Australian marine sponge alkaloids as a new class of glycine-gated chloride channel receptor modulator

Article abstract of DOI:10.1016/j.bmc.2013.04.061

Chemical analysis of a specimen of the sponge Ianthella cf. flabelliformis returned two new sesquiterpene glycinyl lactams, ianthellalactams A (1) and B (2), the known sponge sesquiterpene dictyodendrillin (3) and its ethanolysis artifact ethyl dictyodend

Full text of DOI:10.1016/j.bmc.2013.04.061

Accepted Manuscript
Australian marine sponge alkaloids as a new class of glycine-gated chloride
channel receptor modulator
Walter Balansa, Robiul Islam, Daniel F. Gilbert, Frank Fontaine, Xue Xiao, Hua
Zhang, Andrew M. Piggott, Joseph W. Lynch, Robert J. Capon
PII:
S0968-0896(13)00387-8
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.04.061
BMC 10801
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
25 February 2013
10 April 2013
18 April 2013
Please cite this article as: Balansa, W., Islam, R., Gilbert, D.F., Fontaine, F., Xiao, X., Zhang, H., Piggott, A.M.,
Lynch, J.W., Capon, R.J., Australian marine sponge alkaloids as a new class of glycine-gated chloride channel
receptor modulator, Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.04.061
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Walter Balansa, Robiul Islam, Daniel F. Gilbert, Frank Fontaine, Xue Xiao, Hua Zhang, Andrew M. Piggott,
Joseph W. Lynch* and Robert J. Capon*

Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Australian marine sponge alkaloids as a new class of glycine-gated chloride
channel receptor modulator
a§
b§
b
a
a
a
Walter Balansa, Robiul Islam, Daniel F. Gilbert, Frank Fontaine, Xue Xiao, Hua Zhang,
a
b,c
a
Andrew M. Piggott, Joseph W. Lynch * and Robert J. Capon 
a
Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD, 4072, Australia
Queensland Brain Institute, The University of Queensland, St. Lucia, QLD, 4072, Australia
School of Biomedical Sciences, The University of Queensland, St. Lucia, QLD, 4072, Australia
b
c
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Chemical analysis of a specimen of the sponge Ianthella cf. flabelliformis returned two new
sesquiterpene glycinyl lactams, ianthellalactams A (1) and B (2), the known sponge
sesquiterpene dictyodendrillin (3) and its ethanolysis artifact ethyl dictyodendrillin (4), and five
known sponge indole alkaloids, aplysinopsin (5), 8E-3-deimino-3-oxoaplysinopsin (6), 8Z-3-
deimino-3-oxoaplysinopsin (7), dihydroaplysinopsin (8) and tubastrindole B (9). The
equilibrated mixture 6/7 exhibited glycine-gated chloride channel receptor (GlyR) antagonist
activity with a bias towards 3 over 1 GlyR, while tubastrindole B (9) exhibited a bias towards

