Damipipecolin and damituricin, novel bioactive bromopyrrole alkaloids from the Mediterranean sponge Axinella damicornis

Article abstract of DOI:10.1016/j.bmc.2007.05.074

Two new bromopyrrole alkaloids, damipipecolin (1) and damituricin (2), have been isolated from the Mediterranean sponge Axinella damicornis, and their structures established through spectroscopic methods. Compounds 1 and 2 extend the structural variety of

Full text of DOI:10.1016/j.bmc.2007.05.074

Bioorganic & Medicinal Chemistry 15 (2007) 5877–5887
Damipipecolin and damituricin, novel bioactive
bromopyrrole alkaloids from the Mediterranean
sponge Axinella damicornis
a
a,
a
a
*
Anna Aiello, Ernesto Fattorusso, Antonella Giordano, Marialuisa Menna,
b
b
b
Werner E. G. M u¨ ller, Sanja Perovi c´ -Ottstadt and Heinz C. Schr o¨ der
a
Dipartimento di Chimica delle Sostanze Naturali, Universit a` di Napoli ‘‘Federico II’’, via D. Montesano 49, 80131 Napoli, Italy
b
Abteilung f u¨ r Angewandte Molekularbiologie, Institut f u¨ r Physiologische Chemie, Johannes Gutenberg-Universit a¨ t Mainz,
Duesbergweg 6, 55099 Mainz, Germany
Received 30 January 2007; revised 14 May 2007; accepted 30 May 2007
Available online 7 June 2007
Abstract—Two new bromopyrrole alkaloids, damipipecolin (1) and damituricin (2), have been isolated from the Mediterranean
sponge Axinella damicornis, and their structures established through spectroscopic methods. Compounds 1 and 2 extend the struc-
tural variety of the so far known pyrrole alkaloids; in these compounds, the 4-bromopyrrole 2-carboxylic acid is directly condensed
0
with a non-protein cyclic a-amino acid, the (2R, 4R)-trans-4-hydroxypipecolic acid and (2R, 4R)-cis-N,N -dimethyl-4-hydroxypro-
line (D-turicine) in 1 and 2, respectively. Compounds 1 and 2 were found to display a modulating effect of the serotonin receptor
activity in vitro.
ꢀ
2007 Elsevier Ltd. All rights reserved.
4
1
. Introduction
most important biological function, but the variety of
pharmacological activities that they show make these
compounds important leads for the development of
Bromopyrrole alkaloids constitute a family of exclu-
sively marine alkaloids and represent a fascinating
example of the large variety of secondary metabolites
formed by marine sponges. Most of these compounds
2
fall under the oroidin class of alkaloids, defined by
the signature bromopyrrole carboxamide and aminoim-
idazole moieties connected through a propyl chain. Over
the last 30 years, about 140 derivatives, with various
structures and interesting biological activities, have been
isolated from more than 20 different sponges of various
genera, essentially (but not exclusively) belonging to
Agelasidae, Axinellidae, and Halichondridae families.
It is currently admitted that these alkaloids are taxon-
specific of at least Agelasida order and play the role of
chemical markers of these phylogenetically related
5
–10
new drugs and tools for cell biology.
1
In our search for bioactive natural products from Med-
iterranean sponges, we have investigated two Axinella
species collected in the bay of Calvi (Corsica): Axinella
damicornis and Axinella verrucosa. These sponges were
shown to produce complex mixtures of structurally
diverse bromopyrrole alkaloids. The analysis of
A. verrucosa allowed us to isolate twenty pyrrole-imidaz-
5
ole alkaloids; among them, four were new compounds.
The same study performed on A. damicornis led to the
isolation of a new bromopyrrole alkaloid, daminin,
which was shown to be a potent neuroprotective agent
that might represent a new therapeutic tool for the treat-
ment of CNS diseases such as Parkinson’s and Alzhei-
mer’s diseases. The synthesis of this compound has
3
sponges. From the ecological point of view, the antipre-
datory role of these alkaloids can be considered their
1
1
also been performed.
A deeper investigation of the methanolic extract of
A. damicornis has now led to the isolation, in addition
to a number of already known bromopyrrole alkaloids
Keywords: Marine sponges; Natural products; Bromopyrrole alka-
loids; Axinella damicornis; Serotonin receptor activity.
*
Corresponding author. Tel.: +39 081 678503; fax: +39 081 678552;
e-mail: fattoru@unina.it
1
2
(4-bromopyrrole-2-carboxylic acid, 4,5-dibromopyr-
0
968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.05.074

5878
A. Aiello et al. / Bioorg. Med. Chem. 15 (2007) 5877–5887
2
2
10
role-2-carboxylic acid, oroidin, and agelongine ), of
two new bromopyrrole alkaloids, damipipecolin (1)
and damituricin (2).
composed of three methylenes, four methines, and four
unprotonated carbons, according to DEPT and HSQC
experiments. The combined NMR and mass spectral
data of 1 were compatible with the molecular formula
C H N O Br, which implied six degree of unsatura-
tion. From C NMR data it was evident that four of
the elements of unsaturation were present as double
bonds, two of them being carbonyl groups, one as part
of a conjugated ester and the other as a free acid; the
molecule must be thus bicyclic.
1
1
13
2
4
1
3
Br
4
Br
4
3
3
H
3'
6
5
COOH
H
2'
H
3'
COO-
6
O4
5
2
6 O4'
N
'
'
N
H
2
H
H
2'
O
NH
5
N
O
'
5'
1
2
1
The H NMR spectrum (CD OD) of 1 revealed the pres-
3
ence of two aromatic signals at d 6.92 and 7.01, a
deshielded methylene signal (d 3.17, Ha; 3.20, Hb), two
sp deshielded methine signals (d 3.74 and 5.31), and
In these compounds, the 4-bromopyrrole 2-carboxylic
acid is directly condensed with a non-protein cyclic a-
amino acid, the (2R, 4R)-trans-4-hydroxypipecolic acid
3
0
two partially overlapped methylene resonances in the
region 1.9–2.4 ppm. The HSQC experiment allowed us
to assign all these resonances to the relevant carbon
and (2R, 4R)-cis-N,N -dimethyl-4-hydroxyproline (D-
turicine) in 1 and 2, respectively. The D-and L-forms of
the betaine turicine, where the carboxylate and hydroxyl
functions are cis to each other, and L-betonicine, where
these functions are in the trans relationship, are natu-
rally occurring derivatives of 4-hydroxyproline. They
have been studied in the acetylcholine esterase system
and betonicine, though not turicine, is a competitive
inhibitor of the same order of magnitude of choline
1
3
atoms in the C NMR spectrum. Interpretation of the
COSY spectrum illustrated the proton connectivities in
1
and revealed the presence of two distinct spin systems
belonging to two nitrogen-containing rings. The pres-
ence of a 4-bromopyrrole-2-carboxylic acid derivative
moiety was evident from the chemical shifts and the
multiplicity of the aromatic proton signals, which consti-
1
3
itself. Pipecolic acids (hexahydropiperidine-2-carbox-
ylic acids) are frequently encountered in nature with
their hydroxylated derivatives and often display interest-
1
3
tuted one of the two spin systems, and from a C NMR
pattern of resonances (d 124.5, 97.8, 117.7, 123.6, 159.9).
