Norterpenoids and related peroxides from the formosan marine sponge Negombata corticata

Article abstract of DOI:10.1021/np100353x

Six norterpenes including negombatoperoxides A and B (4 and 5), the inseparable epimers negombatoperoxides C and D (6 and 7), negombatodiol (8), and negombatolactone (9), in combination with three known compounds, (+)-nuapapuin B (1), (+)-nuapapuin B methyl ester (2), and (+)-aikupikoxide C (3), were isolated from the Formosan marine sponge Negombata corticata. In addition, 6,6-dimethylundecane-2,5,10-trione (10) was isolated for the first time from a natural source. Their structures, including relative configurations, were elucidated on the basis of interpretation of spectroscopic data and by the application of the empirical rule established by Capon and MacLeod. The absolute configurations of 8 and 9 were established by the application of Mosher's method and comparison of CD data with known lactones, respectively. Cytotoxicity of these isolates against human breast carcinoma, human liver carcinoma, and human lung carcinoma cell lines was evaluated.

Full text of DOI:10.1021/np100353x

1538
J. Nat. Prod. 2010, 73, 1538–1543
Norterpenoids and Related Peroxides from the Formosan Marine Sponge Negombata corticata
Chih-Hua Chao,† Kuei-Ju Chou,† Guey-Horng Wang,‡ Yang-Chang Wu,§ Li-Hsueh Wang,⊥,| Jeng-Ping Chen,∇
Jyh-Horng Sheu,*,†,4 and Ping-Jyun Sung*,⊥,|
Department of Marine Biotechnology and Resources, National Sun Yat-sen UniVersity, Kaohsiung 804, Taiwan, Republic of China,
Department of Cosmetic Science, Chia-Nan UniVersity of Pharmacy and Science, Tainan 717, Taiwan, Republic of China, Graduate Institute of
Natural Products, Kaohsiung Medical UniVersity, Kaohsiung 807, Taiwan, Republic of China, National Museum of Marine Biology and Aquarium,
Checheng, Pingtung 944, Taiwan, Republic of China, Graduate Institute of Marine Biotechnology, National Dong Hwa UniVersity, Checheng, Pingtung
944, Taiwan, Republic of China, Taiwan Ocean Research Institute, National Applied Research Laboratories, Taipei 106, Taiwan, Republic of China, and
Asia-Pacific Ocean Research Center, National Sun Yat-sen UniVersity, Kaohsiung 804, Taiwan, Republic of China
ReceiVed May 26, 2010
Six norterpenes including negombatoperoxides A and B (4 and 5), the inseparable epimers negombatoperoxides C and
D (6 and 7), negombatodiol (8), and negombatolactone (9), in combination with three known compounds, (+)-nuapapuin
B (1), (+)-nuapapuin B methyl ester (2), and (+)-aikupikoxide C (3), were isolated from the Formosan marine sponge
Negombata corticata. In addition, 6,6-dimethylundecane-2,5,10-trione (10) was isolated for the first time from a natural
source. Their structures, including relative configurations, were elucidated on the basis of interpretation of spectroscopic
data and by the application of the empirical rule established by Capon and MacLeod. The absolute configurations of 8
and 9 were established by the application of Mosher’s method and comparison of CD data with known lactones,
respectively. Cytotoxicity of these isolates against human breast carcinoma, human liver carcinoma, and human lung
carcinoma cell lines was evaluated.
Cyclic peroxides are of great interest due to their varied
biological activities including antiparasitic activity and cyto-
toxicity against cancer cells.1-3 Norterpene-related cyclic per-
oxides have been isolated from marine sponges of the genera
Prianos,4-6 Sigmosceptrella,6-8 Latrunculia,9-11 Mycale,12-17
and Diacarnus.18-24 Among them, some have been shown to
exhibit antimicrobial,9 antiviral,14 cytotoxicity,14 and antimalarial
activities.18,25 The wide spectrum of biological activities of cyclic
peroxides prompted us to investigate the bioactive compounds
from a Formosan marine sponge, Negombata corticata, as this
organism was found to possess the known norditerpene cyclic
peroxide 219 by our preliminary study. The present study has
led to the isolation of three known cyclic peroxides (1-3), four
new norterpene-related cyclic peroxides (4-7), and three new
norterpene-related compounds (8-10). The cyclic peroxides were
characterized by a 2-substituted propionic acid or the corre-
sponding methyl propionate functionality attached to a 1,2-
dioxane ring at the C-3 position, like compounds 1-7, of which
the relative configurations at C-2, C-3, and C-6 were elucidated
by applying the empirical rule introduced by Capon and
MacLeod.26 The cytotoxic activity of compounds 1-3 and 5-10
against breast carcinoma (MDA-MB-231 and MCF-7), human
liver carcinoma (HepG2 and HepG3), and human lung carcinoma
(A-549) cell lines was studied.
Results and Discussion
The sponge N. corticata was subjected to extraction with EtOH.