1 over 3 GlyR. At low- to sub-micromolar concentrations, 9 was also a selective potentiator
Keywords:
of 1 GlyR, with no effect on 3 GlyR – a pharmacology that could prove useful in the
treatment of movement disorders such as spasticity and hyperekplexia (pronounced startle
response). Our investigations into the GlyR modulatory properties of 1–9 were further supported
by the synthesis of a number of structurally related indole alkaloids.
glycine-gated chloride channel receptor modulators
sesterterpene tetronic acid glycinyl lactams
Ianthella cf. flabelliformis
marine natural products chemistry
2
009 Elsevier Ltd. All rights reserved.
1
. Introduction
2
. Results and Discussion
A portion of the EtOH extract of Ianthella cf. flabelliformis
Glycine-gated chloride channel receptors (GlyRs) are
members of the Cys-loop ion channel receptor family comprising
subunits 1–4 and . They play a pivotal role in orchestrating
inhibitory neurotransmission in the spinal cord, brainstem and
was decanted, dried in vacuo and subjected to solvent
partitioning and trituration. The GlyR-modulatory CH Cl - and
2
2
MeOH-soluble materials were further fractionated by reversed
phase HPLC to yield two new sesquiterpene lactams
1
retina, with functional GlyRs being formed either as pentameric
homomers or as  heteromers. GlyR subunits exhibit
differential central nervous system distributions that are
particularly evident in the superficial dorsal horn of the spinal
ianthellalactams
A (1) and B (2), the known sponge
13
sesquiterpene dictyodendrillin (3), its ethanolysis artifact ethyl
dictyodendrillin (4), and five known sponge indole alkaloids,
2
3-6
1
4
15
cord and the retina.
The physiological consequences of
aplysinopsin (5), 8E-3-deimino-3-oxoaplysinopsin (6), 8Z-
15
16
differential distribution patterns are difficult to establish as there
are currently few pharmacological probes that can selectively
3
-deimino-3-oxoaplysinopsin (7), dihydroaplysinopsin (8)
17
and tubastrindole B (9) (Figure 1). The compounds 3–9 were
characterized by detailed spectroscopic analysis and comparison
with literature data (see Supporting Information).
7
inhibit different GlyR isoforms, and therefore small molecule
isoform-selective GlyRs modulators (agonists, antagonists,
potentiators) would have significant value as both pharmaco-
2
,8
High resolution ESI(+)MS analysis of 1 and 2 revealed quasi-
molecular ions [M+H] consistent with isomeric molecular
logical tools and as lead compounds for inflammatory pain,
+
9
10
opioid-induced breathing disorders, epilepsy and movement
1
1
formulae (C H NO ) incorporating six double bond equivalents
disorders. In an attempt to address this need, we recently
screened a library of >2,500 southern Australian and Antarctic
marine invertebrates and algae for metabolites displaying GlyR-
modulating properties. This approach successfully identified a
17 25
3
(DBE). Analysis of the 1D and 2D NMR (methanol-d ) data for 1
4
and 2 (Table 1 and Figure 2) revealed resonances and
correlations consistent with common geranyl and glycinyl
residues, and differing trisubstituted ,-unsaturated esters/
lactones. These observations account for five DBE and require
that both 1 and 2 be monocyclic. Diagnostic deshielded C-19
1
2
number of promising extracts, and in a preliminary report we
described sesterterpene tetronic acid glycinyl-lactams from
several Ircinia spp., as a new class of isoform selective GlyR
modulators. This report extends on our earlier findings and
details the chemical analysis of a specimen of the GlyR-active
sponge Ianthella cf. flabelliformis.
7
,8
resonances supported assignment of E  configurations for 1
 16.3) and 2 ( 16.3), further confirmed by comparison to
(
C
C
authentic samples of the closely related dictyodendrillin (3) and
—
——
 Corresponding authors. JWL: Tel: +61-7-3346-6375; fax: +61-7-3346-6301; e-mail: j.lynch@uq.edu.au; RJC: Tel.: +61-7-3346-2979; fax:
+61-7-3346-2090; e-mail: r.capon@uq.edu.au; § These authors contributed equally to this work

Figure 1. Ianthella cf. flabelliformis metabolites 1–9, and dendrolasin (13)
dendrolasin (13). HMBC correlations and significant deshielding
of H-3 in 1 ( 6.88) compared to H-2 in 2 ( 5.85) confirmed
H
H
the presence of regioisomeric glycinyl lactam moieties. Thus the
complete structures for ianthellalactams A (1) and B (2) were
assigned as shown.
Although 1 and 2 possess a rare glycinyl lactam moiety in
1
2
common with the ircinialactams (marine sponge metabolites
previously reported by us as potent GlyR modulators), neither 1
nor 2 exhibited any effect against either 1 or 3 GlyRs. Indeed,
following a comprehensive testing of all metabolites the GlyR
modulatory activity detected in the crude Ianthella cf.
flabelliformis extract was attributed solely to 6/7, and 9 (Figure 3
and Table 2). Whereas the (photo-activated) equilibrating
mixture of geometric isomers 6/7 exhibited weak GlyR
antagonist activity with a modest bias towards 3 over 1, the
dimeric analog tubastrindole B (9) proved to be a more potent
GlyR antagonist with a bias towards 1 over 3. Of interest, 9
was found to be a selective potentiator of 1 GlyR at low- to sub-

M concentrations (126  5% at 1 M), while exhibiting no such
effect on 3 GlyR – a pharmacology that could prove useful as a
treatment for movement disorders such as spasticity and
hyperekplexia (pronounced startle response). This unusual
difference in activity at lower and high concentrations suggests 9
may have two distinct 1 GlyR binding sites.
Figure 3. Electrophysiological analysis of 6/7, 9 and 11a/b on 1 and 3
GlyRs. (a) Sample dose-responses of 9 and 11a/b on currents activated by
EC50 glycine at 1 GlyR. (b) Averaged dose-responses of the 3 compounds at
1 GlyR and (c) 3 GlyR. Mean parameters of best fit to individual
inhibitory dose-response relationships are presented in Table 1. Curve fits
represent Hill equation fits to averaged data only and these parameters were
not used for analysis. An exception is the biphasic curve fit to the 11a/b data
at the 1 GlyR, which was fit by the sum of two Hill equations.
Figure 2. Diagnostic NMR (methanol-d
and 2
4
) resonances and correlations for 1

