A comparison of sesquiterpene scaffolds across different populations of the tropical marine sponge Acanthella cavernosa

Article abstract of DOI:10.1021/np070156d

Specimens of the Indo-Pacific sponge Acanthella cavernosa Dendy collected from locations along the Eastern coastline of Australia have been shown to contain a range of sesquiterpenes including isothiocyanate 1, isocyanide 10, and the isocyanates 15 and 22

Full text of DOI:10.1021/np070156d

J. Nat. Prod. 2007, 70, 1725–1730
1725
A Comparison of Sesquiterpene Scaffolds across Different Populations of the Tropical Marine
Sponge Acanthella caWernosa
Pinus Jumaryatno,†,‡ Bronwin L. Stapleton,† John N. A. Hooper,§ Douglas J. Brecknell,† Joanne T. Blanchfield,† and
Mary J. Garson*,†
School of Molecular and Microbial Sciences, The UniVersity of Queensland, Brisbane QLD 4072, Australia, Islamic UniVersity of Indonesia,
Yogyakarta, Indonesia, and Queensland Museum, P.O. Box 3300, South Brisbane QLD 4101, Australia
ReceiVed April 5, 2007
Specimens of the Indo-Pacific sponge Acanthella caVernosa Dendy collected from locations along the Eastern coastline
of Australia have been shown to contain a range of sesquiterpenes including isothiocyanate 1, isocyanide 10, and the
isocyanates 15 and 22. These metabolite studies have provided a basis for chemical comparisons between sponge
populations from different geographic locations and between individual specimens collected from a single location.
The bright orange sponge Acanthella caVernosa (order Hali-
chondrida, family Dictyonellidae), which is commonly encountered
on tropical reefs in the Indo-Pacific region, including Australia,
shows remarkable chemical diversity. Chemical studies on the
sponge, and on related species such as Acanthella pulcherrima and
Acanthella klethra, have revealed numerous metabolites character-
ized by the presence of nitrogen-containing functionality, typically
in the form of isocyanide, isothiocyanate, isocyanate, or formamide
substituents attached to a terpene skeleton. Diterpenes of the
kalihinane series of metabolites or sesquiterpenes with a diverse
range of skeletons have both been identified.1,2
Great Barrier Reef populations of A. caVernosa that we have
studied have generally been characterized by the presence of
sesquiterpene compounds alone.3,4 Diterpene (i.e., kalihinane-
derived) metabolites have been encountered only once, in a
collection of sponges from the Wistari channel at Heron Island.3
In contrast, A. caVernosa from Indo-Pacific locations such as Japan,
Guam, the Philippines, Thailand, or Fiji more commonly contains
diterpene metabolites, although sesquiterpene metabolites are also
found.1,2 It may be that there are taxonomic differences between
the sesquiterpene- and diterpene-containing sponges that are not
yet fully apparent using the current suite of depauperate morpho-
logical characters used to differentiate sibling species of Acanthella.
Alternatively, the differing chemistry may result from a response
to environmental factors such as habitat or perhaps is influenced
by the microbial community that may be associated with the sponge.
In preparation for biosynthetic studies on A. caVernosa, we
evaluated the chemical diversity of this common species with
respect to collection site. In this paper we explore subtle chemical
differences between specimens of A. caVernosa collected at the
same location and between samples from different collection sites.
We also provide full characterization of some metabolites from this
sponge species.
described in the Experimental Section, and the resulting crude
extracts were subjected to GC-MS and H NMR analysis. On the
1
basis of the similarity of the GC traces, six of the 10 samples were
combined to give an extract coded as TR1 and the remaining four
extracts combined to give extract TR2. These two organic extracts
were then carefully purified by SiO2 flash chromatography followed
by SiO2 HPLC to afford an array of sesquiterpene compounds
whose structures were assigned on the basis of extensive spectro-
scopic analysis and by comparison with literature data. Sample TR1
afforded 10-isothiocyanatoguai-6-ene (1) along with the known
axisothiocyanate-2 (2),5,6 1-isothiocyanatoaromadendrane (3),7 the
4,8
epimeric 10-isothiocyanato-4-cadinenes 4 and 5,
acanthene B
(6),9 7-isothiocyanato-7,8-dihydro-R-bisabolene (7),10 10R-isothio-
cyanatoalloaromadendrane (8),11 and 10-isothiocyanato-4-amor-
phene (9).12 Also isolated were the new isocyanide 7-isocyano-
7,8-dihydro-R-bisabolene (10) together with the known isocyanides
axisonitrile-3 (11),13,14 1-isocyanoaromadendrane (12),7 and 11-
isocyano-7ꢀH-eudesm-5-ene (13).11 The isothiocyanate fraction of
sample TR2 afforded axisothiocyanate-3 (14),3,6,13,14 the guai-6-
ene 1, and the epimeric cadinenes 4 and 5, together with aroma-
dendranes 3 and 8. This fraction also provided the novel isocyanate
metabolite axisocyanate-3 (15), which we reported recently.15 The
isocyanide metabolites identified in extract TR2 were axisonitrile-3
(11) and 1-isocyanoaromadendrane (12).
