Callyspongisines A-D: Bromopyrrole alkaloids from an Australian marine sponge, Callyspongia sp.

Article abstract of DOI:10.1039/c4ob00091a

An extract of the Great Australian Bight marine sponge Callyspongia sp. (CMB-01152) displayed inhibitory activity against the neurodegenerative disease kinase targets casein kinase 1 (CK1), cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3 (GSK3β). Chemical investigation, employing HPLC-DAD-MS single ion extraction protocols, facilitated identification of the new bromopyrrole alkaloids, callyspongisines A-D (1-4), and two known co-metabolites, hymenialdisine (5) and 2-bromoaldisine (6). Structure elucidation of 1-6 was supported by detailed spectroscopic analysis and chemical interconversion, as well as biosynthetic and synthetic considerations. Callyspongisine A (1) is only the second reported example of a natural imino-oxazoline, and the first to feature a spiro heterocyclic framework, while callyspongisines B-D (2-4) were speculated to be storage and handling artefacts of 1. The kinase inhibitory activity detected in Callyspongia sp. (CMB-01152) was attributed to 5. This journal is The Royal Society of Chemistry 2014.

Full text of DOI:10.1039/c4ob00091a

Organic &
Biomolecular Chemistry
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Callyspongisines A–D: bromopyrrole alkaloids
from an Australian marine sponge,
Callyspongia sp.†
Cite this: Org. Biomol. Chem., 2014,
2, 1579
1
Fabien Plisson, Pritesh Prasad, Xue Xiao, Andrew M. Piggott, Xiao-cong Huang,
Zeinab Khalil and Robert J. Capon*
An extract of the Great Australian Bight marine sponge Callyspongia sp. (CMB-01152) displayed inhibitory
activity against the neurodegenerative disease kinase targets casein kinase 1 (CK1), cyclin-dependent
kinase
5 (CDK5) and glycogen synthase kinase 3 (GSK3β). Chemical investigation, employing
HPLC-DAD-MS single ion extraction protocols, facilitated identification of the new bromopyrrole alka-
loids, callyspongisines A–D (1–4), and two known co-metabolites, hymenialdisine (5) and 2-bromo-
aldisine (6). Structure elucidation of 1–6 was supported by detailed spectroscopic analysis and chemical
interconversion, as well as biosynthetic and synthetic considerations. Callyspongisine A (1) is only the
second reported example of a natural imino-oxazoline, and the first to feature a spiro heterocyclic frame-
work, while callyspongisines B–D (2–4) were speculated to be storage and handling artefacts of 1. The
kinase inhibitory activity detected in Callyspongia sp. (CMB-01152) was attributed to 5.
Received 13th January 2014,
Accepted 14th January 2014
DOI: 10.1039/c4ob00091a
www.rsc.org/obc
plaques. Likewise, tau pathology is believed to be caused by a
host of kinases, including casein kinase 1 (CK1), cyclin-depen-
Introduction
Natural products can be viewed as bioactive chemicals pro- dent kinase 5 (CDK5) and glycogen synthase kinase 3 (GSK3β),
duced by organisms, and optimized by evolution, to deliver a abnormally hyperphosphorylating the highly soluble micro-
survival advantage. Knowledge of natural product chemistry, tubule-associated tau protein to generate neurofibrillary
1
ecology and biology has inspired generations of researchers, tangles (NFT). The development of clinically useful treat-
informing our understanding of the natural world, and inspir- ments for AD (and other NDs) has included a search for safe,
ing the development of many of the most successful medi- potent and selective inhibitors of amyloid and tau pathology.
cines. Notwithstanding past successes, there remains an
In collaboration with the Spanish pharmaceutical company
urgent need to continue natural products discovery, to deliver Noscira, we screened a library of ∼2600 southern Australian
new and improved therapeutics capable of treating a broad and Antarctic marine organisms to successfully detect extracts
array of existing and emerging diseases. For example, the very that inhibited BACE (27, 1%), CK1 (44, 1.7%), CDK5 (26, 1%)
fact that healthcare programs successfully extend life has the and GSK3β (69, 2.6%), and that contained rare and new
alarming corollary of increasing the incidence of neurodegene- natural product scaffolds. A particularly promising extract,
rative disorders (NDs) in an aging population. Patients from a Great Australian Bight marine sponge Callyspongia sp.
suffering from the archetypal ND, Alzheimer’s disease (AD), (CMB-01152), displayed inhibitory activity against all three
experience severe and irreversible deterioration of cognitive kinases, with preliminary chemical profiling (HPLC-DAD-MS
1
ability and loss of quality of life, driven by a highly distinctive and H NMR) indicating the presence of brominated alkaloids.
amyloid and tau brain pathology. Existing wisdom has
amyloid pathology caused by the protease β-secretase (BACE)
abnormally processing the essential neuronal transmembrane
amyloid precursor protein (APP) to generate amyloid (Aβ) Results and discussion
A portion of the aqueous EtOH extract of Callyspongia sp.