1
13
Table 1. H (600 MHz) and C (150 MHz) NMR data for ianthellalactams A (1) and B (2) in methanol-d4
ianthellalactam A (1)
H, mult. (J in Hz)
ianthellalactam B (2)
H, mult. (J in Hz)
Position
a
a
C
C
1
2
3
4
5
6
7
8
9
174.4
139.7
138.9
53.1
27.1
27.2
124.8
137.3
16.3
175.0
121.5
164.6
56.8
30.8
27.4
124.3
137.9
16.3
5.85, tt (1.4, 1.4)
6.88, tt (1.6, 1.6)
4.05, dt (1.6, 1.6)
4.11, d (1.4)
b
2.29
2.27
2.48, td (7.4, 1.4)
2.30, br td (7.4, 7.1)
5.16, tq (7.1, 1.3)
b
5.16, tq (6.9, 1.3)
1.60, br s
1.64, br s
1
1
1
1
1
1
1
2
0
1
2
3
4
5

1.99, br t (7.6)
2.08, br td (7.6, 7.0)
5.09, tqq (7.0, 1.4, 1.4)
41.0
27.9
125.6
132.3
17.9
2.00, br t (7.6)
2.08, br td (7.6, 7.0)
5.08, tqq (7.0, 1.4, 1.4)
40.9
27.8
125.4
132.4
17.9
26.0
44.4
172.9
1.60, br s
1.67, br s
4.21, s
1.60, br s
1.66, br s
4.17, s
26.0
44.9

173.0
a 13
b
C NMR assignments supported by HSQC data; overlapping resonances.
To further probe the indole alkaloid GlyR pharmacophore
Table 2. Automated electrophysiology (concentration dependence)
of 6/7, 9 and 11a/b on stably expressed 1 and 3 GlyRs
exemplified by 6/7 and 9, we employed aldol condensations to
couple indole-3-carboxaldehyde with hydantoin, methyl
hydantoin and creatinine, to synthesize the analogs 10, 11a/b and
Compound
1 GlyR
IC50 (M)
>200
3 GlyR
1
2a/b respectively (Figure 4). Significantly, whereas the
IC50 (M)
a
unmethylated hydantoin derived alkaloid 10 was prepared as the
stable E isomer, the methylated analogs 11a and 11b derived
from methyl hydantoin, and 12a and 12b derived from creatinine,
existed as photo-labile equilibrating mixtures of geometric
isomers. These analogs were exposed to UV light for 3 h prior to
bioassay to generate ~1:1 mixtures of the E:Z isomers. Of note,
only the mixture 11a/b exhibited significant GlyR modulatory
activity, being a more a potent GlyR antagonist than either 6/7 or
6/7
9
67  16
*
b
25.9  5.3
>300
33.9  7.0
*
c
*d
11a/b
8.8  0.5
*
a
b
c
d
p< 0.01; nH = 0.5  0.1; nH = 0.8  0.1; nH = 1.0  0.1; n =
H
0
.8  0.1. All results averaged from 4–10 cells.
A structure activity relationship analysis inspired by the
monomeric alkaloid natural products 5–8, and further informed
by the synthetic alkaloid analogs 10, 11a/b and 12a/b, provides a
sense of the molecular characteristics needed for GlyR potency
and isoform selectivity. For example, conversion of the 3-imino
to a 3-oxo moiety (5 to 6/7, and 12a/b to 11a/b) leads to a
dramatic increase in potency. Likewise, the level of N-
methylation plays a critical role in both potency and isoform
selectivity with the unmethylated 10 being inactive, the 2-N-
methylated 11a/b being selectively potent against 1 GlyRs, and
the 2,4-N,N-dimethylated 6/7 being selectivity potent against 3
9
, with a bias favoring 1 over 3 (Figure 3 and Table 2).
8
,1´
GlyRs. Interestingly, reduction of  (8) abolished all activity
against both 1 and 3 GlyRs.
3
. Conclusion
In summary, our investigations confirm that marine
biodiscovery offers a valuable approach to discover new classes
of GlyR modulators. Following the screening of a library of
marine extracts, we identified a southern Australian marine
sponge, Ianthella cf. flabelliformis as a priority extract for the
discovery of small molecule GlyR modulators. Bioassay guided
fractionation yielded the structurally diverse sesquiterpenes 1–4
and indole alkaloids 5–9, with the GlyR modulatory activity
detected in Ianthella cf. flabelliformis being attributed to the
alkaloids 6/7 and 9. That these sponge alkaloids inhibited GlyR
with apparently ―tunable‖ selectivity for either 1 or 3 GlyRs,
prompted the one-pot synthesis of alkaloids 10, 11a/b and 12a/b,
leading to the discovery of the readily accessible and more potent
1 selective GlyR antagonists.
Figure 4. Synthesis of indole alkaloid analogs. (a) hydantoin; (b)
methylhydantoin; (c) creatinine.