Fractionation of the lipophilic extract by repeated silica gel column
chromatography and final purification using normal-phase HPLC
afforded six new (4-9) and three known compounds (1-3) along
with 6,6-dimethylundecane-2,5,10-trione (10), which was isolated
for the first time from a natural source.27 The new compounds were
* To whom correspondence should be addressed. Tel: 886-7-5252000,
ext. 5030. Fax: 886-7-5255020. E-mail: sheu@mail.nsysu.edu.tw (J.-H.S).
† National Sun Yat-sen University.
‡ Chia-Nan University of Pharmacy and Science.
§ Kaohsiung Medical University.
⊥ National Museum of Marine Biology and Aquarium.
| National Dong Hwa University.
∇ Taiwan Ocean Research Institute.
4 Asia-Pacific Ocean Research Center.
given the trivial names negombatoperoxides A-D (4-7), negom-
batodiol (8), and negombatolactone (9). The known compounds
10.1021/np100353x  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/18/2010

Norterpenoids from Negombata corticata
Journal of Natural Products, 2010, Vol. 73, No. 9 1539
uration, comprising a carboxylic acid (δC 179.1, C), a conjugated
methyl enone (δC 198.7, C, 143.4, CH, 134.3, CH, 26.7, CH3), and
a 1,2-dioxane ring (δC 81.4, CH, 79.7, C). The gross structure of
5 was established by the assistance of extensive 2D NMR analysis
(1H-1H COSY, HMQC, and HMBC). The 1H-1H COSY spectrum
was used to establish the proton sequences from H3-12 to H-2, from
H-2 to H2-5, and from H2-7 to H-9 (Figure 1). The methyl group
(δH 1.21 d, J ) 7.5 Hz) attached at C-2 was confirmed by the
HMBC correlations from H3-12 to C-1, C-2, and C-3. The H3-13
attached at C-6 was deduced by the HMBC correlations from H3-
13 to C-5, C-6, and C-7. Thus, the planar structure of 5 was
established.
The E geometry of the C-8/C-9 double bond was deduced from
a 16.0 Hz coupling constant between H-8 and H-9. By applying
the aforementioned empirical rule, the equatorial orientation for
6-Me was readily assigned on the basis of its 13C NMR shift (δC
24.3, C-13), the axial 6-Me being expected to resonate at δC 20.5
to 20.9. The large coupling constant J3,4ax (9.5 Hz) of the H-3 signal
established an equatorial carbon substituent at C-3. The carbon
resonance for the C-2 methyl group at δC 12.7 (C-12) might require
a C-2/C-3-erythro configuration; however, its proton resonance at
δH 1.21 (H3-12) suggested a C-2/C-3-threo configuration. This result
disclosed that the C-2/C-3 relative configuration could not be
determined by this empirical rule for this case. On inspection of
the empirical rule, it was found that most model compounds are
the methyl esters.26 Thus, the methyl ester 5a was prepared using
a mild and modified esterification method.28 The carbon and proton
resonances of the 2-Me (δC 12.7; δH 1.16) in the methyl ester 5a
required a C-2/C-3-erythro configuration (Experimental Section).
Figure 1. Selected 1H-1H COSY (s) and HMBC (f) correlations
of 4-7 and 9.
were identified as (+)-nuapapuin B (1),19 (+)-nuapapuin B methyl
ester (2),19 and (+)-aikupikoxide C (3).21
Negombatoperoxide A (4) was obtained as a colorless oil. The
HRESIMS of 4 exhibited a pseudomolecular ion peak at m/z
431.2776 [M + Na]+ and established a molecular formula of
Compounds 6 and 7 were isolated as an inseparable epimeric
mixture that gave a [M + Na]+ peak at m/z 335.1472 in the
HRESIMS spectrum, corresponding to a molecular formula of
C16H24O6. It is difficult to clearly distinguish between these two
epimers from the 1H NMR spectrum; however, the 13C NMR
spectrum displayed two sets of signals with partial overlap, and
the intensity indicated that the epimers were present in a ratio close
to 1:1. Analysis of the NMR data revealed that compounds 6 and
7 shared the same C-1-C-6 moiety. In addition, the carbon
resonances around C-10 were observed in pairs at δ 125.2 (CH)/
125.0 (CH), 135.6 (CH)/135.7 (CH), 26.8 (CH3)/26.6 (CH3), and
34.2 (CH2)/34.1 (CH2), suggesting that they might be C-10 epimers.
