 10 and 2.32 in 11) and the germinal
methyl groups (Me-10 δ 1.38 in 10 and 1.18 in 11; Me-11 δ 1.08
in 10 and 1.14 in 11). Furthermore, in their 13C NMR spectra, it
was noticed that the resonances for C-4, C-9, Me-10, and Me-11
appeared at δ 38.5, 51.0, 29.1, and 22.4 in 10, while the
corresponding carbons in 11 resonated at δ 36.3, 45.1, 22.5, and
26.2, respectively. These differences suggested that 10 is a
diastereomer of 11 with opposite A/B ring junction. A significant
NOE correlation between H-4 and H-9 in the NOESY spectrum of
10 confirmed the cisoid rings’ linkage. The other NOE correlations
between H-4/H-3R, H-9/Me-10, H-12/Me-10, and H-3R/H-12
supported that the orientations of H-4 and H-9 in 10 were R instead
of ꢀ as in 11, and the configuration of C-2 was the same as that of
11. On the basis of the above observations, the structure of
compound 10 was determined as a 4,9-epimer of dysetherin, named
isodysetherin.
The relative stereochemistry of 12 was established by NOESY
experiment. The NOE correlations of Me-13/Me-15 and Me-13/
H-4 showed that the rings A/B were cis fused and the configuration
of H-4 was ꢀ. The NOESY correlation between the oxygen-bearing
proton signals (δ 3.86, H-12 and 5.48, H-11) revealed the H-11
and H-12 were cis oriented to each other. The relative configuration
of the spirocarbon (C-8) was tentatively determined as S* by a
NOESY cross-peak observed between H-12 and H-7b (Figure 4).
In conclusion, 12 sesquiterpenes belonging to six different
structural classes were isolated and structurally characterized from
the Hainan sponge D. septosa. It is interesting to note that
dihydropallescensin-2 (9)8 had been previously isolated from the
nudibranch Cadlina luteomarginata collected near San Diego,
California, and furodysinin 3ꢀ,4ꢀ-epoxy lactone (13)11 had been
previously isolated from the nudibranch Chromodoris funereal
collected in Iwayama Bay, Palau. In particular, interestingly,
pelseneeriol-1 (4) and pelseneeriol-2 (5),14 two furanosesquiterpenes
structurally closely related to compounds 1, 2, and 6-8, had been
recently reported from the Portuguese nudibranch Doriopsilla
pelseneeri, and they were hypothetically suggested to originate from
its dietary sponge of genus Dysidea even though no direct
experimental observation supported their hypothesis. This study
provides the indirect experimental evidence that the sponge D.
septosa might be the potential prey of the above-mentioned
nudibranchs. Further study should be conducted to collect these
nudibranchs in the same region of South China Sea, where the title
sponge was found, and to investigate these nudibranchs chemically
so as to unambiguously confirm their prey-predator relationship.
The discovery of these new sesquiterpenes, particularly spirol-
ingshuiolide (12), has added to an extremely diverse and complex
assay of sponge sesquiterpenes, which is rapidly expanding. Now,
there is a strong interest in performing further studies aimed at
Spirolingshuiolide (12)18 was isolated as an optically active,
colorless oil with [R]2D0 -31 (c 0.16, CHCl3). Its molecular formula
was deduced as C15H20O3 from the HREIMS data {m/z 248.1401
[M]+, ∆ ) +1.1 mmu}, the same as 10. Nevertheless, the
spectroscopic properties of 12 were somewhat different from those
of 10. The structural determination of spirolingshuiolide was aided
by the previous experience acquired during the structural elucidation
of isodysetherin. The 13C NMR data of 12 displayed three sp3
methines, two sp2 methines, three sp3 methylenes, three methyls,
and four quaternary carbons. All the protons were connected to
carbons by HMQC experiment. The presence of one disubstituted
double bond [δH 5.32 (br d, J ) 10.0 Hz), 5.80 (ddd, J ) 10.0,
4.8, 2.4 Hz); δC 130.8 (CH), 127.9 (CH)], one epoxy group [δH
3.86 (d, J ) 2.2 Hz), 5.48 (d, J ) 2.2 Hz); δC 59.4 (CH), 78.0
(CH)], and an ester carbonyl [δC 177.0 (q C)] were easily recognized
by characteristic 1H and 13C NMR resonances. These data accounted

1402 Journal of Natural Products, 2008, Vol. 71, No. 8
Huang et al.
m/z 266 [M]+ (5), 248 (45), 230 (35), 204 (55), 121 (80), 84 (100);
HREIMS m/z 266.1510 (calcd for C15H22O4, 266.1518).
experimentally proving the true biogenetic origin and the effective
biological and ecological role that spirolingshuiolide (12) and related
sesquiterpenes play in the life cycle of the sponge and finally at
confirming their structural peculiarities by synthesis.