4
4
. Experimental
74.6 mg yields respectively. A portion of the CH Cl soluble
2 2
fraction (130 mg) was subjected to HPLC fractionation (Zorbax
5 m 250  9.4 mm column, 4 mL/min, gradient elution of
0–100% MeCN/H O over 25 min, with an isocratic 0.01%
.1. General Experimental Details
C
8
1
2
Optical rotations were measured on a JASCO P-1010
TFA/H O modifier) to yield ianthellalactam A (1) (t = 12.9 min;
2
R
polarimeter. UV-vis spectra were obtained on a Cary 50 UV-
visible spectrophotometer. NMR experiments were performed on
a Bruker Avance DRX600. Chemical shifts were calibrated
internally against residual solvent signals (DMSO-d :  = 2.50,
C = 39.5; CDCl :  = 7.26,  = 77.0; CD OD:  = 3.30,  =
4
2
0
.6 mg, 0.056%), ianthellalactam B (2) (t = 13.3 min; 1.7 mg,
R
.032%), dictyodendrillin (3) (t = 13.9 min; 0.2 mg, 0.003%),
R
aplysinopsin (5) (t = 7.0 min; 2.4 mg, 0.041%), 8E-3-deimino-
R
6
H
3-oxoaplysinopin (6) (t = 10.1 min; 2.7 mg, 0.051%), 8Z-3-
R
3
H
C
3
H
C
deimino-3-oxoaplysinopin (7) (t = 10.6 min; 3.5 mg, 0.067%),
R
9.2). ESIMS were recorded using an Agilent 1100 Series
dihydroaplysinopsin (8) (tR = 6.4 min; 1.8 mg, 0.034%) and
separations module equipped with an Agilent 1100 Series
LC/MSD mass detector in both positive and negative ion modes.
HRESIMS measurements were obtained on a Bruker micrOTOF
mass spectrometer by direct infusion in MeCN at 3 L/min flow
using sodium formate clusters as an internal calibrant. HPLC was
performed using an Agilent 1100 series HPLC controlled using
ChemStation Rev.B.02.01. All chemicals were purchased from
Merck, Sigma-Aldrich or Fluka. Solvents used for general
purposes were of at least analytical grade, and solvents used for
tubastrindole B (9) (t = 5.8 min; 1.7 mg, 0.029%). Since the
MeOH soluble fraction exhibited a similar HPLC-DAD and H
R
1
NMR profile to the CH Cl soluble fraction, the remaining
2
2
CH Cl (153.0 mg) and MeOH solubles (74.6 mg) were
2
2
combined and fractionated using the method outlined above, to
yield 1–3 and 5–9 together with a new minor metabolite/artifact,
ethyl dictyodendrillin (4) (tR = 14.2 min; 1.7 mg, 0.029%).
Although
the
geometric
isomers
8E-3-deimino-3-
oxoaplysinopsin (6) and 8Z-3-deimino-3-oxoaplysinopsin (7)
were well resolved by HPLC, the isomers rapidly isomerized
during handling (and on exposure to light). Isolation in a
darkened laboratory, followed by rapid data acquisition, did
permit independent characterization of these photolabile isomers.
Note – yields for all isolated compounds were calculated as a
weight-to-weight estimate against the crude n-BuOH solubles
HPLC were of HPLC grade. Agilent Zorbax C and C 250  9.4
3
8
mm columns were used for HPLC analyses and separations.
4
.2. Marine Extracts Library
A library of >2500 marine invertebrate and alga samples,
collected from intertidal, coastal and deep sea locations across
southern Australia and Antarctica, were processed to generate an
extract library suitably formatted for medium to high throughput
bioassay. A portion (7 mL) of the archived EtOH extract of each
marine sample was decanted, concentrated in vacuo, weighed and
4
4
.5. Characterization of Natural Products
.5.1. ianthellalactam A (1)
Pale yellow oil. UV-vis (MeOH) max (log ) 203 nm (4.33);
partitioned into n-BuOH (2 mL) and H O (2 mL). This pre-
2
NMR (600 MHz, methanol-d ) see Table 1 and Supporting
4
processing achieves a >10 fold concentration of ―drug-like‖ small
molecules, while simultaneously desalting and simplifying the
solubility characteristics of, the n-BuOH solubles. Aliquots (1
Information Figures S1a–b and Table S1; HRESI(+)MS m/z
3
14.1721 (calcd. for C H NO Na 314.1727).
17 25 3
mL) of both n-BuOH and H O phases were transferred to deep
4.5.2. ianthellalactam B (2)
2
9
6-well plates, to generate a set of extract library plates. These
Pale yellow oil. UV-vis (MeOH) max (log ) 202 nm (4.40);
plates were subsequently used to prepare 10- and 100-fold
dilution plates.
NMR (600 MHz, methanol-d ) see Table 1 and Supporting
4
Information Figures S2a–b and Table S2; HRESI(+)MS m/z
3
14.1725 (calcd. for C H NO Na 314.1727).
1
7
25
3
4
.3. Sponge Collection and Taxonomy
13
4
.5.3. dictyodendrillin (3)
1
Sponge specimen CMB-03322 was collected by SCUBA from
Colorless oil. H NMR (600 MHz, CDCl ) see Supporting
3
+
Lonsdale Wall, The Rip, Port Phillip Heads, Victoria (38 17
5 S, 144 37.46 E). A description of the specimen is as
Information Figures S3a–b. ESI(+)MS m/z 251 [M+H] , ESI(–)
–
5
MS m/z 249 [M–H] . Identical with an authentic sample of
1
3
follows: Growth form massive-thickly flabelliform; colour in
EtOH likely to be an aerophobic colour change to dark purple;
texture barely compressible, harsh, fibrous; oscules scattered,
sunken; surface opaque, glossy, conulose; spicules none;
ectosome thick collagen with fibres tangential or protruding the
surface; choanosome a reticulation of fibres strongly laminated
and pith visible. Large primary fibres cored by single column of
sand grains, smaller fibres clear of detritus. The sponge was
identified as Ianthella cf. flabelliformis (Order Verongida,
Family Ianthellidae) and a type specimen registered with
Museum Victoria (Registry No. MVF166222).
dictyodendrillin.
4
.5.4. ethyl dictyodendrillin (4)
Colorless oil. UV-vis (MeOH) max (log ) 204.9 (4.27), 201
4.27); NMR (600 MHz, CDCl ) see Supporting Information
(
3
Figures S4a–b and Table S4.
1
4
4
.5.5. aplysinopsin (5)
Yellow solid. UV-vis (MeOH) max (log ) 217 (4.0), 279
3.49), 391 (3.67); NMR (600 MHz, DMSO-d ) see Supporting
(
6
Information Figures S5a–b and Table S5; HRESI(+)MS m/z
55.1247 (calcd. for C H N O 255.1240). Identical with an
2
1
4
15
4
1
4
authentic sample of aplysinopsin.
4
.4. Extraction and Fractionation
1
5
4
.5.6. 8E-3-deimino-3-oxoaplysinopsin (6)
Yellow solid. UV-vis (MeOH) max (log ) 203 (4.01), 278
3.49), 367 (3.83) nm; NMR (600 MHz, DMSO-d ) see
Supporting Information Figures S6a–b and Table S6; HRESIMS
m/z 278.0909 (calcd. for C H N O Na 278.0900).
A portion (350 mL) of the aqueous EtOH extract of the
Ianthella cf. flabelliformis was decanted and concentrated in
vacuo to return a black solid (1.74 g), which was subsequently
partitioned between n-BuOH (3  50 mL) and H O (50.0 mL).
(
6
2
1
4
13
3
2
GlyR bioassays (see below) established that all activity was
concentrated in the n-BuOH solubles (519.7 mg), which were
sequentially triturated with 2  15 mL aliquots of hexane, CH Cl
1
5
4
.5.7. 8Z-3-deimino-3-oxoaplysinopsin (7)
Yellow solid. UV-vis (MeOH) max (log ) 203 (4.01), 218
(4.23), 278 (3.49), 367 (3.83) nm; NMR (600 MHz, DMSO-d )
2
2
and MeOH, and concentrated in vacuo to return 9.0, 283.0 and
6