1
The H NMR data (Table 1) for isothiocyanate 1 showed an
olefinic proton as a broad singlet at δH 5.16, while methyl doublets
at δH 0.96 and 0.95 coupled to a methine signal at δH 2.25 suggested
the presence of an isopropyl group. A methyl singlet at δH 1.32
corresponded to a methyl adjacent to the –NCS group, while a
doublet signal at δH 1.04 suggested a secondary methyl group. The
13C NMR spectrum showed 12 protonated carbons; there were two
alkene carbons at δC 118.9 (CH) and 149.3 (qC) and a quaternary
carbon at δC 68.0 assigned to the carbon adjacent to the –NCS
group. These data suggested a guai-6-ene structure.16–18 gHMBC
correlations showed the signal for the methyl singlet at δH 1.32
had cross-peaks to δC 54.1, 34.6, and 68.0, which could be assigned
as C-1, C-9, and C-10, respectively, and confirmed the position of
Me-15 at C-10. gHMBC correlations from H-11, H-, and H-13
placed the isopropyl group at C-7, adjacent to the olefinic carbons.
The DQFCOSY spectrum showed connectivities from the olefinic
proton (H-6) at δH 5.16 to the methine proton (H-5) at δH 2.65 and
the methine proton of the isopropyl group (H-11) at δH 2.25.
Additional DQFCOSY correlations were from H-5 to H-1, H-4,
and H-6, and between H-1 and H-6, consistent with a W coupling.
The remaining proton and carbon assignments shown in Table 1
were based on analysis of 2D data.
Results and Discussion
In our study, sponges were collected from three dive sites (Tani’s
Reef, Coral Gardens, both at the Gneerings Reef, and Mudjimba
Island) offshore from Mooloolaba, South-East Queensland, at a
depth of 10–20 m. We first investigated the chemistry of individual
sponge specimens. Ten sponges were collected individually from
approximately the same depth during a single dive at the Tani’s
Reef site. Each sponge was extracted with CH2Cl2/MeOH as
* To whom correspondence should be addressed. Tel: +61-7-3365-3605.
Fax: +61-7-3365-4273. E-mail: m.garson@uq.edu.au.
† The University of Queensland.
‡ Islamic University of Indonesia.
§ Queensland Museum.
10.1021/np070156d CCC: $37.00
2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/23/2007

1726 Journal of Natural Products, 2007, Vol. 70, No. 11
Table 1. NMR Data for Isothiocyanate 1a
Jumaryatno et al.
position
δC
δH (J in Hz)
gHMBCb
NOESY
H-2, H-4, Me-15
H-1, H-2a
1
54.1 CH
23.7 CH2
2.09 (ddd, 12.0, 6.9, 4.0)
1.32 (m)
C-5, C-6, C-9, C-10
2a
2b
3a
3b
4
5
6
7
8a
8b
9a
9b
10
11
12
13
14
15
NCS
1.50 (m)
1.25 (m)
H-1, H-2a, H-3b, Me-15
29.1 CH2
C-1, C-4, C-5
C-3
1.69 (dddd, 14.0, 6.9, 1.5, 1.5)
2.01 (m)
38.9 CH
42.6 CH
118.9 CH
149.3 qC
24.8 CH2
H-3b, Me-14
H-1, H-4, H-8b, Me-14
H-5, H-11, Me-12, Me-13, Me-14
2.65 (m)
5.16 (br, s)
C-1, C-8, C-11
C-6, C-7, C-9, C-10, C-11
1.95 (dddd, 15.6, 6.7, 1,8, 1.8)
2.35 (dddd, 15.6, 13.9, 2.0, 2.0)
1.52 (m)
H-8b, Me-12, Me-13
H-8a, H-9b
H-9b
34.6 CH2
C-7, C-9
C-1, C-7, C-8, C-10, C-15
1.73 (m)
H-9a
68.0 qC
38.0 CH
21.2 CH3
21.2 CH3
15.9 CH3
2.25 (m)
C-6, C-7, C-8, C-11, C-12
C-7, C-11, C-13
C-7, C-11, C-12
C-3, C-4, C-5
Me-12, Me-13
H-6, H-8a, H-11
H-6, H-8a, H-11
H-4, H-5, H-6
H-1, H-2b
0.96 (3H, d, 6.5)
0.95 (3H, d, 7.0)
1.04 (3H, d, 7.0)
1.32 (3H, s)
30.4 CH3
C-1, C-9, C-10
c
a Chemical shifts referenced to CDCl3 (δH 7.25; δC 77.0 ppm). b Shown as 1H–13C; J 1H–13C ) 135 Hz, long-range nJ 1H–13C ) 8 Hz. c Signal not
detected.