A γ-lactone was preliminarily deduced according to the carbon
resonances at δ 85.5 (C) and 176.9 (C)29 as well as the IR
absorption band appearing at 1769 cm-1. The planar structure of
6/7, established as shown in Figure 1, was further confirmed by
1
C24H40O5, implying five degrees of unsaturation. Its H and 13C
NMR spectra clearly revealed the presence of a carboxylic acid
(δC 178.5, C), a trisubstituted double bond (δC 135.9, C and 123.9,
CH), and a tetrasubstituted epoxide containing a methyl group (δC
69.6, C; 63.7, C; 22.1, CH3, and δH 1.28, s). In addition, the above
data and the characteristic 13C NMR signals at δC 81.3 (CH) and
80.2 (C) suggested 4 to be a member of the norterpene peroxide
class.19,23,24,26
The gross structure of 4 was established by 2D NMR spectro-
scopic analysis. To establish the proton sequences in 4, the 1H-1H
COSY spectrum showed the connectivities from H-2 to H3-19, H-2
to H2-5, H2-7 to H-9, H2-11 to H2-12, and H2-15 to H2-17. These
results, together with the 1H-13C long-range correlations observed
in an HMBC experiment (Figure 1), established the planar structure
of 4. Furthermore, according to the empirical rule introduced by
Capon and MacLeod26 and previous related studies,19,21,22 the
configuration at C-2 relative to C-3 for the cyclic peroxy analogues
1
the interpretation of H-1H COSY and HMBC spectra. The E
geometry for the C-8/C-9 double bond was deduced by a 15.8 Hz
coupling constant (measured in C6D6, Experimental Section)
between H-8 and H-9. Compounds 5 and 6/7 were suggested to
share the same relative configuration at C-2, C-3, and C-6 due to
a high degree of similarity in 13C NMR shifts between these
compounds. In addition, according to the aforementioned empirical
rule,26 the orientation of 6-Me (δC 24.2, C-15) was assigned as
1
can be determined on the basis of the H and 13C NMR chemical
1
shifts of 2-Me. If the H NMR shift for 2-Me is observed at δH
1.12-1.15 and δC 12.4-12.8, the C-2/C-3-erythro configuration
is assigned,19,21,22,26 while 2-Me of a corresponding threo config-
uration should resonate at δH 1.22-1.28 and δC 13.2-13.5.22,26
The orientation for 6-Me is determined to be axial if the 13C NMR
shift of this methyl appears at δC 20.5-20.9, while an equatorial
6-Me is assigned if this methyl carbon resonates between δC 23.5
and 24.0.26 The axial or equatorial nature of H-3 in the peroxide
ring was determined by the 1H-1H coupling constant value of 3J3,4ax
(J ) 8-9 Hz for axial H-3; J ) 3-4 Hz for equatorial H-3).22,24,26
Although the multiplicity of H-3 in 4 was not elucidated, a
comparison of the 1H and 13C NMR spectroscopic data of the
C-1-C-6 moiety in 4 (Table 1) with those in diacarperoxide D23
and on the basis of the above rule, allowed the establishment of a
C-2/C-3-erythro configuration and an equatorial 6-Me.
1
equatorial in 6/7. However, similar to compound 5, the H NMR
shift of 2-Me (δH 1.19, J ) 6.6 Hz, H3-14) resonated at an indistinct
region while applying this rule on 6/7. Thus, a mixture of 6 and 7
was esterified with methanol to afford the corresponding methyl
esters 6a/7a. Consequently, the relative configuration for C-2/C-3
1
was readily assigned as erythro on the basis of the H NMR shift
of 2-Me (δH 1.15 d, J ) 6.8 Hz, H3-14) in 6a/7a.
Compound 8 was isolated as an amorphous, white powder. Its
molecular formula was found to be C20H36O4, as deduced from
HRESIMS and 13C NMR data, indicating three degrees of unsat-
uration. The NMR spectroscopic data of 8 were similar to those of
2.19 However, the shifts for C-3 from δC 81.0 in 2 to the upper
field, δC 73.8, in 8 and for H-3 from δH 4.23 in 2 to δH 3.66 in 8
indicated that the cyclic peroxide in 2 was reduced to a C-3/C-6
Negombatoperoxide B (5) was isolated as a colorless oil. Its
HRESIMS spectrum exhibited a pseudomolecular ion peak at m/z
279.1207 [M + Na]+, corresponding to a molecular formula of
C13H20O5. The molecular formula requires four degrees of unsat-

1540 Journal of Natural Products, 2010, Vol. 73, No. 9
Table 1. 13C NMR Spectroscopic Data of Compounds 4-10
Chao et al.
position
4a, δC, mult.
5b, δC, mult.
6/7a, δC, mult.
8a, δC, mult.
9a, δC, mult.
10a, δC, mult.