see Supporting Information Figures S6a–b and Table S7;
HRESIMS m/z 278.0909 (calcd. for C H N O Na 278.0900).
a yellow fluorescent protein-based anion flux assay as previously
12,21
described.
In this bioassay, 90 L of each 100-fold diluted n-
1
4
13
3
2
1
6
BuOH partition was transferred into one well of a 384-well plate,
and was dried under nitrogen. The plates containing dried n-
BuOH partitions were protected from light and stored at 30 °C
until required. On the day of the experiment an aliquot (30 L) of
4
.5.8. dihydroaplysinopsin (8)
2
3
Yellow solid. []D 86 (c 0.05 MeOH); UV-vis (MeOH)
max (log ) 202 (3.98), 218 (3.93), 280 (3.12) nm; NMR (600
MHz, DMSO-d ) see Supporting Information Figures S8a–b and
6
extracellular solution [NaCl (140 mM), KCl (5 mM), CaCl (2
2
Table S8; HRESI(+)MS m/z 257.1393 (calcd. for C H N O
1
4
17
4
mM), MgCl (1 mM), HEPES (10 mM), and glucose (10 mM)
2
2
57.1397).
(pH 7.4 using NaOH)] was added to each well and
1
7
mixed/dissolved using a multi-channel pipette. Subsequently, an
aliquot (10 L) of this solution was transferred into the
corresponding well of a 384-well plate containing approx. 5000
cells and 15 L of extracellular solution per well, and incubated
at room temperature for 30 min. The 384-well plates were then
4
.5.9. tubastrindole B (9)
2
3
Yellow solid. []D 44 (c 0.1 MeOH); UV-vis (MeOH) max
log ) 202 (4.21), 217 (4.27), 205 (4.24), 268 (3.55) nm; NMR
600 MHz, DMSO-d ) see Supporting Information Figures S9a–b
and Table S9; HRESI(+)MS m/z 509.2415 (calcd. for C H N O
(
(
6
2
8
29
8
2
21
placed onto the motorized stage of an in-house imaging system
5
09.2408).
and cells were imaged twice: once in 25 L of extracellular
solution containing the n-BuOH partition and once 10 s after the
injection of 50 L NaI solution supplemented with 500 M
glycine. The NaI solution was similar in composition to NaCl
control solution except the NaCl was replaced by equimolar NaI.
Images of fluorescent cells were segmented and quantitatively
4
4
.6. Synthesis of Analogs
.6.1. General Synthetic Procedures
Solutions of indole-3-carboxaldehyde (1.0 g, 6.9 mmol) and
either hydantoin, methyl hydantoin or creatinine (17 mmol, 2.5
equiv.) in piperidine (5 mL) were refluxed for 45 min under
argon. The solutions were then cooled to room temperature and
diluted with acetone (10 mL). The resulting yellow precipitates
were filtered and washed with cold acetone (10 mL) to yield the
essentially pure 8E isomers (10, 11a and 12a). Suspensions of
2
2
analyzed using a modified version of DetecTIFF software. In
brief, images were segmented using an iterative size and
intensity-based thresholding algorithm and the fluorescence
signal of identified cells was calculated as the mean of all pixel
values within the area of a cell. GlyR activation response was
used as a measure of extract potency and was defined as
1
1a and 12a in methanol were exposed to UV light for 3 h,
yielding ~1:1 mixtures of the 8E and 8Z (11b and 12b) isomers.
(
Finit−Ffinal)100/F , where F and Ffinal were the initial and
init
init
final values of fluorescence, respectively. All experiments were
performed twice in neighboring wells. Hits were selected by
visual inspection of color maps generated by analysis software.
For hit confirmation, 24 L of the corresponding 10-fold diluted
n-BuOH partition was transferred into a well of a 384-well plate,
and was dried and stored as described above. On the day of the
experiment the dried 10-fold diluted n-BuOH partitions were
resuspended in 80 L extracellular solution as described above
and two aliquots each of 17 and 10 L respectively were then
transferred in neighboring wells containing ~5000 cells and 15
1
8
4
.6.2. 8E-3-deimino-2,4-bisdemethyl-3-oxoaplysinopsin
(10)
Yellow solid (0.90 g, 57%). UV-vis (MeOH) max (log )
27.1 (4.03), 277.9 (3.55), 364 (4.09) nm; NMR (600 MHz,
methanol-d ) see Supporting Information Figures S10a–b and
Table S10; HRESIMS m/z 250.0593 (calcd. for C H N O Na
2
4
1
2
9
3
2
2
50.0587).
1
9
4
.6.3. 8E-3-deimino-4-demethyl-3-oxoaplysinopsin (11a).
Yellow solid (0.76 g, 46%). UV-vis (MeOH) max (log )
28.9 (4.01), 276 (3.49) 336.9 (3.83) nm; NMR (600 MHz,
methanol-d ) see Supporting Information Figure S11a and Table
S11a; HRESIMS m/z 264.0746 (calcd. for C H N O Na