Table 2. NMR Data for Isocyanide 10a
position
δC
δH (J in Hz)
1.32 (overlapping)
1.82 (m)
gHMBCb
1ax
1eq
2ax/2eq
3
23.6 CH2
30.7 CH2
133.9 qC
119.8 CH
26.3 CH2
2.00 (2H, m)
C-4, C-6, Me-14
4
5.36 (dd, 1.5, 3.5)
1.92 (m)
2.12 (m)
C-2, C-5, C-6, Me-14
C-4
5ax
5eq
6
7
8a
41.9 CH
63.4 (t) qC
38.3 CH2
1.65 (m)
C-2, C-4, Me-15
C-9, C-10, Me-15
1.65 (m)
8b
1.50 (m)
9a/b
10
11
12
13
14
15
NC
22.5 CH2
122.9 CH
132.7 qC
17.7 CH3
25.7 CH3
23.2 CH3
23.5 CH3
153.7 (t) qC
2.10 (2H, m)
5.07 (dddd, 7.1, 7.1, 1.4, 1.4)
C-8, C-10, C-11
C-8, C-9, Me-12, Me-13
1.61 (3H, s)
1.67 (3H, d, 1.0)
1.63 (3H, s)
C-10, C-11, Me-13
C-10, C-11, Me-12
C-2, C-3, C-4
1.32 (3H, t, 2.0)
C-6, C-7, C-8
b
a Chemical shifts referenced to CDCl3 (δH 7.25; δC 77.0 ppm). Shown as H–13C; J H–13C ) 135 Hz, long-range J H–13C ) 8 Hz.
1
1
n
1
Tada et al. reported 10-isothiocyanatoguai-6-ene (16) from an
unidentified Japanese sponge, but the compound was poorly
characterized by NMR, and there was no proof of relative
stereochemistry other than X-ray crystallography analysis of a co-
occurring isocyanide.16 The diastereomeric isothiocyanate 17 was
later isolated from a Palauan specimen of Axinyssa aplysinoides
by He et al. and the relative stereochemistry secured by a difference
NOE experiment.17 In the NOESY spectrum of 1, there were
correlations from H-1 to H-5, confirming cis stereochemistry at
the ring junction, and from H-1 to H-4, which confirmed the
configuration at C-4. Consistent with this, H-4 had a NOESY
correlation to H-5, and H-6 showed a correlation to Me-14. There
was a NOESY correlation from Me-15 to H-1, which first suggested
that 1 was epimeric to the Tada compound at C-10. However
detailed conformational analysis using PC-Model has revealed that
in each of the two isomers 1 and 16 Me-15 is pseudoequatorial,
and so it could be expected to show an NOE to H-1. Consequently
the relative configuration at C-10 could not be determined.
at δC 153.7 (t, J ) 4.5 Hz, coupled to 14N) confirmed the presence
of the –NC function, while a signal at δC 63.4 (t, J ) 4.6 Hz) was
assigned to a carbon bearing a nitrogen substituent. There were
four alkene carbons at δC 133.9 (qC), 132.7 (qC), 122.9 (CH), and
119.8 (CH), consistent with the presence of two trisubstituted double
bonds. Comparison of these data with the literature suggested this
compound had a bisabolene structure.10,19 The alkene signal at δH
5.07 could be assigned to H-10 since it showed correlations to two
methyl groups (δH 1.61 and 1.67) in the DQF COSY spectrum.
The other alkene proton (H-4) at δH 5.36 showed a correlation to
1
the methyl at δH 1.63 assigned to Me-14. Some H NMR signals
were not well resolved either by DQF COSY or by HSQC; therefore
a 1D TOCSY experiment was undertaken. When the signal at δH
1.82 for H-1 was irradiated, there were correlations to H-2 at δH
2.0, to H-6 at δH 1.65, and to a signal at δH 1.32, also assigned to
H-1. The alkene carbons at δC 133.9 and 132.7 could be assigned
to C-3 and C-11, respectively, on the basis of gHMBC correlations
to their methyl groups, while C-7 at δC 63.4 showed a correlation
from the methyl signal at δH 1.32 (H-15). The remaining assign-
ments shown in Table 2 were based on detailed analysis of 2D
NMR data.