1
2
3
4
5
6
7
8
178.5, C
42.6, CH
81.3, CH
22.6, CH2
32.5, CH2
80.2, C
34.9, CH2
22.1, CH2
123.9, CH
135.9, C
36.5, CH2
28.2, CH2
69.6, C
179.1, C
179.1, C
42.7, CH
81.4, CH
176.5, C
45.4, CH
73.8, CH
28.8, CH2
37.8, CH2
72.6, C
41.0, CH2
20.5, CH2
54.6, CH
149.4, C
32.4, CH2
23.7 CH2
36.1, CH2
35.0, C
177.0, C
30.1, CH3
207.6, C
37.0, CH2
31.1, CH2
214.3, C
42.6, CH
81.4, CH
22.7, CH2
32.6, CH2
79.7, C
37.9, CH2
143.4, CH
134.3, CH
198.7, C
29.2, CH2
33.4, CH2
87.0, C
39.7, CH2
20.4, CH2
54.2, CH
148.9, C
32.2, CH2
23.6, CH2
36.0, CH2
35.0, C
25.5, CH3
109.5, CH2
28.4., CH3
26.6, CH3
22.7, CH2
32.0, CH2
79.9, C
37.7, CH2
125.2/125.0, CH
135.7/135.6, CH
85.5, C
34.2/34.1, CH2
29.1, CH2
176.9, C
12.8, CH3
24.2, CH3
26.6/26.8, CH3
47.3, C
39.4, CH2
19.0, CH2
43.9, CH2
208.8, C
30.0, CH3
24.5, CH3
24.5, CH3
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
OMe
26.7, CH3
12.7, CH3
24.3, CH3
63.7, C
30.4, CH2
17.2, CH2
36.2, CH2
34.7, C
12.7, CH3
24.0, CH3
16.1, CH3
22.1, CH3
25.9, CH3
25.4, CH3
14.3, CH3
26.9, CH3
109.1, CH2
28.4, CH3
26.5, CH3
51.8, CH3
a Spectra were measured in CDCl3 (75 MHz). b Spectra were measured in CDCl3 (125 MHz).
effect (∆ε -0.1) at 219 nm attributed to the nfπ* electronic
transition of the γ-lactone.32,33 Consequently, the absolute config-
uration at C-4 was assigned as R, while the configuration at C-7,
the asymmetric carbon in the 2,2-dimethyl-6-methylenecyclohexyl
moiety, remains unknown. In view of the fact that compound 8
has a 2R,3R,6R configuration and the known compounds 1 and
2 both possess a 2R,3R,6R configuration, and on the basis of
biogenetic considerations, the absolute configurations of com-
pounds 4-7 are tentatively assigned to be the same as those of
1, 2, and 8.
1
Figure 2. H NMR chemical shift differences of MTPA esters
The HRFABMS data of 10 established a molecular formula of
C13H22O3, implying three degrees of unsaturation. The IR spectrum
of 10 showed the presence of a carbonyl group (1715 cm-1).
Resonances for three sp2 quaternary carbons at δ 207.6, 208.8, and
214.3 were ascribed to the presence of three ketones. The remaining
10 carbons were composed of four methyls, five methylenes, and
one quaternary carbon. The spectroscopic data suggested the acyclic
nature of 10. Detailed interpretation of HMBC and 1H-1H COSY
spectra of 10 led to the establishment of acyclic triketone 10.
Biogenetically, 10 might be derived from oxidative cleavage of
epimuqubilin A19 at the C-9/C-10 and C-13/C-14 double bonds.
This compound was isolated for the first time from a natural
source.27
of 8.
diol in 8. In order to confirm the above elucidation and the relative
configuration of 8, a reductive cleavage was applied to compound
2, a cyclic peroxide for which the absolute configuration is known,19
to afford the corresponding diol. The 1H and 13C NMR spectroscopic
data of this synthetic diol were identical to those of 8. In addition,
the specific rotation of the synthetic diol ([R]25D +5) prepared from
2 showed the same positive sign as that of the isolate 8 ([R]25
D
+2); however, the values of both natural and synthetic compounds
were too small to be used for conclusive determination of the
absolute configuration of 8. The absolute configuration of 8 was
further determined by the application of Mosher’s method.30,31
1
The cytotoxicity of compounds 1-3 and 5-10 against MDA-
MB-231, MCF-7, HepG2, Hep3B, and A-549 cancer cell lines was
evaluated. The data revealed that 1 was cytotoxic toward MDA-
MB-231, MCF-7, HepG2, Hep3B, and A-549 cancer cell lines with
IC50 values of 0.3, 5.9, 0.9, 41.3, and 0.6 µM, respectively, while
its methyl ester 2 showed activity against the above cancer cell
lines with IC50 values of 3.5, 38.5, 2.9, 23.7, and 5.38 µM,
respectively. Compound 3 showed weaker cytotoxicity toward three
of the above cancer cell lines with IC50 values ranging from 11.9
to 36.7 µM. The other tested compounds were not cytotoxic (IC50
> 50 µM) toward the above five cancer cell lines. The diol 8 and
lactone 9, with the loss of the 1,2-dioxane ring, were inactive. In
the case of 1, 5, and 6, it was found that enhancement of the
lipophilicity at C-6 in norterpene cyclic peroxides, like compound
1, can increase the cytotoxicity.34
Analysis of the H NMR data of esters 8a and 8b resulted in the
determination of a 3R configuration (Figure 2). Consequently, the
absolute configuration of 8 was determined as 2R, 3R, and 6R.
However, the configuration at C-9 remains unknown.