Lingshuiperoxide (6): colorless oil; [R]2D0 -7.3 (c 0.41, CHCl3);
1
IR (KBr) νmax 2921, 1745, 1461, 1376 cm-1; H NMR (CDCl3, 400
MHz) 1H NMR (CDCl3, 400 MHz) δ 6.92 (1H, br s, H-10), 6.59 (1H,
dd, J ) 6.1, 8.2 Hz, H-3), 6.39 (1H, br d, J ) 8.2 Hz, H-2), 6.11 (1H,
br s, H-11), 4.33 (1H, m, H-4), 4.11 (1H, br s, OH), 2.38 (1H, m,
H-8b), 2.33 (1H, m, H-8a), 2.29 (1H, m, H-5), 1.99 (1H, m, H-7b),
1.66 (1H, m, H-7a), 1.30 (3H, s, Me-13), 0.83 (3H, s, Me-15), 0.82
(3H, d, J ) 6.6 Hz, Me-14); 13C NMR (CDCl3, 100 MHz) see Table
1; ESIMS m/z 303 [M + Na]+; HRESIMS m/z 303.1209 (calcd for
C15H22O5Na, 303.1208).
It has been discovered that protein tyrosine phosphatase 1B
(PTP1B) is involved both physiologically and pathologically in
regulating the signaling of the insulin receptor. Knockout studies
in mice have shown that PTP1B-deficient mice displayed enhanced
insulin sensitivity and resistance to diet-induced obesity. PTP1B
thus becomes a promising target in the treatment of type-II diabetes
and obesity.19 For this reason, the development of small-molecule
inhibitors of PTP1B has attracted considerable attention in both
academic research and pharmaceutical investigation. In fact, a
number of reports describing natural products as PTP1B inhibitors
have appeared in the past decade.20 In light of this observation, all
the new and known compounds isolated in this study were evaluated
for their inhibitory activity against PTP1B. The bioassay results
showed that compounds 7-9 had moderate inhibitory activities,
with IC50 values of 8.8, 11.6, and 6.8 µg/mL, respectively, while
compound 15 had the strongest PTP1B inhibitory effect, with an
IC50 value of 1.9 µg/mL.
Isodysetherin (10): colorless oil; [R]2D0 +81 (c 0.26, CHCl3); IR
(KBr) νmax 2966, 1776, 1461, 1070 cm-1; 1H NMR (CDCl3, 400 MHz)
δ 5.47 (1H, d, J ) 2.3 Hz, H-13), 5.34 (1H, br s, H-5), 3.60 (1H, d, J
) 2.3 Hz, H-12), 2.85 (1H, m, H-4), 2.09 (1H, dd, J ) 8.2, 14.0 Hz,
H-3R), 1.93 (1H, m, H-3ꢀ), 1.89 (2H, m, H2-7), 1.64 (2H, m, H2-8),
1.66 (1H, m, H-9), 1.64 (3H, s, Me-15), 1.38 (3H, s, Me-10), 1.08
(3H, s, Me-11); 13C NMR (CDCl3, 100 MHz) see Table 1; EIMS m/z
248 [M]+ (50), 233 (35), 203 (50), 135 (45), 119 (100); HREIMS m/z
248.1415 (calcd for C15H20O3, 248.1412).
Spirolingshuiolide (12): colorless oil; [R]2D0 -31 (c 0.16, CHCl3);
1
IR (KBr) νmax 2962, 1772, 1460, 1068 cm-1; H NMR (CDCl3, 400
MHz) δ 5.80 (1H, ddd, J ) 2.4, 4.8, 10.0 Hz, H-2), 5.48 (1H, d, J )
2.2 Hz, H-11), 5.32 (1H, br d, J ) 10.0 Hz, H-1), 3.86 (1H, d, J ) 2.2
Hz, H-12), 2.18 (1H, m, H-7a), 2.02 (1H, m, H-3b), 1.98 (1H, m, H-7b),
1.96 (1H, m, H-6b), 1.94 (1H, m, H-4), 1.82 (1H, m, H-3a), 1.76 (1H,
m, H-6a), 1.03 (3H, s, Me-15), 0.95 (3H, d,J ) 6.6 Hz, Me-14), 0.79
(3H, s, Me-13); 13C NMR (CDCl3, 100 MHz) see Table 1; EIMS m/z
248 [M]+ (5), 219 (20), 203 (25), 121 (100); HREIMS m/z 248.1401
(calcd for C15H20O3, 248.1405).