1
The H NMR of isocyanide 10 showed two olefinic protons at
δH 5.36 (1 H, br dd, J ) 3.5 Hz, 1.5 Hz) and 5.07 (1 H, br m),
respectively, three olefinic methyl signals at δH 1.67 (3H, s), 1.63
(3H, s), and 1.61 (3H, s), and a signal at δH 1.32 (3H, t, J ) 2.0
Hz, coupled to 14N) that was assigned to the methyl group adjacent
to an isocyanide group (Table 2). In the carbon spectrum, a signal
Sullivan et al. reported 7-isothiocyanato-7,8-dihydro-R-bisabolene
7 from a Halichondria sp. and deduced the stereochemistry as (6R,
7S) by chemical correlation with (6R,7E)-R-bisabolene.10 This

Sesquiterpene Scaffolds of the Sponge Acanthella caVernosa
Journal of Natural Products, 2007, Vol. 70, No. 11 1727
Table 3. Collection Sites and Chemistry for Different Populations of Acanthella caVernosa
compounds isolated
location
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
20
21
22
23
a
Tani Reef TR1 G322184
24-10-04
X
X
X
X
X
X
X
X
X
X
X
X
X
Tani Reef TR2 G322184
24-10-04
Coral Gardens G319567
16-1-02
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Coral Gardens 20-9-00
Mudjimba Island
1-12-04
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
a Detected by GC-MS, but not isolated.
compound has also been isolated from P. pustulosa by Kassühlke
et al.20 Gulavita et al. isolated the bisabolene isocyanide 18 from
a Hawaiian specimen of Ciocalypta sp.; however the stereochem-
istry of their compound was not rigorously proven.19 More recently,
this compound has been reported without spectroscopic data from
the nudibranchs Phyllidia pustulosa21 and Phyllidia ocellata,22 and
those reported elsewhere7 for isocyanide 12. Samples of A.
caVernosa collected at Mudjimba Island provided isocyanate 22,
together with isothiocyanates 3, 8, 17, and 20 and the cadinene
isothiocyanate 23.29 The isocyanides isolated were 11–13.
From a chemical perspective, the most valuable aspects of this
study are that all the collections analyzed contained aromadendrane
isothiocyanates 3 and 8, the spiroaxane isocyanide 11, and
aromadendrane isocyanide 12, while four out of the five collections
contained the spiroaxane isothiocyanate 14 (Table 3). Although the
amounts of the major compounds isolated from each collection
differed, we consider the presence of these four compounds to be
taxonomically representative of A. caVernosa at Mooloolaba. The
five specimens collected at Mudjimba Island were all chemically
identical, whereas sponges collected at Tani’s Reef or Coral Gardens
showed greater chemical diversity.
from Acanthella caVernosa.22 The H NMR data cited for 18 by
1
Gulavita et al. are unreliable; when the 1H NMR data for compounds
7 and 18 were compared to each other or to other bisabolene
compounds reported in these two papers, there were significant
differences in the reported chemical shifts for the alkene protons.
In contrast, the 13C NMR data reported for 7 and 18 agreed apart
from the expected differences in chemical shift in the vicinity of
C-6 and C-7. The NMR data for compound 10 matched closely
that of 7 apart from minor chemical shift differences caused by
the change in substituent at C-7. The optical rotation of 10 was
+60.8, compared to the value of –49.9 for isocyanide 18 and +60.5
for isothiocyanate 7. Since literature precedent shows that R–NC
and the corresponding R–NCS compounds have closely similar
optical rotation values,4,13 the new isocyanide was proposed to have
a (6R, 7S) configuration. Isolation of the isothiocyanate 7 from the
same extract strongly supported this stereochemical conclusion. On
treatment with elemental sulfur and with catalytic amounts of
selenium and Et3N in THF, isocyanide 10 was converted to an
isothiocyanate product whose 1H and 13C NMR data were identical
with those of 7.
Cadinene 4 was first described by our group from a Heron Island
collection of A. caVernosa.4 The same compound was subsequently
reported from a Fijian collection of Phakellia carduus by Wright,23
but the NMR data of the two samples differ. Notably, the 13C NMR
signal for C-10 was reported at δC 64.7 for the Heron Island
compound and at δC 61.1 for the Fijian compound. The corre-
sponding isocyanide 19 has δC 60.7 for C-10.24 In RNC/RNCS
pairs, the chemical shift of an isothiocyanato-substituted carbon is
usually between 3 and 4 ppm downfield of the value for the
equivalent isocyano-substituted carbon.7,11 On this basis, the Heron
island data better match the suggested structure. Although axisoni-
trile-3 11 and axisothiocyanate-3 14 are frequently reported from
Acanthella spp., they remain poorly characterized in the literature.13
Consequently, full NMR assignments are reported for both com-
pounds in the Experimental Section.