The molecular formula of 9 was found to be C16H26O2, as
deduced from HRESIMS and 13C NMR data. Its IR absorption
bands showed the absence of a hydroxy group and the presence of
a γ-lactone (1770 cm-1). The latter was confirmed by the carbon
resonances at δC 87.1 (C) and 177.0 (C).29 A comparison of the
NMR spectroscopic data of 9 with those of 8 disclosed that both
shared the same 2,2-dimethyl-6-methylenecyclohexyl moiety. The
above functionality fulfilled the total four degrees of unsaturation
computed from the molecular formula of 9. Consequently, 9 was
suggested to be composed of a 2,2-dimethyl-6-methylenecyclohexyl
moiety linked through a C2H4 chain to a γ-lactone. This was
confirmed by the interpretation of the H-1H COSY and HMBC
experiments, and the planar structure of 9 was thus determined as
shown in Figure 1. Its CD spectrum exhibited a negative Cotton
Previous studies and our present work revealed that the empirical
rule developed by Capon and MacLeod is quite successful for the
analysis of C-2, C-3, and C-6 relative configurations of norterpene
1

Norterpenoids from Negombata corticata
Journal of Natural Products, 2010, Vol. 73, No. 9 1541
column chromatography with gradient elution (n-hexane-EtOAc, 20:1
to 10:1). Compounds 1 (7.6 mg) and 3 (1.7 mg) were obtained from
subfraction 9A using normal-phase HPLC (n-hexane-CH2Cl2-acetone,
88:7:5). Similarly, compound 4 (1.2 mg) was afforded from subfraction
9B using normal-phase HPLC (n-hexane-CH2Cl2-acetone, 88:7:5).
Subfraction 9C was further purified using normal-phase HPLC (n-
hexane-CH2Cl2-acetone, 80:7:13) to yield compounds 8 (1.8 mg) and
10 (2.3 mg). Fraction 12 was fractionated on a Sephadex LH-20 column
(acetone as eluent) and subsequently purified by normal-phase HPLC
(n-hexane-CH2Cl2-acetone, 75:7:18) to afford compound 5 (4.6 mg)
and an inseparable epimeric mixture of 6/7 (5.4 mg).
peroxide methyl esters. However, this rule showed that it is not
always reliable for elucidation of the C-2/C-3 relative configuration
of norterpene peroxide acids, as the H NMR shift of 2-Me could
1
be near or higher than 1.20 ppm, such as in the case of 5, 6/7, and
(+)-muqubilone B (δH 1.20 for 2-Me),24 exceeding the range of
δH 1.12-1.15 for a C-2/C-3-erythro configuration and close to the
range of δH 1.22-1.28 for a C-2/C-3-threo configuration, although
these compounds all have the C-2/C-3-erythro configuration. Prior
studies on the determination of the C-2/C-3 relative configuration
for carboxylic acids of this type were accomplished mostly by
preparation of the methyl ester, followed by analysis of the NMR
spectroscopic data using the above empirical rule. By careful
examination of the 13C NMR data, we found that all of the 2-Me
shifts of C2/C3-threo norterpene peroxide carboxylic acids appeared
at δC 13.2-13.5, while the corresponding C-2/C-3-erythro acids
showed signals at δC 12.4-12.8. Consequently, the aforementioned
results revealed that the 13C NMR shifts are more reliable for the
determination of the C-2/C-3 relative configuration of the indicated
acids than the 1H NMR shifts. An overview of the literature revealed
that structures of cyclic peroxide-related norterpenoids are quite
diverse among different sponge species,4-24 including the organism
of our present study. Prior investigation of a Red Sea sponge N.
corticata resulted in the isolation of a sphingolipid35 and a
latrunculin-type compound.36 This is the first report of cyclic
peroxides from the marine sponge N. corticata. In addition,
nuapapuin A methyl ester was isolated and assigned an equatorial
H-3 in 1984;5 however, the H-3 relative configuration of this
compound was eventually revised to an axial H-3 in 1999.8
Accordingly, none of the cyclic norterpene peroxides of this class
have been found to possess an equatorial H-3 to date.
(+)-Nuapapuin B (1): [R]24D +54 (c 0.65, CHCl3); lit. [R]D +45 (c
0.46, CHCl3).19
(+)-Nuapapuin B methyl ester (2): [R]24 +60 (c 0.35, CHCl3);
D
lit. [R]D +39 (c 1.74, CHCl3).19
(+)-Aikupikoxide C (3): [R]24D +71 (c 0.72, CH2Cl2); lit. [R]D +88
(c 2.00, CH2Cl2).21
Negombatoperoxide A (4): colorless oil; [R]24 -71 (c 0.52,
D
CHCl3); IR (KBr) νmax 3000-3500, 1716 cm-1
;
13C and 1H NMR data,
see Tables 1 and 2; ESIMS m/z 431 [M + Na]+; HRESIMS m/z
431.2776 [M + Na]+ (calcd for C24H40O5Na, 431.2775).
Negombatoperoxide B (5): colorless oil; [R]24 +80 (c 0.64,
D
CHCl3); UV (MeOH) λmax (log ε) 224 (3.75) nm; IR (KBr) νmax
3000-3500, 1715, 1667 cm-1
;
13C and H NMR data, see Tables 1
1
and 2; ESIMS m/z 279 [M + Na]+; HRESIMS m/z 279.1207 [M +
Na]+ (calcd for C13H20O5Na, 279.1209).