L extracellular solution per well. Imaging and image analysis
2
were conducted as described above. The concentration
dependence of the drug effects was used as measure for extract
potency and level of prioritization. From these experiments we
identified Ianthella cf. flabelliformis (CMB-03322) as an extract
with antagonist activity on 1 and 3 GlyRs.
4
1
3
11
3
2
2
64.0743).
1
9
4
.6.4. 8Z-3-deimino-4-demethyl-3-oxoaplysinopsin (11b).
Yellow solid. UV-vis (MeOH)max (log ) 228.9 (4.01), 276
3.49) 336.9 (3.83) nm; NMR (600 MHz, methanol-d ) see
4.7.2. Automated Electrophysiology
(
4
Electrophysiological screening was performed using an
automated planar patch-clamp device (Patchliner, Nanion
Technologies GmbH, Munich, Germany). Stable 1 and 3
GlyR expressing HEK293 cell lines, produced as previously
Supporting Information Figure S11b and Table S11b; HRESIMS
m/z 264.0746 (calcd. for C H N O Na 264.0743).
1
3
11
3
2
2
0
4
.6.5. 8E-4-demethylaplysinopsin (12a).
1
2,22
described,
were maintained in Minimal Essential medium.
Yellow solid. (0.70 g, 42%). UV-vis (MeOH) max (log )
28.9 (3.93), 277.9 (3.49), 387 (3.90) nm; NMR (600 MHz,
DMSO-d ) see Supporting Information Figure 12a and Table
S12a; HRESIMS m/z 241.1085 (calcd. for C H N O 241.1084).
After 2–3 days in culture, cells were trypsinized and suspended
2
in extracellular solution [NaCl (140 mM), KCl (4 mM), CaCl (2
2
6
mM), MgCl (1 mM), HEPES/NaOH (10 mM) and glucose (5
2
1
3
13
3
6
mM) (pH 7.4 adjusted with NaOH)] at a density of 110
2
0
4
.6.6. 8Z-4-demethylaplysinopsin (12b).
Yellow solid. UV-vis (MeOH) max (log ) 228.9 (3.93), 277.9
3.49), 387 (3.90) nm; NMR (600 MHz, DMSO-d ) see
cells/mL before injecting into the Patchliner chip. This standard
extracellular solution was employed in all Patchliner
experiments. The internal solution contained KCl (50 mM), NaCl
(10 mM), KF (60 mM), MgCl2 (2 mM), EGTA (20 mM),
HEPES/KOH (10 mM) (pH 7.2). All experiments were
performed at room temperature. 1 M glycine (Ajax Finechem,
Seven Hills, NSW, Australia) stocks were prepared in water and
stored at –20 °C. All test compounds (marine natural products
and synthetics) were dissolved in DMSO (Sigma-Aldrich) and
stored at –20 °C. From these stocks, solutions for experiments
were prepared on the day of recording.
(
6
Supporting Information Figure 12b and Table S12b; HRESIMS
m/z 241.1085 (calcd. for C H N O 241.1084).
1
3
13
4
4
4
.7. Bioassays
.7.1. Primary GlyR Screening of Marine Extracts
The initial screen of >2500 marine extracts against
recombinantly expressed 1 and 3 GlyRs was performed using