This hypothesis was fully corroborated by comparison of
the NMR data of 1 with those reported in the literature
1
4–18
ing and potent biological activity.
Pipecolic acid
derivatives are also useful synthetic intermediates for
the preparation of medicinally important compounds
2
3
for other bromopyrrole alkaloids.
1
9
20
such as peptides,
inhibitors,
immunosuppressants,
22
or NMDA antagonists.
enzyme
1
5,21
The identified moiety accounted for two double bonds,
the ester function and one ring. The remaining part of
the molecule, consisting of C H NO , thus contained
6
10
2
Compounds 1 and 2 were found to display a modulating
effect on the serotonin receptor activity in vitro.
3
three methylene groups, two deshielded sp methine
groups, and a carboxyl function. Following the COSY
correlations in the spectrum recorded in CD OD, start-
3
ing from the deshielded methine signal at d 3.74, the
other large spin system was observed, corresponding
2
. Results
0
0
to the segment H-2 /2H-6 of the molecule. In the HSQC
0
2
.1. Isolation of compounds 1 and 2
spectrum, the proton signal at d 5.31 (H-4 ) of this spin
system was correlated to a signal at d 67.8, clearly indi-
cating that it was due to a proton linked to an oxygen-
bearing carbon. A nitrogen atom was inferred to be
Specimens of A. damicornis (Esper, 1794) were collected
in the bay of Calvi (Corsica) in November 2001. The
sponge was homogenized and extracted with methanol
and, subsequently, with chloroform. The combined
extracts were concentrated in vacuo. The resulting aque-
0
0
13
attached to both C-2 and C-6 from their C chemical
shifts (d 56.4 and 40.2), and a cross peak, present in
the HMBC spectrum of 1 between the resonance for
ous suspension was partitioned between H O and buta-
2
0
H-2 (adjacent to N) and the carbonyl resonating at d
nol. The fractionation of the butanol layer was achieved
by MPLC over a reversed-phase RP-18 column and a
fraction mainly composed of polar alkaloids was ob-
tained. Separation and purification of this fraction was
achieved by repeated reverse-phase HPLC, to give com-
pounds 1 and 2 as pure products.
1
79.9, suggested the carboxylic function to be located
0
at C-2 . On the basis of the combination of these
NMR results, a framework of 4-substituted piperidine-
2
HMBC spectrum showed a long-range correlation be-
-carboxylic acid (pipecolic acid) was identified. The
0
0
tween the oxymethine proton at d 5.31 (C-4 ) with C-6
carbon (d 159.9), thus indicating that C-4 was con-
nected with the bromopyrrolecarboxylic acid moiety
through an ester linkage.
2
.2. Structure elucidation
The planar structure of damipipecolin (1) was deter-
mined by spectroscopic methods. The ESI (positive ions)
mass spectrum of 1 showed two intense pseudomolecu-
The relative stereochemistry of the two substituents on
the pipecolic acid ring of damipipecolin could be defined
by H– H coupling constants analysis. The C-2 proton,
resonating at d 3.74, appeared as a double doublet with
a large coupling of 12 Hz and a small coupling of 3 Hz
+
1
1
0
lar ion peaks at m/z 317 and 319 [M+H] in the ratio
1
:1, suggesting that 1 is a monobromo compound. The
C NMR spectrum (CD OD) contained 11 signals,
1
3
3

A. Aiello et al. / Bioorg. Med. Chem. 15 (2007) 5877–5887
5879
0
to the protons of the adjacent C-3 methylene, whereas
0
AB systems due to methylene groups (2H : d 3.92, Ha;
5
3
H-4 (brs) showed small J values to the protons of the
3.98, Hb. 2H : d 2.64, Ha; 3.10, Hb), and two sp
3
0
0
adjacent C-3 and 5 methylenes. Assuming that the
piperidine ring is in a chair-like conformation, a trans
relative configuration of the 4-substituted pipecolic acid,
where H-2 and H-4 occupy an axial and an equatorial
position, respectively, was consistent with the observed
coupling pattern. The absolute stereochemistry of the
amino acid portion could be clarified through acid
deshielded methine signals (d 4.19 and 5.56). These sig-
nals were connected, by interpretation of COSY spec-
trum, in an only large spin system belonging, also
according to mass data, to an oxygenated nitrogen-con-
taining ring, as deduced by the following HMBC results.
In the HMBC spectrum, both the N-methyl protons
were long-range correlated to the methylene carbon at
0
0
0
0
hydrolysis of 1 performed with 1 N HCl in H O at
2
0 ꢁC overnight. The reaction mixture was evaporated
d 72.0 (C-5 ) and the methine carbon at d 75.7 (C-2 );
thus, the same nitrogen atom was inferred to be linked
to these carbons and to methyl carbons. A further key
cross peak was observed in the HMBC spectrum be-
8
under nitrogen and then subjected to HPLC purifica-
tion, yielding 1 mg of pure 4-bromopyrrole 2-carboxylic
acid (3) and (+)-trans-4-hydroxypipecolic acid (4). Both
compounds could be easily identified by comparison of
their spectroscopic data with those reported in the
literature; in particular, the NMR data of 4-hydroxy-
pipecolic acid were identical to those reported for the
0
tween the proton resonating at d 4.19 (H-2 ) and the car-
bonyl resonating at d 169.0; it clearly indicated that a
carboxylate function was linked at C-2 . On the basis
0
of these results, and according to the whole series of
0
COSY, HSQC, and HMBC correlations, an N,N -di-
2
4
(
2R, 4R)-trans isomer, thus confirming the relative
stereochemistry proposed for damipipecolin. Both the
sign and value of the optical rotation of the amino acid
methyl-4-hydroxypyrrolidine-2-carboxylic
acid
(4-
hydroxyproline betaine) unit was deduced to be present
in 2. An HMBC correlation between the oxymethine
0
4
matched well with those of (2R, 4R)-4-hydroxypipe-
proton at d 5.56 (H-4 ) and the ester carbonyl at d
1
8
colic acid previously reported thus indicating that the
absolute stereochemistry of the amino acid portion of
damipipecolin is (2R, 4R).