Institute for Molecular Bioscience, The University of Queensland, St. Lucia,
QLD 4072, Australia. E-mail: r.capon@uq.edu.au; Fax: +61-7-3346-2090;
Tel: +61-7-3346-2979
(CMB-01152) was decanted, concentrated in vacuo and sub-
jected to solvent partitioning and trituration. Analytical
HPLC-DAD-MS analysis (Fig. 1) revealed two major products
†
Electronic supplementary information (ESI) available: Full details of sponge
(
5 and 6), while careful analysis of the very minor co-metab-
taxonomy, bioassays, synthetic transformations, tabulated 2D NMR data and
NMR spectra. See DOI: 10.1039/c4ob00091a
olites (utilizing single ion extraction protocols) detected four
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analysis, hydrolysis of a portion of 1 yielded a sufficient quan-
tity of 2 to permit assignment of the structure, as shown in
Fig. 2. The known bromopyrroles 5 and 6 were identified by
detailed spectroscopic analysis with comparison to literature
2
,3
data,
below.
HRESI(−)MS analysis of 1 revealed a quasi-molecular
while the structure elucidation of 1–4 is outlined
−
ion [M
14BrN
DMSO-d ) data (Table 1) permitted assembly of structure frag-
−
H] consistent with the molecular formula
C
12
H
5 5
O S (Δmmu −1.5), while analysis of the NMR
(
6
ments A, B and C (Fig. 3). Fragment A (N-1 to C-10) was identi-
fied as a 2-bromopyrrole fused to a 7-membered lactam, with
the 2-bromo regiochemistry supported by comparison of NMR
chemical shifts in 1 (δH 6.13, s, H-3; δC 110.5, C-3) with those
for the known 2-bromopyrrole co-metabolites hymenialdisine
(
5) (δ
H C H
6.63, s, H-3; δ 111.2, C-3), 2-bromoaldisine (6) (δ 6.68,
s, H-3; δ 113.3, C-3) and spongiacidin D (7) (δ 6.53, s, H-3; δC
C
H
Fig. 1 HPLC-DAD-MS analysis of Callyspongia sp. (CMB-01152) crude
extract showing HPLC-DAD trace at 254 nm and extracted ion
mass chromatograms corresponding to quasi-molecular ions for 1, 2, 5,
4
1
11.7, C-3), and the known 3-bromopyrrole analogues debro-
5
mostevensine (8) (δ
hymenin (9) (δH 7.08, d, H-2; δ 122.1, C-2) and spongiacidin
H C
7.20, d, H-2; δ 123.0, C-2), debromo-
5
6
and 3.
C
4
H C
B (10) (δ 7.28, H-2; δ 123.2, C-2) (Fig. 4). Fragment B (N-15
to C-17, plus C-11) was identified as a taurinyl residue fused to
2
an sp hybridized deshielded quaternary carbon (δC 181.0,
C-11), with the latter more consistent with an imidazoline (e.g.
6
nagelamide X (11), δ
C
179.1) than an amide (e.g. mauritamide
7
D (12), δC 158.8) (Fig. 4). The presence of a taurinyl moiety
was further supported by comparison of NMR data with an
array of known sponge taurinyl-2-aminoimidazoles (ESI Fig. S2
and Table S1†). A diagnostic HMBC correlation from 15-NH to
C-10 supported assembly of fragments A and B (Fig. 3), and
2
necessitated that fragment C (CH NO) be incorporated into
1
3
1
as a spiro heterocyclic moiety. The deshielded C NMR shift
for C-10 in 1 (δ
C
91.1) favoured substitution by oxygen (e.g.
82.3, and indolmycin (14), δ 85.3), versus
nitrogen (e.g. 5-phenyl-hydantoin (15), δC 61.2) (Fig. 4).
Diagnostic HMBC correlations from 14-NH to C-11 and from
H-9 to C-13 supported assembly of fragments A, B and C
Fig. 3). Thus callyspongisine A (1) was identified as the first
pemoline (13), δ
C
C
8
2
(
example of a natural product bearing a spiro-imino-oxazoline
moiety, and was assigned the structure as shown (Fig. 2).