The chemistry of Tani’s Reef samples was next compared with
those collected at two other dive sites, Coral Gardens and Mudjimba
Island. Samples of A. caVernosa collected at Coral Gardens provided
isothiocyanates 8 and 2025 and isocyanides 11 and 12. A second
collection from this site provided isothiocyanates 3, 8, 17, 20, and
21,11 isocyanides 11–13, and the aromadendrane isocyanate 22. This
last metabolite was first reported by Braekman et al. without any
detailed spectroscopic characterization,26 and the relative stereo-
chemistry initially proposed, along with that for the corresponding
isocyanide, deduced by chemical comparison with the terpene
alcohol palustrol.27,28 A subsequent stereochemical reconsideration
for palustrol28 led to the revised stereochemistry shown here for
22. The 1H and 13C NMR data of the Braekman isocyanide27 match
Intraspecies variation in sponge chemistry represents a significant
challenge to the reisolation of pharmacologically useful lead
compounds for development studies. The reasons for chemical

1728 Journal of Natural Products, 2007, Vol. 70, No. 11
Jumaryatno et al.
from light to moderately heavy. Megascleres consist of axial strongyles,
sinuous or curved (287-609 × 2-11.5 µm), and extra axial styles,
straight or curved (271-453 × 3-12 µm). Variability within popula-
tions of this species mainly concern the density of the axial skeletal
compression (looser in specimen G322184; more compressed in
G319567); development of collagen in the mesohyl (lighter in G322184
and heavier in G319567); and thickness or degree of silicification of
megascleres (thinner in G322184 and thicker in G319567). Neverthe-
less, this variation is not considered to be significant or to differentiate
either as not conspecific.
Extraction and Isolation of Metabolites: Tani’s Reef. Ten frozen
sponges (8.3 g each) were chopped finely and extracted with CH2Cl2/
MeOH, 1:1 (3 × 50 mL each). The extracts were filtered through a
cotton wool plug, evaporated to aqueous suspension, and partitioned
between H2O (20 mL) and ethyl acetate (3 × 70 mL). The organic
layers were combined, dried with anhydrous MgSO4, and evaporated
to give dark orange crude extracts. On the basis of GC/MS analysis
and 1H NMR data, six crude extracts were combined into a single
sample named TR1 (501 mg), and four crude extracts were combined
into a single sample named TR2 (281 mg). Each sample was then
subjected to SiO2 flash chromatography using a solvent gradient (100%
hexanes to 5:1, 1:1, 1:5 hexanes/CH2Cl2 to 100% CH2Cl2 to 5:1, 1:1,
1:5 CH2Cl2/EtOAc to 100% EtOAc to 100% MeOH).
diversity within a given sponge species are likely complex and can
be related to genetic30 and environmental factors (including light
and depth),31,32 a response to predation33 or infection,34 competition
for nutrients,35 or the involvement of microbial symbionts.30,32,36
Transplant experiments have shown that chemical variation in
diterpene composition in the tropical marine sponge Rhopaloeides
odorabile may be related to environmental factors such as light
rather than to genetic differences per se.31 However a study on the
diterpene chemotype of A. caVernosa that was kept in aquaria for
extended periods showed little chemical difference from wild
sponges.37 Speculation on a role for microorganisms in the
biosynthesis of terpenes in Acanthella sponges is premature for two
reasons. First, the symbiont populations of these sponges have not
been adequately described, and second, the ability to synthesize
terpenes has not been ascribed to marine microorganisms. In a
number of sponge genera, terpenes have been shown to be localized
within sponge cells rather than within symbionts.36,38,39 It is clear
from our study that, while there are major metabolites consistently
produced by Acanthella caVernosa, there are as yet unknown factors
that trigger the production of other metabolites in some specimens
and not in others. Whether these represent genetic variants of the
sponge or a response to subtle triggers such as co-located organisms,
predation, trace elements or nutrient content of the water, water
temperature, or sunlight exposure is a matter for further investiga-
tion. Finally, seasonal variation is a factor that could not be
addressed in the current study, but which has been documented to
impact on sponge chemistry or toxicity.40–42
TR1 Sample. The 100% hexanes fraction contained sesquiterpene
hydrocarbons including 9-aristolene (9.9 mg). The combined fractions
eluting with 100% hexanes and hexanes/CH2Cl2 (5:1) were further
purified by NP HPLC (two columns in series, 0.25% EtOAc/hexanes,
2 mL/min) to give the new 10-isothiocyanatoguai-6-ene (1) (0.2 mg)
and a mixture of axisothiocyanate-2 (2) and 1-isothiocyanatoaroma-
dendrane (3) (9.7 mg). The next fractions eluting with hexanes/CH2Cl2
(5:1) were further purified by NP HPLC (two columns in series, 0.25%
EtOAc/hexanes, 2 mL/min) to give a mixture of the two 10-
isothiocyanato-4-cadinenes (4 and 5) (6.9 mg), a mixture of acanthene
B (6) and 7-isothiocyanato-7,8-dihydro-R-bisabolene (7) (4.6 mg), 10R-
isothiocyanatoalloaromadendrane (8) (1.3 mg), and 10-isothiocyanato-
4-amorphene (9) (3.8 mg). NP HPLC (two columns in series, 0.5%
EtOAc/hexanes, 2 mL/min) of a portion of the hexanes/CH2Cl2 (1:1)
flash column fraction gave axisonitrile-3 (11) (0.6 mg), 1-isocyanoaro-
madendrane (12) (0.9 mg), 7-isocyano-7,8-dihydro-R-bisabolene (10)
(3.0 mg), and 11-isocyano-7ꢀH-eudesm-5-ene (13) (0.8 mg).