Negombatoperoxide C (6/7): colorless oil; [R]24 +62 (c 0.10,
D
CHCl3); IR (KBr) νmax 3200-3600, 1769, 1720 cm-1
;
13C and 1H NMR
data (CDCl3), see Tables 1 and 2; selected 1H NMR (C6D6, 300 MHz)
5.65 (1H, m, H-8), 5.34 (1H, d, J ) 15.8 Hz, H-9), 4.26 (1H, m, H-3),
2.55 (1H, m, H-2), 1.09 (3H, s, H3-16), 0.98 (3H, d, J ) 6.1 Hz, H3-
14), 0.91 (3H, d, J ) 6.1 Hz, H3-15); ESIMS m/z 335 [M + Na]+;
HRESIMS m/z 335.1472 [M + Na]+ (calcd for C16H24O6Na,335.1471).
Negombatodiol (8): amorphous powder; [R]24D +2 (c 0.14, CHCl3);
IR (KBr) νmax 1736 cm-1
;
13C and H NMR data, see Tables 1 and 2;
1
Experimental Section
ESIMS m/z 363 [M + Na]+; HRESIMS m/z 363.2510 [M + Na]+ (calcd
General Experimental Procedures. Optical rotations were measured
on a JASCO P-1020 polarimeter. IR spectra were recorded on a JASCO
FT/IR-4100 Fourier transform infrared spectrophotometer. The NMR
spectra were recorded on a Bruker AVANCE 300 FT-NMR (or Varian
Unity INOVA 500 FT-NMR) instrument at 300 MHz (or 500 MHz)
for C20H36O4Na, 363.2513).
Negombatolactone (9): colorless oil; [R]24 +10 (c 0.52, CHCl3);
D
CD (2.4 × 10-3 M, MeOH) λmax (∆ε) 219 (-0.1); IR (KBr) νmax 1770
cm-1; H NMR (CDCl3, 300 MHz) 4.76 (1H, s, H-14a), 4.52 (1H, s,
1
H-14b), 2.59 (2H, m, H2-2), 2.04 (1H, m, H-3a), 2.02 (2H, m, H2-9),
1.98 (1H, m, H-3b), 1.55 (2H, m, H-6a and H-7), 1.54 (1H, m, H-5a),
1.51 (2H, m, H2-10), 1.45 (1H, m, H-11a), 1.44 (1H, m, H-5b), 1.43
(1H, m, H-6b), 1.40 (3H, s, H3-13), 1.25 (1H, m, H-11b), 0.92 (3H, s,
H3-15), 0.85 (3H, s, H3-16); 13C NMR (CDCl3, 75 MHz) see Table 1;
ESIMS m/z 273 [M + Na]+; HRESIMS m/z 273.1828 [M + Na]+ (calcd
for C16H26O2Na, 273.1834).
1
for H (referenced to TMS, δH 0.0 ppm) and 75 MHz (or 125 MHz)
for 13C in CDCl3 (referenced to the center line of CDCl3, δC 77.0 ppm).
LRMS and HRMS spectra were obtained by ESI on a Bruker APEX II
mass spectrometer. Silica gel 60 (Merck, 230-400 mesh) was used
for column chromatography. Precoated silica gel plates (Merck Kie-
selgel 60 F254 0.2 mm) were used for TLC analysis. High-performance
liquid chromatography (HPLC) was performed on a Shimadzu LC-
10AT apparatus equipped with a Shimadzu SPD-10A UV detector. A
Varian Dynamax normal-phase column (Si gel, 250 × 21.4 mm, 100
Å, 5 µm) was used. S-(+)-R-Methoxy-R-(trifluoromethyl)phenylacetyl
chloride, R-(-)-R-methoxy-R-(trifluoromethyl)phenylacetyl chloride,
and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDC·HCl) were purchased from Aldrich, and 4-(dimethylamino)py-
ridine (4-DMAP) was purchased from Lancaster. 4-DMAP ·HCl was
prepared by adding concentrated HCl to a solution of 4-DMAP in
THF.37
6,6-Dimethylundecane-2,5,10-trione (10): colorless oil; IR (KBr)
ν
max 1715 cm-1; 1H NMR (CDCl3, 300 MHz) 2.75 (2H, m, H2-4), 2.68
(2H, m, H2-3), 2.41 (2H, t, J ) 6.4 Hz, H2-9), 2.20 (3H, s, H3-1), 2.13
(3H, s, H3-11), 1.50 (2H, m, H2-8), 1.47 (2H, m, H2-7), 1.13 (6H, s,
H3-12 and H3-13); 13C NMR (CDCl3, 75 MHz) 214.3 (C, C-5), 208.8
(C, C-10), 207.6 (C, C-2), 47.3 (C, C-6), 43.9 (CH2, C-9), 39.4 (CH2,
C-7), 37.0 (CH2, C-3), 31.1 (CH2, C-4), 30.1 (CH3, C-1), 30.0 (CH3,
C-11), 24.5 (CH3, C-12) 24.5 (CH3, C-13), 19.0 (CH2, C-8); ESIMS
m/z 249 [M + Na]+; HRESIMS m/z 249.1470 [M + Na]+ (calcd for
C13H22O3Na, 249.1468).