Further support for this assignment is outlined below.
−
HPLC-DAD-MS analysis of a sample of 1 (m/z 418 [M − H] )
stored for >12 months at −30 °C in DMSO revealed ∼10% con-
−
version to a less polar analogue 2 (m/z 419 [M − H] ) with a
near identical UV-vis spectrum. HPLC-HRESI(−)MS analysis
on this mixture established
a molecular formula for
2
(C H BrN O S, Δmmu −0.6) indicative of a hydrolysis
Fig. 2 Callyspongia sp. (CMB-01152) chemistry.
12 13
4 6
product of 1. Supportive of this hypothesis, a sample of 1 in
.5% TFA–MeOH maintained at 65 °C for 3 days was observed
0
additional brominated co-metabolites (1–4). Subsequent semi- to undergo conversion to >60% 2, with a plausible mechanism
preparative reversed-phase HPLC fractionation yielded the new for this transformation outlined in Fig. 5. Analysis of the NMR
minor bromopyrrole alkaloids, callyspongisines A (1) and C–D (DMSO-d ) data for 2 (Table 1 and Fig. 3) confirmed the pres-
6
(
3–4), and the known major bromopyrrole alkaloids, hymenial- ence of structure fragments A and B, while the absence of res-
2
3
disine (5) and 2-bromoaldisine (6) (Fig. 2). Although onances associated with 14-NH and a more upfield resonance
2
callyspongisine B (2) was only detected in the crude extract at for C-13 (δC 165.2 in 2 vs. 172.3 in 1) indicated the presence
levels that precluded isolation and detailed spectroscopic of a spiro-oxazolone in place of the spiro-imino-oxazoline in 1.
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1
13
Table 1 H (600 MHz) and C (150 MHz) NMR data for callyspongisines A–D (1–4) in DMSO-d
Callyspongisine A (1) Callyspongisine B (2) Callyspongisine C (3)
, mult (J in Hz) , mult (J in Hz) , mult (J in Hz)
12.43, s
6
Callyspongisine D (4)
, mult (J in Hz)
12.29, s
Pos.
c
c
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
1
2
3
4
5
6
7
8
-NH
12.71, s
6.13, s
12.25, s
6.13, s
105.1
110.5 5.86, s
118.8
127.3
161.1
104.5
110.1
122.1
126.5
161.4
103.6
103.6
112.4
124.1
125.7
161.6
112.6 6.13, s
124.6
125.4
161.7
-NH
8.27, t (5.4)
3.30
8.10, dd (6.9, 2.8)
36.1 3.28
7.99, t (1.0)
a 3.28, m
8.00, s
a
a
a
36.3
37.3
35.5 a 3.26, m
35.5
34.4
a
b 3.15, ddd (7.7, 7.7, 1.0)
a 2.31, dd (15.0, 7.7)
b 2.16, dd (15.0, 7.7)
b 3.16, m
9
a 2.47, m
b 2.39, m
36.6 a 2.26, dd (14.8, 10.0)
34.7 a 2.32, dd (15.0, 7.7)
b 2.12, dd (14.8, 6.8)
b 2.16, dd (15.0, 7.7)
1
1
1
1
0
1
3
91.1
181.0
172.3
82.5
181.8
165.2
79.3
172.4
79.7
172.7
4-NH
2
a 10.16, s
b 9.74, s
10.34, t (5.4)
3.66, ddd (7.0, 7.0, 5.4)
a 2.74, dt (13.0, 7.0)
b 2.67, dt (13.0, 7.0)
1
1
1
5-NH
6
7
8.79, t (5.5)
41.1 3.50, m
48.8 a 2.68, m
b 2.59, m
b
39.6
49.31
CO
OCH
2
CH
2
CH
3
a 4.15, dq (14.3, 7.0)
b 4.11, dq (14.3, 7.0)
a 3.45, dq (14.2, 7.0)
61.2
60.0
2
CH
3
a
b 3.30
CO
OCH
CO CH
2
CH
2
CH
3
3
1.17, t (7.0)
1.06, t (7.0)
14.2
15.8
2
CH
2
3
3.67, s
3.16, s
52.6
52.2
OCH
3
a
b
c
Overlapped by H
2
O resonance. Overlapped by DMSO resonance. Assignments supported by 2D NMR correlations.