159.2 suggested that this unit was connected to the
bromopyrrolecarboxylic acid moiety through an ester
linkage.
Crystal structures of the two diastereoisomeric betoni-
0
Br
cine (trans-N,N -dimethyl-4-hydroxy-L-proline, levoro-
0
4
3
H
3'
H
COO-
H
2'
COOH
H
2'
3'
5
HO4'
HO4'
tatory) and turicine (cis-N,N -dimethyl-4-hydroxy-D-
proline, dextrorotatory) had been previously determined
6
OH
2
N
H
5'
NH
N
O
5'
25
6
'
as their hydrochlorides; the NMR characterization of
the trans diastereomer, isolated from the extracts of
5
3
4
2
6
leaves of Melaleuca species, had been also reported.
Taking into account these reported data, the relative
cis-stereochemistry to the betaine unit present in the
molecule of 2 could be assigned on the basis of a
The ESI (positive ions) mass spectrum of damituricin (2)
showed two intense pseudomolecular ion peaks at m/z
+ 13
31 and 333 [M+H] in the ratio 1:1. The C NMR
3
spectrum (CD OD) contained 12 carbon signals, which
ROESY experiment (CD OD) results. In fact, an intense
3
0
0
cross peak was observed between H-2 (d 4.19) and H-4
3
were identified, through DEPT and HSQC experiments,
as two methyls, two sp methylenes, two sp methines,
(d 5.56), which was consistent only with a relative cis-
orientation of the carboxylate group and the acylic por-
tion. The absolute stereochemistry of the amino acidic
portion of 2 could be clarified through acid hydrolysis
of 2, performed following the same procedure reported
for 1, which gave 4-bromopyrrole 2-carboxylic acid (3)
3
2
3
two deshielded sp methines, and four unprotonated
carbons. These NMR and mass spectral data were com-
patible with the molecular formula C H N O Br,
1
2
15
2
4
1
3
implying six elements of unsaturation. From
C
0
NMR data, the presence of two carbon–carbon double
bonds and two carbonyl groups was evident; thus, also
molecule 2 must be bicyclic.
and (+)-cis-N,N -dimethyl-4-hydroxy-D-proline (turi-
cine, 5). The optical rotation value and sign of com-
2
7
pound 5 were identical to those reported for turicine;
thus, on the basis of this result, the (2R, 4R) absolute
stereochemistry was assigned to the amino acidic por-
tion of damituricin.
1
13
The comparison of H and C NMR spectra of 1 and 2
revealed some similarities. Particularly, the aromatic
proton and carbon resonances present in the spectra of
2
almost identical to those of 1, indicating in 2 the pres-
(d : 124.3, 97.1, 117.5, 122.2; d : 6.95, 7.05) were
2.3. Bioactivities of damipipecolin (1) and damituricin (2)
C
H
ence of the same 4-bromopyrrole-2-carboxylic acid
moiety as in 1, connected via an ester bond (d 159.2)
2.3.1. Cytotoxic effect. The cell viability tests using the
2
8
MTT assay revealed that compounds 1 and 2 are not
toxic at a concentration of 10 lg/ml or less for all cell
lines tested (PC12, HeLa, and L5178y cells).
C
1
to a different cyclic unit. Analysis of H NMR spectrum
(
CD OD) of 2 was integrated with data obtained from
3
the HSQC experiment. In addition to pyrrole signals,
the proton spectrum contained two three-proton-
singlets at d 3.43 and 3.34, attributable, also on the basis
of their deshielded carbons (d 52.3 and 46.7), to N-
linked methyls of a quaternary ammonium compound.
2.3.2. Effect of compound 1, compound 2, and serotonin on
Ca -level in primary neurons. As shown in Figure 1A
addition of 200 lM serotonin (5-hydroxytryptamine; 5-
2
+
HT) and 2.5 mM CaCl to primary neuronal cells (rat
cortical cell cultures) resulted in a marked increase in
2
1
Additional features of H NMR spectrum were two

5880
A. Aiello et al. / Bioorg. Med. Chem. 15 (2007) 5877–5887
2
+
Figure 1. Effect of damipipecolin (1) on 5-HT (A, B) and L-Glu (C, D)-induced increase in [Ca
with fura-2-AM and analyzed by the fluorescence ratio-imaging system as described in Experimental procedures. Fluorescence images were recorded
at time zero (a), after 5 (b) and 10 min (c). In (d) the cells inspected are shown by Nomarski phase contrast interference optics. (A, B) Effect on 5-HT-
i
] level in primary neuronal cells. Cells were loaded
2+
induced increase in [Ca
i
] level. (A-a) Start of measurement (time: 0 min). (A-b) Addition of 0.1% (v/v) DMSO (control; 5 min). (A-c) Addition of
2+
2+
2
00 lM 5-HT and 2.5 mM Ca (10 min). (B-a) Time zero. (B-b) Addition of 0.1 lg/ml of compound 1 (5 min). (B-c) Addition of 5-HT/Ca
2+
(10 min). (C, D) Effect on L-Glu-induced increase in [Ca
]
i
level. (C-a) Time zero. (C-b) Addition of 0.1% (v/v) DMSO (control; 5 min). (C-c)
2+
Addition of 200 lM L-Glu and 2.5 mM Ca (10 min). (D-a) Time zero. (D-b) Addition of 0.1 lg/ml of compound 1 (5 min). (D-c) Addition of L-
2
Glu/Ca (10 min). The spectrum colour scale ranges from blue (low [Ca
+
2+
2+
i i
] ) to red (high [Ca ] ). Magnification, 400-fold.