Experimental Section
General Experimental Procedures. Optical rotations were recorded
on a Perkin-Elmer 241-MC polarimeter. One- and two-dimensional
NMR spectra were acquired using Bruker AMX-400, Bruker DRX-
500, or Bruker DMX-750 instruments. NMR spectra were obtained in
deuterochloroform or deuterated methanol at room temperature. Samples
were internally referenced to CHCl3 at δH 7.25 and δC 77.0 or MeOH
at δH 3.30. High- and low-resolution electron impact mass spectrometry
(EIMS) were recorded on a Kratos MS25RFA mass spectrometer with
an ionizing voltage of 70 eV. Gas chromatography/mass spectrometry
(GC/MS) were recorded on a Hewlett-Packard 5890A gas chromato-
graph, carrying a DB5 capillary column in tandem with a Hewlett-
Packard 5970 mass selective detector or a Shimadzu GCMS-QP5050A
gas chromatograph mass spectrometer, carrying a Zebron ZB-5 capillary
column (30 cm × 0.32 mm i.d.; × 0.25 µm df; 5% phenyl polysiloxane)
with a Shimadzu AOC-20i auto injector. Retention times were obtained
using the following temperature ramping program: initial oven tem-
10-Isothiocyanatoguai-6-ene (1): colorless oil; [R]D +18.4 (c 0.043,
1
13
CHCl3); IR (CHCl3) νmax 2110 (–NCS) cm-1; H and C NMR see
Table 1; GC/MS m/z [M]+ 263 (28), 248 (7), 205 (9), 161 (86), 105
(100); HREIMS m/z 263.1706 [M]+ (calcd for C16H25NS, 263.1708).
7-Isocyano-7,8-dihydro-r-bisabolene (10): yellow oil; [R]D +60.8
(c 0.049, CHCl3); IR (CHCl3) νmax 2136 (–NC) cm-1; 1H and 13C NMR
in CDCl3 see Table 2; 1H NMR (C6D6) δ 5.26 (1H, dd, 1.5, 4.0, H-4),
5.04 (1H, br m, H-10), 2.13 (1H, m, H-9a), 2.02 (1H, m, H-9b), 1.96
(1H, m, H-5eq), 1.80 (1H, m, H-5ax), 1.75 (2 × 1H, m, H-2), 1.65
(3H, s, H-12), 1.57 (3H, s, H-14), 1.53 (3H, s, H-13), 1.52 (1H, m,
H-1eq), 1.46 (1H, m, H-8a), 1.40 (1H, m, H-6), 1.27 (1H, m, H-8b),
1.12 (1H, m, H-1ax), 0.94 (3H, t, 2.0, H-15); 13C NMR (C6D6) δ 133.4
(C-3), 132.2 (C-11), 123.6 (C-10), 120.4 (C-4), 63.1 (C-7), 42.1 (C-
6), 38.6 (C-8), 30.8 (C-2), 26.5 (C-5), 26.0 (C-12), 23.7 (C-1), 23.30
(C-15), 23.27 (C-14). 22.9 (C-9), 17.5 (C-13); GC/MS m/z [M]+ 231
(2), 216 (7), 205 (2), 204 (12), 121 (56), 93 (100); HREIMS m/z
231.1976 [M]+ (calcd for C16H25N, 231.1987).
perature 50 or 100 °C; isothermal time 3.0 min, ramp 16 °C min-1
final oven temperature 270 °C.
;
Animal Material. Specimens of Acanthella caVernosa Dendy 1922
(order Halichondrida, family Dictyonellidae) were collected at a depth
of 10–20 m from Tani’s Reef or Coral Gardens dive sites, both part of
the Gneerings reef, Mooloolaba, or from Mudjimba Island, offshore
from Maroochydore (Australia). The Tani’s Reef and Coral Gardens
sites are approximately 3 km offshore and less than 1 km apart from
each other, whereas Mudjimba Island is 1 km offshore and 5 km distant
from the Gneerings Reef sites. The samples were transported to the
University of Queensland on ice, where they were stored at -20 °C
until analysis. Voucher samples held at The Queensland Museum are
for specimens collected at Tani’s Reef (QM G322184) and Coral
Gardens Dive Site (QM G319567). Although already well-described
in the literature, a brief taxonomic description of the Queensland
populations is appropriate here. Growth form is subspherical to
flabellate, digitate or globular, with a honeycombed, reticulate construc-
tion. Color varies from bright orange, pale orange, or light brown alive.