6
of NMR (DMSO-d ) data (Table 1) confirmed that 3–4 share
fragment A in common with 1 (Fig. 3). This analysis also
revealed that where 3 features 11-OEt (δ 1.06 and 3.30/3.45;
15.8 and 60.0) and 11-CO Et (δ 1.17 and 4.11/4.15; δ 14.2,
1.2 and 172.4) moieties, 4 features 11-OMe (δH 3.16; δC 52.2)
and 11-CO Me (δ 3.67; δ 52.6 and 172.7) moieties. These
H
δ
C
2
H
C
6
2
H
C
considerations permitted assignment of structures to cally-
spongisines C (3) and D (4) as shown (Fig. 2).
A plausible biosynthetic/chemical relationship linking 1–6
1
0,11
(
Fig. 6) invokes oxidative processing of Δ
in 5 to yield 6
and the hydroxy acid precursor (i) that can later undergo con-
jugation with guanidine to yield a spiro-imino-oxazoline inter-
mediate (ii), which in turn forms the taurinyl Schiff base 1.
Supportive of the proposed guanidine conjugation step, treat-
ment of mandelic acid ethyl ester with guanidine. HCl (2 eq.)
and NaOH (2 eq.) delivered a high yield (82%) of the known
imino-oxazoline CNS stimulant pemoline (13). Subsequent
efforts to form a pemoline-taurinyl Schiff base proved unsuc-
cessful, presumably due to unfavourable keto–enol tauto-
merism. During long-term storage in EtOH we propose that 1
undergoes partial hydrolysis to 2, and (i) undergoes ethano-
Fig. 3 Diagnostic 2D NMR correlations and structure fragments for
–4.
1
These observations permitted assignment of the structure for lysis to 3. As 4 was only detected/isolated following HPLC
callyspongisine B (2) as shown in Fig. 2.
(H O–MeOH–TFA) fractionation we propose it is a methanoly-
2
HRESI(+)MS analysis of the co-metabolites 3 and 4 revealed sis artefact of 3. In summary, we present callyspongisine A (1)
quasi-molecular and adduct ions consistent with the mole- as a natural product, and callyspongisines B–C (2–3) and cally-
+
cular formulae C H BrN O ([M + H] , Δmmu +0.2) and spongisine D (4) as possible storage and isolation artefacts
1
3
17
2 4
+
11 2 4
C H13BrN O
([M + Na] , Δmmu −0.6) respectively. Analysis respectively.
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Fig. 4 Known model compounds 7–15 with key carbons highlighted.
1
3
0.07 μM (GSK3β)), which is a well known kinase inhibitor.
During cytotoxicity screening, 5 displayed moderate growth-
inhibitory activity against human colon adenocarcinoma cell
line SW620 (IC50 3.1 μM) and epidermoid carcinoma cell line
KB-3-1 (IC50 2.0 μM), while all tested compounds were inactive
(
IC50 > 30 μM) against human multiform glioblastoma cell line
Fig. 5 Mechanism for hydrolysis of 1 to 2.
SF-295 and large cell lung cancer cell line NCI-H460. None of
the tested compounds inhibited the efflux properties of the
transmembrane transporter P-glycoprotein (P-gp) in the adria-
mycin and vinblastine selected multidrug-resistant cell lines,
SW620 Ad300 and KB-V1, nor did they inhibit growth of the
Gram-positive bacteria Staphylococcus aureus (ATCC 9144 and
ATCC 25923), Bacillus subtilis (ATCC 6051 and ATCC 6633) and
BCG (ATCC 35733), the Gram-negative bacteria Escherichia coli
(
ATCC 11775) and Pseudomonas aeruginosa (ATCC 10145), or
the fungus Candida albicans (ATCC 90028).
Fig. 6 Plausible biosynthetic/chemical relationship between 1–6.
Conclusions
Imino-oxazolines such as 1 are exceptionally rare in the
natural products literature, with the only reported example In summary, during the course of our chemical investigation
being indolmycin (14), first reported in 1961 from Streptomyces of the Great Australian Bight sponge Callyspongia sp.