2
intracellular free calcium level ([Ca ] ), as documented
+
(p < 0.001) reduced (decrease to 29.7 ± 20.3% and
38.0 ± 19.7%, respectively; mean value of 2–5 experi-
ments) when 0.3 or 1 lg/ml of compound 1 was added
to the cells. At higher or lower concentrations, the com-
pound became less effective (0.03 lg/ml; 71.6%); no
i
by the record of the fluorescence images obtained in
2
+
experiments using the Ca indicator dye fura-2. A
strong shift from dark blue to light-blue, green, orange
and red was seen in all cells immediately (30 s) after
2
+
addition of 5-HT/CaCl (Fig. 1A-c), which was done
2
effect on 5-HT/CaCl -induced change of [Ca ] level
2
i
1
0 min after the beginning of the experiment. Addition
was found at 0.01, 3, and 10 lg/ml of compound 1
(Fig. 2). The increase in the fluorescence ratio (340/
380 nm) in the control samples after addition of 5-HT/
of 0.1 lg/ml of compound 1 strongly reduced the 5-
2
HT-induced elevation of [Ca ] (Fig. 1B-c). In Figure
+
i
1
A-d and B-d, a photo of the cells taken at the end of
CaCl was set to 100%.
2
the experiment by Nomarski phase contrast interference
optics is shown.
Compound 2 showed on primary neurons a similar ef-
fect like compound 1. As shown in Figure 6 preincuba-
tion of the cells for 5 min with 10 lg/ml of compound 2
2
The strongest effect on [Ca ] in neurons was observed
+
i
2
+
after preincubation of the cells for 5 min with 0.1 lg/ml
resulted in a small decrease in the [Ca ] values to
i
of compound 1 (Fig. 2) followed by 5-HT/CaCl appli-
79.3%. In contrast, treatment (5 min) of neurons with
10 and 100 times (1 and 0.1 lg/ml) lower doses of com-
pound 2 induced a significant (p < 0.001) reduction of
2
cation (reduction to 11.2 ± 0.8%; mean value of three
experiments). A quantitative analysis revealed that the
increase in the 340/380 nm ratio was also significantly
2
+
the [Ca ] influx after addition of 5-HT/CaCl2
i

1C addition of L-Glu and 2.5 mM CaCl to neuronal
2
cells resulted in a strong shift from dark blue to green,
orange and red (Fig. 1C-c).
A. Aiello et al. / Bioorg. Med. Chem. 15 (2007) 5877–5887
5881
1
1
1
80
50
20
160
140
120
100
9
6
3
0
0
0
0
80
60
40
20
0
0
.01
0.03
0.1
0.3
1
3
10
Damipipecolin (µg/ml)
Neurons
PC12 cells
HEK cells
2
Figure 2. Incubation of neurons with 5-HT and CaCl in the presence
of various concentrations of compound 1 (damipipecolin). In the first
set of experiments (control) neurons were stimulated with 200 lM 5-
Figure 3. Incubation of primary neurons, PC12 cells and HEK cells
with 200 lM 5-HT and 2.5 mM CaCl in the presence of 1 lg/ml of
compound 1 (damipipecolin; black column) and 2 (damituricin; white
column). The results are expressed as % of control ± standard
deviation of three independent experiments. Further details are given
in Figure 2.
2
HT and 2.5 mM CaCl
the second set of experiments the effect of 5-HT/CaCl
2
only; the values obtained were set to 100%. In
on the calcium
2
level in neurons preincubated for 5 min with 0.01 to 10 lg/ml of
compound 1 was determined. Experiments were performed in Locke’s
2+
solution. In all assays, [Ca
i
] level was measured for 20 min. The
results are expressed as % of control ± standard deviation of 3–5
independent experiments. The cell number per concentration per
experiment was ꢀ90.
2
Fig. 6). The [Ca ] values decreased to 41.8 ± 4.5%
1 lg/ml) and 29.5 ± 9.5% (0.1 lg/ml), respectively.
+
(
(
i
As shown on Figure 4A preincubation (5 min) of the
neuronal cells with 0.1, 1, and 10 lg/ml of 1 induced
2
+
2.3.3. Effect of compound 1, compound 2, and serotonin on
Ca -level in PC12 and HEK cells. Treatment of PC12
no significant decrease in [Ca ]
Glu/CaCl . The fluorescence images showed like in the
control sample a fast change of the color from dark blue
to red after addition of L-Glu/CaCl following preincu-
bation of the neurons with 0.1 lg/ml of 1 (Fig. 1D-c). In
i
after addition of L-
2
+
2
and HEK cells with 200 lM 5-HT and 2.5 mM CaCl
2
2
resulted only in a small increase in [Ca ] (expressed
+
i
2
as 340/380 nm ratio values); D ratio 0.096 (PC12 cells)
and 0.115 (HEK cells). The control values were set to
2
+
comparison with the control, [Ca ] amounted to 83.3%
i
1
00%.
(D ratio 1.524; 0.1 lg/ml), 90.5 (D ratio 1.656; 1 lg/ml),
and to 96.6% (D ratio 1.766; 10 lg/ml), respectively.
As shown in Figure 3 preincubation of PC12 cells and
HEK cells for 5 min with 1 lg/ml of 1 did not induce
Preincubation (5 min) of the neurons with 0.1, 1, and
10 lg/ml of compound 2 (Fig. 6) induced no changes
2
+
a significant change of [Ca ] after addition of 5-HT/
i
2
+
CaCl , while preincubation of primary neuronal cells
2
in the [Ca ]
ison with the control the [Ca ]
ml), 100.2% (1 lg/ml), and 98.9% (10 lg/ml).
i
after addition of L-Glu/CaCl
2
. In compar-
reached 109.7% (0.1 lg/
i
2
+
with the compound under the same condition caused a
strong decrease in 5-HT/CaCl -induced elevation of
2
2
+
[
Ca ] level.
i
2.3.5. Effect of compound 1, compound 2, and N-methyl-
D-aspartic acid on the Ca -level in primary neurons.
Stimulation of primary neuronal cells with 200 lM
2
+
In parallel experiment preincubation of PC12 cells for
min with 1 lg/ml of compound 2 (Fig. 3) generated a
5
significant change of [Ca ] after addition of 5-HT/
2
+
N-methyl-D-aspartic acid (NMDA) and 2.5 mM CaCl
2+
resulted in a significant (p < 0.001) increase in [Ca ]
D ratio 0.868 (set to 100%).
i
2
2
CaCl . The [Ca ] decreased to 43.8 ± 18.4%. At the
+
;
2
i
i
same time preincubation of HEK cells with compound
under the same conditions caused no changes in
2
[
2
Ca ] .