Animal Material. The sponge Negombata corticata was collected
by hand using scuba off the southern Taiwan coast in November 2002,
at a depth of 20 m, and was stored in a freezer. This sponge was
identified by one of the authors (L.-H.W.). A voucher specimen was
deposited in the Department of Marine Biotechnology and Resources,
National Sun Yat-sen University (specimen no. DTSP).
Extraction and Isolation. The sponge N. corticata (1.4 kg, wet wt)
was minced and extracted exhaustively with EtOH (4 × 1 L). The
combined extract was concentrated to an aqueous suspension and was
further partitioned between EtOAc and H2O. The EtOAc extract (13
g) was fractionated by open column chromatography on silica gel using
n-hexane-EtOAc and EtOAc-MeOH mixtures of increasing polarity
to yield 14 fractions. Fraction 4, eluted with n-hexane-EtOAc (7:1),
was further separated by Sephadex LH-20 column chromatography with
acetone as eluent to yield compound 2 (100 mg). Fraction 7, eluted
with n-hexane-EtOAc (3:1), was subjected to normal-phase HPLC (n-
hexane-CH2Cl2-EtOAc, 90:5:5) to obtain compound 9 (2.1 mg). Three
subfractions (9A-9C) were afforded from fraction 9 using silica gel
Esterification of 5 and the Mixture of 6 and 7. To a stirring
solution of compound 5 (3 mg) in CH2Cl2 (0.5 mL) were successively
added DMAP (2 mg), 4-DMAP ·HCl (2 mg), and EDC ·HCl (4 mg).
After the mixture was stirred at 0 °C for 3 h, anhydrous MeOH (0.3
mL) was added. The mixture was then warmed to room temperature
and allowed to react overnight. The reaction was quenched by H2O,
and the mixture was subsequently extracted with EtOAc (5 × 6 mL).
The EtOAc extract was combined and successively washed with 5%
aqueous HCl, saturated aqueous NaHCO3, and brine. The organic layer
was dried over anhydrous Na2SO4 and concentrated to give a residue,
which was chromatographed on silica gel with n-hexane-EtOAc (1:
1) as eluent to afford the corresponding methyl ester 5a (2.9 mg): [R]24
D
+55 (c 0.15, CHCl3); 1H NMR (CDCl3, 300 MHz) of 5a: δ 6.79 (1H,
ddd, J ) 16.3, 8.9, 5.7 Hz, H-8), 6.12 (1H, d, J ) 16.3 Hz, H-9), 4.31
(1H, dd, J ) 9.5, 7.5 Hz, H-3), 3.72 (3H, s, OMe), 2.93 (1H, dd, J )
14.3, 5.7 Hz, H-7a), 2.55 (1H, dq, J ) 7.5, 7.2 Hz, H-2), 2.38 (1H, dd,
J ) 14.3, 8.9 Hz, H-7b), 2.27 (3H, s, H3-11), 1.83 (2H, m, H2-5), 1.75
(2H, m, H2-4), 1.16 (3H, d, J ) 7.2 Hz, H3-12), 1.11 (3H, s, H3-13).13C

1542 Journal of Natural Products, 2010, Vol. 73, No. 9
Table 2. 1H NMR Spectroscopic Data of Compounds 4-8
Chao et al.
position
4, δH (J in Hz)a
5, δH (J in Hz)b
6/7, δH (J in Hz)a
8, δH (J in Hz)a
2
3
4
2.58, m
4.25, m
1.69, m
2.56, dq (8.5, 7.5)
4.31, dd (9.5, 8.5)
1.75, m
2.55, m
4.26, m
1.69, m
2.56, m
3.66, m
a: 1.67, m
b: 1.45, m
a: 1.64, m
b: 1.57, m
a: 1.42, m
b: 1.21, m
1.55, m
5
7
a: 1.78, m
b: 1.67, m
a: 1.90, m
b: 1.55, m
2.04, m
1.83, m
1.67, m
a: 2.95, dd (14.1, 5.7)
b: 2.37, dd (14.1, 9.0)
6.81, ddd (16.0, 9.0, 5.7)
6.13, d (16.0)
a: 2.62, m
b: 2.35, dd (13.7, 7.0)
5.60-5.70c
5.60-5.70c
2.03-2.13c
2.23-2.27c
2.55, m
8
9
11
5.14, br t (7.0)
2.05 m
1.65, m
2.01 m
2.28, s
12
13
a: 1.81, m
b: 1.66, m
1.21, d (7.5)
1.12, s
1.56, m
a: 1.50, m
b: 1.23, m
14
15
1.19, d (6.6)
1.07, s
a: 1.83, m
b: 1.68, m
1.37, m
a: 1.39, m
b: 0.97, m
1.21, d (7.0)
16
17
1.51, s
1.17, s
a: 4.75, s
b: 4.53, s
0.92, s
18
19
20
1.17, d (6.8)
1.12, s
0.85, s
21
1.61, s
22
1.28, s
23
1.02, s
24
1.06, s
OCH
3.69, s
a Spectra were measured in CDCl3 (300 MHz). b Spectra were measured in CDCl3 (500 MHz). c Signals are overlapped with the epimer.