4
.7.3. Data Analysis
Results are expressed as mean  standard error of the mean of
8.
Xiong, W.; Cui, T.; Cheng, K.; Yang, F.; Chen, S. R.; Willenbring, D.;
Guan, Y.; Pan, H. L.; Ren, K.; Xu, Y.; Zhang, L. J. Exp. Med. 2012,
2
09, 1121.
three or more independent experiments. The Hill equation was
used to calculate the half inhibitory concentration (IC ) and Hill
coefficient (n ) values of individual concentrationresponse
relationships. All curves were fitted using a nonlinear least
squares algorithm (Sigmaplot 11.0; Jandel Scientific, San Rafael,
CA, USA). Statistical significance was determined by unpaired
Student’s t-test, with p < 0.05 representing significance.
9
1
1
.
Manzke, T.; Niebert, M.; Koch, U. R.; Caley, A.; Vogelgesang, S.;
Hulsmann, S.; Ponimaskin, E.; Muller, U.; Smart, T. G.; Harvey, R. J.;
Richter, D. W. J. Clin. Invest. 2010, 120, 4118.
5
0
H
0. Eichler, S. A.; Kirischuk, S.; Juttner, R.; Schaefermeier, P. K.;
Legendre, P.; Lehmann, T. N.; Gloveli, T.; Grantyn, R.; Meier, J. C. J.
Cell Mol. Med. 2008, 12, 2848.
1. Chung, S. K.; Vanbellinghen, J. F.; Mullins, J. G.; Robinson, A.;
Hantke, J.; Hammond, C. L.; Gilbert, D. F.; Freilinger, M.; Ryan, M.;
Kruer, M. C.; Masri, A.; Gurses, C.; Ferrie, C.; Harvey, K.; Shiang, R.;
Christodoulou, J.; Andermann, F.; Andermann, E.; Thomas, R. H.;
Harvey, R. J.; Lynch, J. W.; Rees, M. I. J. Neurosci. 2010, 30, 9612.
2. Balansa, W.; Islam, R.; Fontaine, F.; Piggott, A. M.; Zhang, H.; Webb,
T. I.; Gilbert, D. F.; Lynch, J. W.; Capon, R. J. Bioorg. Med. Chem.
2010, 18, 2912.
Acknowledgments
1
We thank L. Goudie for taxonomic identification of marine
sponges and acknowledge the generous support of an Australian
Development Scholarship (W.B) and a University of Queensland
Scholarship (R.I). This research was funded in part by the
Institute for Molecular Bioscience, the Queensland Brain
Institute, The University of Queensland, the National Health and
Medical Research Council (project grant #613448) and the
Australian Research Council (LP120100088).
13. Tran, N. H.; Hooper, J. N. A.; Capon, R. J. Aust. J. Chem. 1995, 48,
757.
1
1
4. Kazlauskas, R.; Murphy, P. T.; Quinn, R. J.; Wells, R. J. Tetrahedron
Lett. 1977, 18, 61.
1
5. Guella, G.; Mancini, I.; Zibrowius, H.; Pietra, F. Helv. Chim. Acta
1988, 71, 773.
16. Okuda, R. K.; Klein, D.; Kinnel, R. B.; Li, M.; Scheuer, P. J. Pure
Appl. Chem. 1982, 54, 1907.
1
7. Iwagawa, T.; Miyazaki, M.; Okamura, H.; Nakatani, M.; Doe, M.;
Takemura, K. Tetrahedron Lett. 2003, 44, 2533.
References and notes
1
8. Rashid, M. A.; Gustafson, K. R.; Cardellina, J. H., II; Boyd, M. R. J.
Nat. Prod. 1995, 58, 1120.
1
2
.
.
Lynch, J. W. Physiol. Rev. 2004, 84, 1051.
Harvey, R. J.; Depner, U. B.; Wassle, H.; Ahmadi, S.; Heindl, C.;
Reinold, H.; Smart, T. G.; Harvey, K.; Schutz, B.; Abo-Salem, O. M.;
Zimmer, A.; Poisbeau, P.; Welzl, H.; Wolfer, D. P.; Betz, H.;
Zeilhofer, H. U.; Muller, U. Science 2004, 304, 884.
Haverkamp, S.; Muller, U.; Harvey, K.; Harvey, R. J.; Betz, H.;
Wassle, H. J. Comp. Neurol. 2003, 465, 524.
Haverkamp, S.; Muller, U.; Zeilhofer, H. U.; Harvey, R. J.; Wassle, H.
J. Comp. Neurol. 2004, 477, 399.
Heinze, L.; Harvey, R. J.; Haverkamp, S.; Wassle, H. J. Comp. Neurol.
19. Jakse, R.; Recnik, S.; Svete, J.; Golobic, A.; Golic, L.; Stanovnik, B.
Tetrahedron 2001, 57, 8395.
20. Djura, P.; Faulkner, D. J. J. Org. Chem. 1980, 45, 735.
21. Kruger, W.; Gilbert, D.; Hawthorne, R.; Hryciw, D. H.; Frings, S.;
Poronnik, P.; Lynch, J. W. Neurosci. Lett. 2005, 380, 340.
22. Gilbert, D.; Esmaeili, A.; Lynch, J. W. J. Biomol. Screen. 2009, 14, 86.
3
4
5
6
7
.
.
.
.
.
Supplementary Material
2
007, 500, 693.
Weltzien, F.; Puller, C.; O'Sullivan, G. A.; Paarmann, I.; Betz, H. J.
Comp. Neurol. 2012, 520, 3962.
Webb, T. I.; Lynch, J. W. Curr. Pharm. Des. 2007, 13, 2350.
Chemical profiling, isolation schemes and spectroscopic data
for compounds 1–13 can be found, in the online version, at
doi:XX.XXXX/j.bmc.2013.XX.XXX.