Preparation of (S)- and (R)-MTPA Ester. The 2S derivative was
obtained by treating 2 (1.5 mg) with (R)-MTPA-Cl in dry pyridine for
ca. 16 h under stirring at RT. The reaction mixture was purified by CC
(silica gel) to afford pure 2S (1.2 mg). In a similar manner, 2R (1.3
mg) was prepared from (S)-MTPA-Cl.
Experimental Section
General Experimental Procedures. Optical rotations were measured
on a Perkin-Elmer 241MC polarimeter. UV spectra were recorded on
a Varian Cary 300 Bio spectrophotometer. IR spectra were recorded
on a Nicolet-Magna FT-IR 750 spectrometer. NMR spectra were
measured on a Bruker DRX-400 spectrometer with the residual CHCl3
(δH 7.26 ppm, δC 77.0 ppm) as an internal standard. EIMS and HREIMS
data were obtained on a Finnigan-MAT-95 mass spectrometer. ESIMS
and HRESIMS spectra were recorded on a Q-TOF Micro LC-MS-
MS mass spectrometer. Reversed-phase HPLC (Agilent 1100 series
liquid chromatography using a VWD G1314A detector at 210 nm and
a semipreparative ODS-HG-5 [5 µm, 10 mm (i.d.) × 25 cm] column)
was also employed. Commercial Si gel (Qing Dao Hai Yang Chemical
Group Co., 200-300 and 400-600 mesh) was used for CC, and
precoated Si gel plates (Yan Tai Zi Fu Chemical Group Co., G60 F-254)
were used for analytical TLC.
Biological Material. The specimens of D. septosa identified by
Professor J.-H. Li of Institute of Oceanology, Chinese Academy of
Sciences, were collected by scuba at Lingshui Bay, Hainan Province,
China, in November 2001, and were frozen immediately after collection.
A voucher sample (SS-38) is available for inspection at the Herbarium
of Shanghai Institute of Materia Medica, CAS.
Extraction and Isolation. The frozen sample (500 g dry weight)
was lyophilized and exhaustively extracted with acetone. The extract
was concentrated in Vacuo, and the resulting residue was extracted with
Et2O and n-BuOH, respectively. The Et2O-soluble portion was repeat-
edly subjected to silica gel column chromatographies (using increasing
concentrations of EtOAc in petroleum ether as the eluent) to give 1
(50.0 mg), 2 (55.0 mg), 6 (4.8 mg), 7 (18.7 mg), 8 (18.7 mg), 9 (18.7
mg), 12 (0.8 mg), 13 (10.9 mg), 14 (10.7 mg), and 15 (41.7 mg),
respectively. The portion eluted with EtOAc/petroleum ether (95:5) was
further purified by semipreparative RP-HPLC (MeOH/H2O, 70:30; 2.5
mL/min; UV 210 nm) to give 10 (1.5 mg, tR ) 37.7 min) and 11 (3.2
mg, tR 38.8 min).
2S: 1H NMR (CDCl3, 400 MHz) δ 7.06 (1H, br s, H-11), 6.81 (1H,
br s, H-10), 5.46 (1H, br s, H-2), 5.53 (1H, m, H-3), 2.26 (1H, m,
H-8b), 1.98 (1H, m, H-8a), 1.85 (1H, m, H-4R), 1.83 (1H, m, H-5),
1.67 (3H, s, Me-13), 1.63 (2H, m, H2-7), 1.51 (1H, m, H-4ꢀ), 0.91
(3H, s, Me-15), 0.89 (3H, d, J ) 6.5 Hz, Me-14).