Oscules are large, located between surface conules, surrounded by
membranous “lip” flush with surface. Texture is rubbery. Surface
ornamentation consists of prominent conules, sharpish, interconnected
by ridges, and cavernous. Ectosomal skeleton is membranous, collag-
enous, but with tips of styles protruding singly, regularly spaced.
Choanosomal skeleton consists of axially compressed fibers, with degree
of compression varying among specimens cored by strongyles (light
fibers nearly fully cored) with extra-axial styles embedded in the axial
skeleton and ascending to the surface. Collagen in the mesohyl varies
Preparation of Synthetic 7-Isothiocyanato-7,8-dihydro-r-bisa-
bolene (7). A sample of 7-isocyano-7,8-dihydro-R-bisabolene 10 (1.3
mg, 5.6 µmol) in dry THF 0.5 M (0.011 mL) was added into a mixture
of elemental S (0.2 mg, 6.7 µmol), a catalytic amount of Se (5 mol
%), and freshly distilled triethylamine (1.36 mg, 0.0134 mmol).43 The
mixture was refluxed for 2 h at room temperature, then the deposited
Se was removed by filtration. Removal of triethylamine and THF in
Vacuo afforded the desired 7-isothiocyanato-7,8-dihydro-R-bisabolene
(7) (1.4 mg, 93%) as an amber oil: [R]D +23.7 (c 0.015, CHCl3), lit.10
+60.5 (c 6.8, CHCl3); 1H and 13C NMR identical to data for the natural
compound.10
Axisonitrile-3 (11):
13
pale white solid; mp 99–102 °C, lit.13
101–103 °C; [R]D +43.4 (c 0.006, CHCl3), lit.13 [R]D +68.4 (c 1.0,
CHCl3); 1H NMR (CDCl3) δ 5.11 (1H, br s, H-4), 3.57 (1H, br s, H-6),
2.21 (2H, m, H-2a, H-2b), 1.93 (2H, m, H-1a, H-1b), 1.77 (2H, m,
H-8a, H-10), 1.72 (3H, br s, H-14), 1.57 (1H, m, H-11), 1.48 (1H, m,
H-9a), 1.33 (1H,ddd, 4.0, 13.0, 13.0, H-8b), 1.13 (1H, m, H-7). 1.04

Sesquiterpene Scaffolds of the Sponge Acanthella caVernosa
Journal of Natural Products, 2007, Vol. 70, No. 11 1729
(1H, m, H-9b), 0.92 (3H, d, 6.6, H-12), 0.89 (3H, d, 6.6, H-13), 0.74
(3H, d, 7.0, H-15); 13C NMR (CDCl3) δ 155.6 (br, C-16), 144.8 (C-3),
123.6 (C-4), 64.5 (C-6), 57.0 (C-5), 43.8 (C-7), 35.8 (C-2), 34.9 (C-
1), 34.3 (C-10), 31.2 (C-9), 29.7 (C-11), 24.9 (C-8), 20.7 (C-12), 20.3
(C-13), 16.9 (C-14), 16.0 (C-15); GC/MS m/z 231 [M+], 216, 205.
Axisothiocyanate-3 (14):.3,6,13 pale yellow oil; [R]D +152.3 (c
0.003, CHCl3), lit.13 [R]D +165.2 (c 1.0, CHCl3); 1H NMR (CDCl3) δ
5.11 (1H, q, 2.1, H-4), 3.67 (1H, br s, H-6), 2.22 (2H, m, H-2a, H-2b),
1.90 (2H, m, H-1a, H-1b), 1.79 (1H, m, H-8a), 1.72 (3H, br s, H-14),
1.68 (1H, m, H-10), 1.52 (2H, m, H-9a, H-11), 1.24 (2H, m, H-7, H-8b),
1.08 (1H, m, H-9b), 0.91 (3H, d, 8.3, H-12), 0.89 (3H, d, 8.3, H-13),
0.75 (3H, d, 8.3, H-15); 13C NMR (CDCl3) δ 144.8 (C-3), 129.2 (C-
16), 123.9 (C-4), 67.3 (C-6), 58.9 (C-5), 45.3 (C-7), 35.9 (C-2), 35.0
(C-1, C-10), 31.3 (C-9), 30.1 (C-11), 25.4 (C-8), 20.8 (C-12), 20.4
(C-13), 16.9 (C-14), 16.1 (C-15); GC/MS m/z 263 [M+], 248, 205.