9
10
albus and again in 1978 from Streptomyces griseus. Renewed (CMB-01152), we identified the major metabolites as the
interest in the biological properties of 14 resurfaced recently, known bromopyrrole alkaloids hymenialdisine (5) and 2-bromo-
with 2001 and 2004 accounts of antibacterial activity against aldisine (6). Through the application of highly sensitive
1
1
Helicobacter pylori and methicillin-resistant Staphylococcus HPLC-DAD-MS single ion extraction protocols, we extended
1
2
aureus (MRSA), and an unpublished 2013 observation in our these discoveries to detect and identify exceptionally minor
laboratory that indolmycin exhibits growth inhibitory activity callyspongisines 1–4. Callyspongisine A (1) is only the second
against the Mycobacterium tuberculosis surrogate Bacillus reported example of a natural imino-oxazoline, and the first to
Calmette–Guérin (BCG). These observations prompted us to feature a spiro heterocyclic framework, while callyspongisines
extend our biological evaluations to assess the kinase-inhibi- B–D (2–4) were speculated to be storage and handling artefacts
tory, cytotoxic and antibiotic properties of 1 and 3–6.
of 1. Structure elucidation of 1–6 was supported by detailed
The kinase inhibitory activity detected in the Callyspongia spectroscopic analysis and chemical interconversion, as well
sp. extract was attributed to the major metabolite hymenialdi- as biosynthetic and synthetic considerations. The potent
sine (5) (IC50 0.03 μM (CK1δ), 0.16 μM (CDK5/p25) and kinase inhibitory activity detected in Callyspongia sp.
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CMB-01152) was attributed to hymenialdisine (5), while the 250 × 9.4 mm semi-preparative column, 4.2 mL min− , iso-
1
(
callyspongisines proved to be non-cytotoxic against a range of cratic 0.01% TFA modifier, gradient elution from 90% H O–
2
prokaryotic, eukaryotic and mammalian cell lines.
MeOH to 75% H
MeOH to 40% H
2
O–MeOH over 8 min, then from 75% H
O–MeOH over 10 min, then from 40% H
2
2
O–
O–
2
MeOH to 100% MeOH over 2 min, then a 3 min hold at 100%
MeOH, to yield callyspongisine A (1) (t = 8.3 min, 1.5 mg),
hymenialdisine (5) (t = 13.4 min, 1.1 mg) and 2-bromoaldi-
sine (6) (t = 16.4 min, 0.7 mg).
Experimental section
Collection
R
R
R
The sponge material identified as Callyspongia sp. (UQ code:
CMB-01152, Museum Victoria Registry no. MVF166266) was
collected in January 1991 during a scientific expedition to the
Great Australian Bight aboard the RV Franklin between
%
yields for 1 (0.41%), 3 (0.49%), 4 (0.38%), 5 (3.1%) and 6
2.7%) are calculated as weight-to-weight estimate against the
crude extract (366.4 mg).
(
Adelaide and Portland, SA. Freshly collected samples were Characterisation of compounds
frozen (−4 °C) for shipping to the laboratory, where they were
thawed, catalogued, diced, and steeped in aqueous EtOH at
Callyspongisine
max (log ε) 204 (3.96), 236 (3.99), 274 (3.61) nm; NMR (600 MHz,
DMSO-d ) see Table 1 and ESI Table S2;† HRESI(−)MS m/z
A
(1). Amorphous solid. UV (MeOH)
λ
−30 °C for prolonged storage.
6
−
79
−
4
17.9841 [M − H] (calcd for C H BrN O S , 417.9826).
12
13
5 5
Extraction and isolation
Callyspongisine B (2). Amorphous solid. UV (MeOH) λmax
An aliquot (100 mL) of the aqueous EtOH extract of specimen (log ε) 224 (3.80), 273 (3.56) nm; NMR (600 MHz, DMSO-d6)
CMB-01152 was decanted, concentrated in vacuo, and the see Table 1 and ESI Table S10;† HRESI(−)MS m/z 418.9665
−
79
−
residue (366.4 mg) partitioned between H
2
O and n-BuOH. The [M − H] (calcd for C H
12 4 6
BrN O S , 418.9666).
1
2
n-BuOH-soluble fraction was concentrated in vacuo (108.1 mg)
Callyspongisine
C
(3). Amorphous solid. UV (MeOH)
and the residue further triturated into light petroleum λmax (log ε) 203 (3.63), 220 (3.45), 274 (3.64) nm; NMR (600 MHz,
18.9 mg), CH Cl (14.0 mg), and MeOH (74.3 mg) solubles. DMSO-d ) see Table 1 and ESI Table S3;† HRESI(+)MS m/z
The H O-soluble fraction was concentrated in vacuo (258.3 mg) 345.0442 [M + H] (calcd for C H BrN O , 345.0444).
and the residue further triturated into MeOH (168.4 mg) and
O (88.4 mg) solubles. Kinase inhibition assays localized λmax (log ε) 203 (3.42), 273 (3.33) nm; NMR (600 MHz,
inhibitory activity in the CH Cl and MeOH solubles.