+
As shown in Figure 4B preincubation (5 min) of the neu-
rons with 10 and 0.1 lg/ml of 1 induced no significant
i
2
+
2
.3.4. Effect of compound 1, compound 2, and L-glutamic
decrease in [Ca ]
comparison with the control the changes in [Ca ] are
i
i
after addition of NMDA/CaCl . In
2
2
+
2+
acid on the Ca -level in primary neurons. In primary
neuronal cells treated with 200 lM L-glutamic acid (L-
between 105.9% (D ratio 0.919; 10 lg/ml) and 114.1%
Glu) and 2.5 mM CaCl a strong (p < 0.001) increase
(D ratio 0.990; 0.1 lg/ml). Only after addition of 1 lg/
2
2
+
2+
ml of 1 a significant (p < 0.01) decrease in [Ca ]
in [Ca ] from 0.704 ± 0.012 to 2.533 ± 0.058 (D ratio
i
was
i
1
.829; set to 100%) was measured. As shown in Figure
measured (70.4%, D ratio 0.611).

5882
A. Aiello et al. / Bioorg. Med. Chem. 15 (2007) 5877–5887
A
B
1
1
20
00
1
1
20
00
8
6
4
2
0
0
0
0
0
8
6
4
2
0
0
0
0
0
0
.1
1
10
0.1
1
10
Damipipecolin (µg/ml)
Damipipecolin (µg/ml)
Figure 4. Incubation of neurons with (A) 200 lM L-Glu and 2.5 mM CaCl
2
2
and (B) 200 lM NMDA and 2.5 mM CaCl in the presence of 0.1, 1, and
10 lg/ml of compound 1 (damipipecolin). Further details are given in Figure 2.
2+
Figure 5. Effect of 5-HT on [Ca
]
i
level on cells of Suberites domuncula. Fluorescence images were recorded at time zero (a) and after 5 (b) and
1
0 min (c). The sponge cells were treated with 200 lM 5-HT and 10 mM CaCl . In (d) the cells inspected are shown by Nomarski phase contrast
2
interference optics. Magnification, 400-fold.
In contrast, pretreatment of neurons with compound 2
induced only in the concentration range of 10 lg/ml a
2.3.6. Effect of compound 1 and serotonin on calcium level
in sponge cells. In parallel to mammalian cells, the effect
2
+
significant decrease in [Ca ] after addition of
2+
of 5-HT on [Ca ] level in primmorphs (aggregates of
i
i
NMDA/CaCl (Fig. 6). The values decreased to 58.8%
2
dissociated cells) from the marine sponge Suberites
domuncula was determined. As shown in Figure 5, addi-
(
D ratio 0.510). No changes were measured after treat-
ment of neurons with 1 (119.52%) and 0.1 lg/ml
95.0%) of compound 2.
tion of 200 lM 5-HT and 10 mM CaCl to S. domuncula
2
2
cells did not result in a significant change of [Ca ] level
+
(
i

A. Aiello et al. / Bioorg. Med. Chem. 15 (2007) 5877–5887
5883
3
6
0
.1
1
10 µg/ml
group of the G protein-coupled receptors. Based on se-
quence data seven subtypes (5-HT –5-HT ) have been
1
7
1
1
1
40
20
00
3
7
distinguished. The 5-HT1 receptors exhibit a high
affinity for serotonin, in contrast to 5-HT and 5-HT
2
4
receptors, and have been implicated in the pathogenesis
of many mental and neurological disorders; therefore
they have become an important target for drug ther-
8
6
4
2
0
0
0
0
0
38
apy. 5-HT -receptors, on the other hand, are increas-
3
ingly used as target for antiemetics to prevent
3
chemotherapy-induced nausea and vomiting.
9
Our studies revealed that compound 1 is a potent anta-
gonist of serotonin in neuronal cells; the effect of serotonin
on neurons was nearly completely blocked at low con-
centrations of 0.1 and 0.3 lg/ml of compound 1 (ꢀ88%
and ꢀ70% reduction). Higher concentrations of com-
pound 1 were interestingly less active. At a concentration
5
-HT
NMDA
L-Glu
Figure 6. Incubation of primary neurons with (i) 200 lM 5-HT and
.5 mM CaCl , (ii) 200 lM NMDA and 2.5 mM CaCl , and (iii) 200 lM
L-Glu and 2.5 mM CaCl in the presence of 0.1, 1, and 10 lg/ml of
compound 2 (damituricin). The results are expressed as % of control ±
standard deviation of 2–3 independent experiments. Further details are
given in Figure 2.
2
+
2
2
2
of 1 lg/ml of 1, Ca influx in neurons was reduced to
2
ꢀ
62%, while preincubation of neurons with P3 lg/ml
of compound 1 did not induce a decrease in Ca influx
2+
after addition of 5-HT/CaCl . In contrast, compound 2 is
2
a slightly less potent agonist of serotonin at the concen-
tration of 1 lg/ml (ꢀ59% reduction) and 0.1 lg/ml of
2
(ꢀ71% reduction). Like compound 1 higher con-
2+
(
1
340/380 nm ratio) after an incubation period of 5 and
centration of compound 2 (10 lg/ml) induces small
2
0 min. No change of [Ca ] level was also found after
+
i
(ꢀ22% reduction) decrease in Ca influx after addition
addition of 0.1 lg/ml or 1 lg/ml of 1 to the cells (data
not shown).
of 5-HT/CaCl .
2
The reason of the reduced efficiency of compounds 1
and 2 at higher concentration is not yet known. It
should be noted that different types of serotonin recep-
tors exist in brain tissue, and it has not been elucidated
which receptor subtype(s) is/are involved in the mode of
action of compounds.
3
. Discussion
The new bromopyrrole alkaloids damipipecolin (1) and
damituricin (2) extend the structural variety of the so far
known pyrrole alkaloids since their structures lack the
commonly found short linear aliphatic segment linking
the bromopyrrole 2-carboxylic acid moiety to the com-
mon imidazole nucleus. Until now, this feature has been
In PC12 cells, 5-HT is known to have neuritogenic ef-
fects, although the signal transduction pathway respon-
4
0
sible for these effects is not understood. Application of
5-HT to PC12 cells enhances nerve growth factor
(NGF)-induced neurite outgrowth in a dose-dependent
2
9
10
found only in manzacidins A–C, in agelongine, and
1
1
in its bromine-free analog daminin isolated from the
same sponge A. damicornis. Many alkaloids comprising
a pyrrole ring linked to an imidazole moiety are biolog-
4
0
manner. Compound 1 did not exhibit a significant
effect on Ca influx in PC12 cells and HEK cells. In
2
+
6
6
ically active, showing antibacterial, antifungal, antitu-
8
mor,
contrast, compound 2 was a potent antagonist of seroto-
nin-induced Ca entry in PC12 cells; the effect of sero-
30
2+
antihistaminergic, and immunosuppressive
9
3
0
activities, or acting as a-adrenergic and serotonergic
receptor antagonists.
tonin on PC12 cells was more then 50% blocked (57%
reduction). These pharmacological tests indicated that
1
0
2
-HT-induced [Ca ] increase in PC12 cells involves
+
5
i
2
+
Damipipecolin and damituricin were found to exhibit a
modulating effect on the serotonin receptor activity
in vitro. Alterations of serotonin (5-hydroxytryptamine,
the mediation by a voltage-dependent Ca channel,
evoked by membrane depolarization via the activation
4
0,41
of cation channel-type receptors, 5-HT receptors.