TR2 Sample. The 100% hexanes fraction contained sesquiterpene
hydrocarbons including 9-aristolene (4.3 mg). The combined fractions
eluting with 100% hexanes and hexanes/CH2Cl2 (5:1) (17.4 mg) were
further purified by NP HPLC (two columns in series, 0.25% EtOAc/
hexanes, 2 mL/min) to give a mixture of axisothiocyanate-3 (14) and
axisocyanate-3 (15) (1.8 mg), the new 10-isothiocyanatoguai-6-ene (1)
(1.1 mg), 1-isothiocyanatoaromadendrane (3) (6.6 mg), a mixture of
10-isothiocyanato-4-cadinenes (4 and 5), and 10R-isothiocyanatoal-
loaromadendrane (8) (0.4 mg) The next fraction eluting with hexanes/
CH2Cl2 (5:1) contained acanthene B (6) (1.1 mg). The fraction eluting
with hexanes/CH2Cl2 (1:1) was further purified by NP HPLC (two
columns in series, 0.5% EtOAc/hexanes, 2 mL/min) to give axisoni-
trile-3 (11) (4.2 mg) and 1-isocyanoaromadendrane (12) (1.0 mg).
Extraction and Isolation of Metabolites: Coral Gardens. Frozen
sponge (22.3 g) was chopped finely and extracted as before to give a
dark orange crude extract (225 mg). When chromatographed on SiO2,
the 100% hexanes fraction contained sesquiterpene hydrocarbons
including (-)-9-aristolene (46.2 mg). The combined fractions eluting
with 100% hexanes and hexanes/CH2Cl2 (5:1) contained a mixture of
10R-isothiocyanatoalloaromadendrane(8)and(1R,5R,6R,8S)-dec[4.4.0]ane-
1,5-dimethyl-8-(1′-methylethyl)-5-isothiocyanate (20) (1.8 mg). The
hexanes/CH2Cl2 (5:1) flash column fraction contained a mixture of
axisonitrile-3 (11) and 1-isocyanoaromadendrane (12) (49.0 mg).
Extraction of a second sample (225 g) collected at the same site yielded
isothiocyanates 3 (1.8 mg), 8 (1.9 mg), 14 (10 mg), 20 (0.2 mg), and
21 (1.1 mg), isocyanides 11 (121 mg), 12 (124 mg), and 13 (40 mg),
and the aromadendrane isocyanate 22 (0.7 mg).
for spectroscopic assistance. K. Rands-Trevor assisted with the prepara-
tion of synthetic 7. P.J. and B.L.S. acknowledge the Islamic University
of Indonesia and the School of Molecular and Microbial Sciences,
respectively, for financial support. We thank two anonymous referees
for their valuable comments on the manuscript.
Supporting Information Available: Figures S1–S6. 1H and 13C
NMR spectra for compounds 1, 10, and 22 and photograph of A.
caVernosa . This material is available free of charge via the Internet at
http://pubs.acs.org.
References and Notes
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1-Isocyanatoaromadendrane (22):26 colorless oil; 1H NMR
(CDCl3) δ 2.19 (1H, m, H-4), 1.91 (2 × 1H, m, H-3a, H-8a), 1.88
(1H, m, H-2a), 1.67 (1H, t, 11.0, H-5), 1.46 (1H, m, H-3b), 1.37 (3 ×
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s, H-13), 1.02 (1H, m, H-8b), 0.99 (3H, s, H-12), 0.98 (3H, d, J ) 7.3,
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0.62 (1H, dd, J ) 11.0, 9.2, H-6); sample decomposed during
acquisition of 13C NMR data; GC/MS m/z [M]+ 247, 232, 219, 204,
176, 161, 122,107, 81, 67, 41; HREIMS m/z 247.1936 [M]+ (calcd for
C16H25NO, 247.1940).
Extraction and Isolation of Metabolites: Mudjimba Island. Five
sponges (8.3 g each) were chopped finely and extracted as before to
give dark orange crude extracts of between 100 and 150 mg in size.
1
On the basis of the GC/MS analysis and H NMR spectra, the five
crude extracts were combined into a single sample, which was then
subjected to SiO2 flash chromatography using the previously described
solvent gradient. The fraction eluting with 100% hexanes and hexanes/
CH2Cl2 (5:1) contained sesquiterpene hydrocarbons including 9-aris-
tolene (4.1 mg). The combined fractions eluting with hexanes/CH2Cl2
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HPLC using 0.25% EtOAc/hexanes with two HPLC columns in series
(flow rate 2 mL/min) to give a mixture of axisothiocyanate-3 (14) and
axisocyanate-3 (15) (1.1 mg), a mixture of 1-isothiocyanatoaromaden-
drane (3) and the cadinene 23 (3.6 mg), 10R-isothiocyanatoalloaro-
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(1:1, 1:5) fraction contained a mixture of axisonitrile-3 (11) and
1-isocyanoaromadendrane (12) (80.3 mg). The hexanes/CH2Cl2 (1:5)
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NP070156D