DMSO-d ) data see Table 1 and ESI Table S4;† HRESI(+)MS
The CH Cl solubles (14.0 mg) were subjected to HPLC frac- m/z 338.9957 [M
(
2
2
6
+
79
18
+
2 4
2
13
Callyspongisine
D (4). Amorphous solid. UV (MeOH)
H
2
2
2
6
+
79
+
2
2
+
Na] (calcd for C H
11 13 2 4
BrN O Na ,
tionation (Agilent Zorbax Phenyl 5 μm, 150 × 4.6 mm analytical 338.9951).
column, 1 mL min− , isocratic 0.01% TFA modifier, gradient
1
Hymenialdisine (5). A yellow solid. NMR (DMSO-d6,
elution from 90% H O–MeCN to 100% MeCN over 18 min, 600 MHz) data see ESI Table S5;† HRESI(+)MS m/z 324.0097
2
+
+
then a 4 min hold at 100% MeCN), to yield 2-bromoaldisine [M + H] (calcd for C H BrN O , 324.0091).
1
1
11
5 2
(
1
6) (t
3.0 min, 0.9 mg).
R
= 9.2 min, 0.3 mg) and callyspongisine C (3) (t
R
=
2-Bromoaldisine (6). A yellow oil. NMR (DMSO-d , 600 MHz)
6
−
data see ESI Table S6;† HRESI(−)MS m/z 240.9627 [M − H]
7
9
−
A portion (37.1 mg) of the MeOH solubles derived from the (calcd for C H BrN O , 240.9618).
8
6
2 2
n-BuOH solubles was subjected to HPLC fractionation (Agilent
Note: Insufficient quantities of 1–4 were available to obtain
stable optical rotation measurements.
Zorbax C 5 μm, 250 × 9.4 mm semi-preparative column, 4 mL
3
−
1
min , isocratic 0.01% TFA modifier, isocratic elution of 90%
O–MeCN for 5 min, then a gradient elution from 90% H O–
MeCN to 40% H O–MeCN over 15 min, then a 4 min hold at
H
2
2
2
1
7
00% MeCN, to yield hymenialdisine (5) (t
R
= 10.6 min,
Acknowledgements
.1 mg) and 2-bromoaldisine (6) (t = 13.0, 5.0 mg).
R
The remainder of the MeOH solubles (37.2 mg) derived We thank Noscira (Madrid, Spain) and M. Conte (UQ, IMB) for
from the n-BuOH solubles was subjected to HPLC fraction- kinase inhibitor screening, A. Blumenthal (UQ, DI) for BCG
ation (Agilent Zorbax Eclipse C 5 μm, 250 × 9.4 mm semi-pre- screening, S. Bates and R. Robey (NCI, NIH) for providing
parative column, 4 mL min , isocratic 0.01% TFA modifier, SW620 and SW620 Ad300 cell lines, M.M. Gottesman (NIH) for
8
−
1
gradient elution from 90% H
over 10 min, then from 64% H
2
O–MeOH to 64% H
O–MeOH to 22% H
2
2
O–MeOH providing KB-3-1 and KB-V1 cell lines, E. Lacey (BioAustralis,
O–MeOH Australia) for providing indolmycin, H. Zhang (UQ, IMB) for
2
over 16.4 min, then from 22% H O–MeOH to 100% MeOH valuable technical advice, the CSIRO Division of Oceanography
2
over 1.6 min, then a 3 min hold at 100% MeOH, to yield hyme- and crew of the R.V. Franklin for the collection, and L. Goudie
nialdisine (5) (t
R
= 10.1 min, 3.0 mg), 2-bromoaldisine (6) (t
R
=
for taxonomic identification of Callyspongia sp. (CMB-01152).
1
3.2 min, 3.9 mg), callyspongisine D (4) (t = 15.8 min, 1.4 mg) F.P., X.H. and Z.K. acknowledge provision of IMB Postgraduate
R
and callyspongisine C (3) (t
R
= 21.6 min, 0.9 mg).
Awards and The University of Queensland International Post-
The MeOH solubles (168.4 mg) derived from H
2
O solubles graduate Scholarships. This work was partially funded by the
were subjected to HPLC fractionation (Agilent Zorbax C 5 μm, Institute for Molecular Bioscience, The University of
3
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Queensland, the Australian Research Council (ARC LP0775547,
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