3
5
-HT) levels are associated with many pathological dys-
functions of CNS, such as affective disorders, schizo-
The effect can be blocked in a dose-dependent manner
4
0,41
with nifedipine
channel blocker), MDL 72222, and metoclopramide
(voltage-dependent L-type calcium
41
3
1
40
phrenia, and migraine. The serotonergic system also
plays a significant role in the development of depression.
5
Some forms of phobia seem to be associated with an in-
crease in serotonin levels; 5-HT receptor antagonists
2
seem to be efficacious in the treatment of psychosis.
(selective 5-HT receptor antagonist), but not by ketan-
3
3
2
40
-HT reuptake inhibitors are effective antidepressants.
serin, a 5-HT(2) receptor antagonist, or thapsigar-
4
0
gin, a specific inhibitor of endoplasmic reticulum
Ca -ATPase. Our results indicate that compound 2 is
2
+
3
3–35
a strong 5-HT receptor antagonist or L-type calcium
3
channel blocker in PC12 cells.
Serotonergic neurons are found in nearly every brain
area. Serotonin receptors can be classified in three differ-
ent groups: (i) the transporter (5-HT ‘uptake site’), (ii)
In addition, no evidence was obtained that compounds 1
and 2 interact with glutamate or NMDA receptors.
L-Glutamate and L-aspartate are the most abundant
the ligand-gated ion-channel (5-HT ) and (iii) the largest
3

5884
A. Aiello et al. / Bioorg. Med. Chem. 15 (2007) 5877–5887
excitatory neurotransmitters in the CNS. The excito-
toxic effect of L-Glu is associated with an influx of
Na , K , and Ca in neurons as a consequence of a sus-
SONS) mass spectrometer. Optical rotations were mea-
sured using a Perkin-Elmer 192 polarimeter. CD spectra
were recorded on a J-710 spectropolarimeter (Jasco,
Tokyo, Japan) equipped with a J-710 for Windows soft-
ware (Jasco). NMR experiments were performed on
aVarian INOVA 500 spectrometer; chemical shifts are
referred to the residual solvent signal. Medium pressure
liquid chromatographies (MPLC) were performed on a
+
+
2+
4
2,43
tained activation of glutamate receptors.
L-Glu
stimulates ionotropic receptors, such as the NMDA
receptor, and metabotropic receptors. In our experi-
ments, preincubation of neurons with 0.1, 1, and
1
icant decrease in Ca entry.
0 lg/ml of compounds 1 and 2 did not induce a signif-
2+
Buchi 861 apparatus with SiO (230–400 mesh) packed
2
columns. High-performance liquid chromatography
(HPLC) separations were achieved on a Knauer 501
apparatus equipped with an RI detector.
Further studies could be performed on compounds 1
and 2 in order to gain information about their mode
of action and structure–activity relationships. Anyway,
some indication can be obtained from the data previ-
ously reported on structurally related compounds.
Experiments performed on pyrrole alkaloids to evaluate
the interactions with the cellular calcium homeostasis
led to a structure–activity relationship underlining both
the importance of the degree of bromination of the
pyrrole moiety and the presence of an aminoimidazole
4.2. Collection, extraction, and isolation of compounds 1
and 2
Specimens of the sponge Axinella damicornis were col-
lected and kept frozen until used. For the extraction,
the fresh thawed sponge (75.2 g dry weight after extrac-
tion) was homogenized and treated at room temperature
with methanol (3· 600 ml) and, subsequently, with chlo-
roform (3· 600 ml). The extracts were combined and
concentrated in vacuo to give an aqueous suspension
4
4
group. Particularly, these studies revealed that the
-bromopyrrole-2-carboxylic acid moiety is not respon-
4
sible alone for the biological effect of the brominated
pyrrole alkaloids since both 4-bromopyrrole-2-car-
boxylic acid and pyrrole-2-carboxylic acid did not de-
crease voltage-dependent calcium elevation in PC12
which was then partitioned between H O and BuOH.
2
The BuOH layer, after evaporation of the solvent, was
subjected to a medium pressure chromatography
(MPLC) over a reversed-phase (RP-18) column using a
gradient elution (H O ! MeOH ! CHCl ). Following
4
4
cells. Besides, the different potency of 4,5-bromopyr-
role-2-carboxylic acid and oroidin (more active), in
which this moiety is linked to the aminoimidazole group
through a short linear aliphatic segment, reveals the
importance of this group. This is further supported by
earlier studies showing a marked antiserotonergic activ-
ity for the pyrrole alkaloids hymenidin, oroidin, and
2
3
this procedure, the fraction eluted with MeOH/H O
2
8:2 was mainly composed of polar alkaloids. This frac-
tion was further separated by HPLC on a preparative
RP-18 column (Kromasil, 10 lm, 250 · 10 mm), with
H O/MeOH 7:3 as the eluent, and gave a mixture of
2
4
5,46
their unbrominated analog clathrodin
of activity of dispacamides, in which the aminoimidazole
and the lack
compounds 1 and 2. Final separation and purification
of the two compounds was achieved by HPLC on a
RP-18 column (Luna, 5 lm, 250 · 4.6 mm), using
8
group is oxidized to an aminoimidazolone moiety.
MeOH/H O 2:8 as the eluent, thus obtaining damipipeco-
2
The bioactivity shown by compounds 1 and 2 suggests
that antiserotonergic effects are associated to bromopyr-
role rings when they are linked both to an aminoimid-
azole group and to other nitrogen heterocycle rings,
such as pyrrolidine and piperidine moieties. In order
to clarify the mode action of 1 and 2, the effect of
lin [1, 4 mg], and damituricin [2, 3 mg], both in a pure
form.