NMR (CDCl3, 75 MHz) of 5a: 198.6 (C, C-10), 174.2 (C, C-1), 143.4
(CH, C-8), 134.2 (CH, C-9), 81.4 (CH, C-3), 79.6 (C, C-6), 52.0 (CH3,
OMe), 42.7 (CH, C-2), 37.9 (CH2, C-7), 32.7 (CH2, C-5), 26.8 (CH3,
C-11), 24.3 (CH3, C-13), 22.8 (CH2, C-4), 12.7 (CH3, C-12); ESIMS
m/z 293 [M + Na]+. The same procedure was applied on a mixture of
6/7 (1.2 mg) to prepare the corresponding methyl esters 6a/7a (1.1
with (S)-MTPA chloride. Selected 1H NMR (CDCl3, 300 MHz) of 8b:
δ 7.380-7.550 (5H, m, Ph), 5.378 (1H, m, H-3), 4.753 (1H, s, H-17a),
4.514 (1H, s, H-17b), 3.639 (3H, s, COOMe), 3.537 (3H, s, MTPA-
OMe), 2.859 (1H, m, H-2), 1.742 (1H, m, H-4a), 1.632 (1H, m, H-4b),
1.182 (3H, d, J ) 7.1 Hz, H3-15), 1.027 (3H, s, H3-16), 0.917 (3H, s,
H3-18), 0.841 (3H, s, H3-19).
Cytotoxicity Testing. Cell lines were purchased from the American
Type Culture Collection. Cytotoxicity assays were performed using the
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
colorimetric method.38,39 Doxorubicin was employed as positive control,
which exhibited cytotoxic activity toward MDA-MB-231, MCF-7,
HepG2, Hep3B, and A-549 cancer cell lines with IC50 values of 0.24,
0.59, 0.19, 0.14, and 0.36 µM, respectively.
mg): [R]24 +47 (c 0.09, CHCl3); 1H NMR (CDCl3, 500 MHz) of
D
mixture 6a/7a: δ 5.65-5.69 (2H, overlapped, H-8 and H-9), 4.26 (1H,
td, J ) 9.0, 3.5 Hz, H-3), 3.71 (3H, s, OMe), 2.60 (1H, m, H-7a), 2.58
(1H, m, H-2), 2.56 (2H, m, H2-12), 2.36 (1H, dd, J ) 14.0, 8.0 Hz,
H-7b), 2.21 (1H, m, H11a), 2.07 (1H, m, H-11b), 1.75 (1H, m, H-5a),
1.67 (2H, m, H2-4), 1.65 (1H, m, H-5b), 1.50/1.49 (3H, s, H3-16), 1.16
(3H, d, J ) 7.0 Hz, H3-14), 1.07 (3H, s, H3-15). 13C NMR (CDCl3,
125 MHz) of 6a/7a: 176.7 (C, C-13), 174.3 (C, C-1), 135.6/135.5 (CH,
C-9), 125.2/125.0 (CH, C-8), 85.3 (C, C-10), 81.5 (CH, C-3), 79.8 (C,
C-6), 52.0 (CH3, OMe), 42.7 (CH, C-2), 37.6 (CH2, C-7), 34.2/34.1
(CH2, C-11), 31.9 (CH2, C-5), 29.0 (CH2, C-12), 26.7/26.5 (CH3, C-16),
24.1 (CH3, C-15), 22.7 (CH2, C-4), 12.9 (CH3, C-14); ESIMS m/z 349
[M + Na]+.
Acknowledgment. This work was supported by grants from the
National Science Council of Taiwan (NSC98-2113-M-110-002-MY3)
and Ministry of Education (97C031702) awarded to J.-H.S.
Supporting Information Available: 1H NMR, 13C NMR, and
HRESIMS spectra for 4-10; 1H and 13C NMR spectra of 1, 2, 5a, and
6a/7a; and 1H NMR spectra of MTPA esters 8a and 8b. This material
is available free of charge via the Internet at http://pubs.acs.org.
Reductive Cleavage of Cyclic Peroxide 2. A mixture of 2 (20 mg),
EtOAc (10 mL), HOAc (1 mL), and zinc (600 mg) was stirred at room
temperature overnight. The solution was filtered, evaporated, and
separated on a short silica gel column with n-hexane-EtOAc (1:1) to
afford the corresponding diol (19.8 mg), which has the identical NMR
References and Notes
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D
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