2R: 1H NMR (CDCl3, 400 MHz) δ 7.06 (1H, br s, H-11), 6.81 (1H,
br s, H-10), 5.33 (1H, br s, H-2), 5.54 (1H, m, H-3), 2.26 (1H, m,
H-8b), 1.98 (1H, m, H-8a), 1.94 (1H, m, H-4R), 1.85 (1H, m, H-5),
1.64 (3H, s, Me-13), 1.62 (2H, m, H2-7), 1.60 (1H, m, H-4ꢀ), 0.91
(3H, s, Me-15), 0.92 (3H, d, J ) 6.5 Hz, Me-14).
Biological Assay. Recombinant PTP1B catalytic domain was
expressed and purified according to a previous report.21 The enzymatic
activities of the PTP1B catalytic domain were determined at 30 °C by
monitoring the hydrolysis of pNPP. Dephosphorylation of pNPP
generates product pNP, which was monitored at an absorbance of 405
nm by the EnVision multilabel plate reader (PerkinElmer Life Sciences,
Boston, MA). In a typical 100 µL assay mixture containing 50 mmol/L
3-[N-morpholino] propanesulfonic acid (MOPs), pH 6.5, 2 mmol/L
pNPP, and 30 nmol/L recombinant PTP1B, activities were continuously
monitored and the initial rate of the hydrolysis was determined using
the early linear region of the enzymatic reaction kinetic curve. The
IC50 was calculated with Prism 4 software (Graphpad, San Diego, CA)
from the nonlinear curve fitting of the percentage of inhibition (%
inhibition) versus the inhibitor concentration [I] by using the following
equation: % Inhibition ) 100/(1 + [IC50/[I]]k), where k is the Hill
coefficient.
Lingshuiolide A (1): colorless oil; [R]2D0 +85.4 (c 0.37, CHCl3); IR
(KBr) νmax 3376, 2968, 1762, 1658, 1444, 1010 cm-1; 1H NMR (CDCl3,
400 MHz) δ 6.85 (1H, br s, H-10), 6.06 (1H, br s, H-11), 5.85 (1H, br
s, H-2), 4.06 (1H, m, H-3), 2.22 (1H, m, H-8b), 1.89 (1H, m, H-8a),
1.82 (1H, m, H-4R), 1.77 (1H, m, H-5), 1.66 (3H, s, Me-13), 1.61
(2H, m, H2-7), 1.39 (1H, m, H-4ꢀ), 0.92 (3H, s, Me-15), 0.90 (3H, d,
J ) 6.5 Hz, Me-14); 13C NMR (CDCl3, 100 MHz) see Table 1; EIMS
m/z 266 [M]+ (5), 248 (10), 203 (10), 128 (36), 121 (100); HREIMS
m/z 266.1532 (calcd for C15H22O4, 266.1518).
Acknowledgment. This research work was financially supported by
the National Marine 863 Projects (Nos. 2006AA09Z447, 2006AA09Z412,
and 2007AA09Z447), the Natural Science Foundation of China (Nos.
30730108, 20721003, and 20772136), STCSM Projects (Nos. 07XD14036
and 06DZ22028), and CAS Key Project (Grant KSCX2-YW-R-18).
Lingshuiolide B (2): colorless oil; [R]2D0 -2.0 (c 0.33, CHCl3); IR
(KBr) νmax 3355, 2964, 1751, 1658, 1446, 1010 cm-1; 1H NMR (CDCl3,
400 MHz) δ 6.82 (1H, br s, H-10), 6.07 (1H, br s, H-11), 5.47 (1H, br
s, H-2), 4.20 (1H, m, H-3), 2.22 (1H, m, H-8b), 1.89 (1H, m, H-8a),
1.82 (1H, m, H-4R), 1.77 (1H, m, H-5), 1.66 (3H, s, Me-13), 1.61
(2H, m, H2-7), 1.39 (1H, m, H-4ꢀ), 0.92 (3H, s, Me-15), 0.90 (3H, d,
J ) 6.5 Hz, Me-14); 13C NMR (CDCl3, 100 MHz) see Table 1; EIMS
Supporting Information Available: 1D and 2D NMR and HREIMS
spectra of compound 12. This information is available free of charge
via the Internet at http://pubs.acs.org.

Sesquiterpenes from the Sponge Dysidea septosa
Journal of Natural Products, 2008, Vol. 71, No. 8 1403
(12) Grode, S. H.; Cardellina, J. H., II J. Nat. Prod. 1984, 47, 76–83.
(13) Schulte, G.; Scheuer, P. J.; McConnell, O. J. HelV. Chim. Acta 1980,
63, 2159.
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