2
0
Damipipecolin (1): ½aꢁ þ 5:4 (H O, c 0.001). HR-FAB-
D
MS (positive ion mode): m/z = 317.013694 [M + H] ;
2
+
the molecular formula C H N O Br requires
4
1
1
14
2
2
+
1
317.013688. H (500 MHz, CD OD) and C NMR
13
compounds 1 and 2 on Ca level in the presence of
selective agonists of 5-HT receptors [e.g., sumatriptan,
3
(125 MHz, CD OD) data are reported in Table 1.
3
47
1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane], could
provide useful information.
2
0
Damituricin (2): ½aꢁ þ 10:7 (H O, c 0.0013). HR-FAB-
D
MS (positive ion mode): m/z = 331.029354 [M + H] ;
2
+
In conclusion, damipipecolin and damituricin seem to be
promising new serotonin antagonists with a potential in
the therapy of disorders associated with changes of sero-
tonin levels, such as psychosis, different phobia, and
mood fluctuation disorders, as well as chemotherapy-in-
duced emesis.
the molecular formula C H N O Br requires
16
1
2
2
4
1
13
331.029338. H (500 MHz, CD OD) and C NMR
3
(125 MHz, CD OD) data are reported in Table 1.
3
4.3. Hydrolysis of damipipecolin
A solution of 1.3 mg of 1 was stirred with 1 N HCl in
methanol 91% at 80 ꢁC overnight. The reaction mixture
was evaporated under nitrogen and then subjected to
4
. Experimental procedures
HPLC purification using
a
RP-18 Luna 5 lm
4
.1. General aspects
(250 · 3 mm) column with H O/MeOH 8:2 as the eluent,
2
yielding 1 mg of pure 4-bromopyrrole 2-carboxylic acid
20
ESI mass spectra were obtained by using an API 2000
mass spectrometer. High-resolution FAB mass spectra
(3) and (+)-trans-4-hydroxypipecolic acid (4, ½aꢁ þ23, c
D
0.001, H O; ESI-MS : m/z 146 [M+H] ; H and
NMR data are reported in Table 2).
+
1
13
C
2
(
glycerol matrix) were recorded on a VG Prospec (FI-

A. Aiello et al. / Bioorg. Med. Chem. 15 (2007) 5877–5887
5885
1 13
Table 1. H and C NMR data (CD
1
13
3
OD) of compounds 1 and 2
Compound 2
Table 2. H and C NMR data (D
and D-turicine (5)
2
O) of 4-hydroxypipecolic acid (4)
Position
Compound 1
Position
Compound 4
Compound 5
d
C
d
H
J in Hz)
(mult.,
d
C
H
d (mult.,
J in Hz)
d
C
d
H
d
C
d
H
(mult.,
(mult.,
J in Hz)
2
3
4
5
6
123.6
—
122.2
—
7.05(d, 1.6)
—
J in Hz)
124.5 7.01 (d, 1.6) 124.3
97.8 97.1
117.7 6.92 (d, 1.6) 117.5
0
—
2
54.1 3.82
(dd, 12, 3)
: 2.11
(dt, 14.3, 3.3)
76.2
36.2
4.05 (dd, 12.5, 8.5)
6.95(d, 1.6)
—
4.19
0
159.9
56.4 3.74
—
159.2
75.7
3
32.7
H
a
H
a
: 2.89 (m)
0
0
2
a
(dd, 12, 3.4)
(dd, 12.3, 8.5)
: 3.10 (m)
H
b
: 1.86
b
H : 2.25
3
32.9
H
a
: 2.40
bd, 14,7)
33.6
H
a
(ddd, 15.3,
12.1, 4.0)
4.63 (m)
(
a
0
0
b
H : 1.91
b
H : 2.64
4
62.0 4.12 (bs)
a
29.0 1.81
65.8
74.5
(
ddd, 15, 12.3, 4.1)
5.56 (m)
: 3.98
bd, 13.5)
: 3.92
dd, 13.5, 6.6)
5
H
a
: 3.62
(bd,13.2)
b
H : 3.67
0
0
4
5
67.8 5.31 (bs)
68.9
72.0
a
a
28.5
40.2
H
a
: 2.01
H
a
(
(dd, 13.2, 6.8)
0
H
b
: 1.98
H
b
6
38.7 3.20 (m)
—
—
170.4
—
—
(
COOH 174.5
N–Me
0
a
a
6
H
H
a
: 3.17
—
—
—
Me
Me
a
: 53.1 Me : 3.26 (s)
b
: 47.8 Me : 3.19 (s)
a
b
: 3.20
b
COOH 179.9
N–Me
—
—
169.0
—
: 52.3 Me
a
Overlapped signals.
—
Me
Me
a
a
: 3.43 (s)
b
: 3.34 (s)
b
: 46.7 Me
a
Overlapped signals.
cells) and 1:10 ratio (HeLa cells). All cell cultures were
kept in a humidified atmosphere of 5% CO /air at 37 ꢁC.
2
Specimens of the marine sponge Suberites domuncula
(Porifera, Demospongiae, Hadromerida) were collected
in the Northern Adriatic near Rovinj (Croatia) and kept
in aquaria in Mainz (Germany) at a temperature of
4
.4. Hydrolysis of damituricin
1
7 ꢁC. Sponge primmorphs were prepared from dissoci-
50
A solution of 2 mg of 2 was stirred with 1 N HCl in
methanol 91% at 80 ꢁC overnight. The reaction mixture
was evaporated under nitrogen and then subjected to
ated sponge cells as described. The primmorphs were
incubated for 1 day in Ca - and Mg -free artificial
seawater (CMFSW)
experiments.
2
+
2+
5
1
at 17 ꢁC until start of the
HPLC purification using
a
RP-18 Luna 5 lm
(
yielding 4-bromopyrrole 2-carboxylic acid (3) and
250·3 mm) column with H O/MeOH 8:2 as the eluent,
2
4.6. MTT assay
0
1
.3 mg of (+)-cis-N,N -dimethyl-4-hydroxy-D-proline
20
(
turicine, 5, ½aꢁ þ 20:3, c 0.001, H O; ESIMS : m/z
To estimate the IC50 values, cells were incubated for
72 h in the presence of various concentrations of com-
pounds 1 and 2. The viability of the cells was determined
using the MTT colorimetric assay system. Evaluation
was performed in 96-well plates at 595 nm using an ELI-
SA plate reader, after overnight incubation at 37 ꢁC.
D
82 [M+Na] ; H and C NMR are reported in
2
+
1
13
1
Table 2).