5
7
12
15
15
3
Figure 1
O
NH2
'
3
4
2
MeN
MeN
N
9
14
8
1
NMe
E/Z
NMe
O
4
7
1'
1
'
ianthellalactam A (1)
5'
3
a
3
2
' CO H
2
O
O
7a N
1
N
H
2
5
7
12
H
O
3
aplysinopsin (5)
8E-3'-deimino-3'-oxoaplysinopsin (6)
8Z-3'-deimino-3'-oxoaplysinopsin (7)
1
N
4
9
14
TFA salt
1
'
ianthellalactam B (2)
CO H
2
2
'
Me
N
NH2
NH2
NMe
O
MeN
RO
NMe NH
O
O
dictyodendrillin (3) R = H
NMe
ethyl dictyodendrillin (4) R = Et
O
NH2
N
H
N
H
N
Me
O
dihydroaplysinopsin (8)
tubastrindole B (9)
O
dendrolasin (13)
TFA salt
TFA salt

δ
6.88
Fig
H
ure 2
N
O
δC 16.3
CO H
2
ianthellalactam A (1)
COSY
HMBC
δH 5.85
O
N
δC 16.3
CO H
2
ianthellalactam B (2)

(a)
α1 GlyR
[
9](μM) 0.1
1.0
3.0
3.0
10
30
30
100
glycine:
3
s
[
11a/b](μM) 1.0
10
100 300
glycine:
3
s
b) 140
(
120
6/7
9
1
100
1a/b
80
60
40
20
0
α1 GlyR
-2
-1
0
1
2
3
10
10
10
10
10
10
[
compound] (μM)
c) 120
00
(
1
80
60
40
20
0
α3 GlyR
-2
-1
0
1
2
3
10
10
10
10
10
10
[
compound] (μM)

O
Figure 4MeN
O
MeN
NH
NH
E/Z
hν
O
O
methanol
3 h
E (11a)
Z (11b)
E (11a)
N
H
N
H
piperidine
reflux, 45 min
(b)
O
3
'
HN
CHO
8
NH
(
a)
4
7
1'
3
a
3
5'
piperidine
reflux, 45 min
O
N
H
1
7
a N E (10)
H
piperidine
reflux, 45 min
(c)
NH
NH
NH
MeN
MeN
NH
E/Z
hν
O
O
methanol
3
h
E (12a)
Z (12b)
N
H
E (12a)
N
H