.5. Mammalian cells and sponge primmorphs
2
8
4
Primary neuronal cells (rat cortical cells) were prepared
from the brains of 17- to 18-day-old Wistar rat embryos,
4.7. Calcium measurements
4
8,49
as described.
The cells were maintained in DMEM/
2
For determination of the intracellular Ca concentra-
+
HG (high glucose; 4.5 g/l glucose) serum-free medium
supplemented with 0.1% (w/v) bovine serum albumin
2
tion ([Ca ] ), cells were cultivated on poly-L-lysine
+
i
(
1
BSA), 2 mM L-glutamine, 100 lg/ml transferrin,
00 mU/l insulin, 16 lg/ml putrescine, 6.3 ng/ml of pro-
gesterone, and 5.2 ng/ml Na SeO .
coated borosilicate coverglass (Nunc). An inverted-stage
Olympus IX70 microscope with objective UApo40X/340
was used for the fluorescence measurements. The cells
were alternately illuminated with light of wavelengths
340 and 380 nm. An additional 0.25 ND filter was used
at 380 nm. The fluorescence emissions at 510 nm were
monitored by an intensified CCD camera, model
C2400-87 (Hamamatsu, Herrsching, Germany). Images
were then digitized with a computerized imaging system
(Argus-50, Hamamatsu).
2
3
PC12 cells were grown in Dulbecco’s modified Eagle’s
medium [DMEM]/10% (v/v) fetal calf serum (FCS)/5%
(
v/v) horse serum. HEK cells were grown in Dulbecco’s
modified Eagle’s medium [DMEM]/10% (v/v) FCS/
.5 g/l glucose. PC12 and HEK cells were passaged twice
4
per week at a 1:10 ratio. L5178y and HeLa cells were
maintained in Roswell Park Memorial Institute medium
[RPMI] 1640 supplemented with 10% (v/v) FCS. The
cells were subcultured twice weekly at a 1:160 (L5178y
2
+
The [Ca ] was determined by measuring the fluores-
i
2
cence ratio of the Ca -indicator dye fura-2-AM at
+

5886
A. Aiello et al. / Bioorg. Med. Chem. 15 (2007) 5877–5887
4
9
3
4
40 and 380 nm. Neurons were loaded in the dark with
lM fura-2-acetoxymethyl (AM) ester (Molecular
References and notes
Probes, Leiden, The Netherlands), and PC12 and
HEK cells with 10 lM fura-2-AM in DMEM/HG ser-
um-free medium, supplemented with 1% (w/v) BSA at
7 ꢁC for 1 h. Sponge cells (primmorphs) were loaded
with 12 lM fura-2-AM in CMFSW containing 1% (w/v)
BSA at 17 ꢁC for 2 h. Subsequently, the cells were
washed twice with medium supplemented with 10%
FCS (mammalian cells) and incubated further at 37 ꢁC
for 1 h; sponge cells were washed with CMFSW and
incubated further at 17 ꢁC for 1 h. A calcium calibration
curve was prepared according to the method of
1. Faulkner, D. J. Nat. Prod. Rep. 2000, 17, 7.
. Forenza, S.; Minale, L.; Riccio, L.; Fattorusso, E. Chem.
Comm. 1971, 1129.
. Braekman, J. C.; Daloze, Q.; Stoller, C.; van Soest, R. W.
Biochem. Syst. Ecol. 1992, 20, 417.
. Lindel, T.; Hoffmann, H.; Hochgurtel, M.; Pawlik, J. R.
J. Chem. Eco. 2000, 26, 1477.
. Aiello, A.; D’Esposito, M.; Fattorusso, E.; Menna, M.;
M u¨ ller, W. E. G.; Perovic-Ottstadt, S.; Schr o¨ der, H. C.
Bioorg. Med. Chem. 2006, 14, 17.
6. Cafieri, F.; Fattorusso, E.; Tagliatatela-Scafati, O. J. Nat.
Prod. 1998, 61, 122.
. Bringmann, G.; Lang, G.; Tsuruta, H.; Fattorusso, E.;
Aiello, A.; D’Esposito, M.; Menna, M.; M u¨ ller, W. E. G.;
Schr o¨ der, H. C.; Perovic-Ottstadt, S. Ger. Offen 2005, 18.
. Cafieri, F.; Carnuccio, R.; Fattorusso, E.; Tagliatatela-
Scafati, O.; Vallefuoco, T. Bioorg. Med. Chem. Lett. 1997,
, 2283.
. Kobayashi, J.; Nakamura, H.; Ohizumi, Y. Experientia
988, 44, 86.
2
3
4
5
3
5
2
7
8
9
Grynkiewicz.
Locke’s solution (154 mM NaCl, 5.6 mM KCl, 3.6 mM
NaHCO , 5.6 mM glucose, and 10 mM Hepes; pH 7.4)
without Ca and Mg was used as incubation medium
in all set of experiments. A stock solution of 10 mg/ml of
compound 1 dissolved in DMSO and of compound 2
3
2
+
2+
7
1
(
5 mg/ml) dissolved in water were used. Serial dilutions
10. Cafieri, F.; Fattorusso, E.; Mangoni, A.; Tagliatatela-
Scafati, O.; Carnuccio, R. Bioorg. Med. Chem. Lett. 1995,
8, 799.
were done in DMSO or water. In experiments with
sponges, Locke’s solution with sea water osmolarity
pH 8.0 was used.
1
1. Aiello, A.; D’Esposito, M.; Fattorusso, E.; Menna, M.;
M u¨ ller, W. E. G.; Perovic-Ottstadt, S.; Tsuruta, H.;
Gulder, T. A. M.; Bringmann, G. Tetrahedron 2005, 61,
In the first set of experiments (control) neurons were
stimulated with 0.1% (v/v) DMSO or water after 5 min
and with 200 lM 5-HT, NMDA or L-Glu and 2.5 mM
7
266.
2. Ackermann, D. H. G. Hoppe-Seylers Z. Physiol. Chem
960, 332, 198.
1
1
CaCl after 10 min from the beginning of the measure-
2
13. Friess, S. L.; Patchett, A. A.; Witkop, B. J. Am. Chem.
Soc. 1957, 79, 459.
14. Moloney, M. G. Nat. Prod. Rep. 1998, 205.
15. Ho, B.; Zabriskie, T. M. Bioorg. Med. Chem. Lett. 1998, 8,
739.
ments. In the second set of experiments the primary neu-
rons were first preincubated with various concentrations
of compound 1 or 2 (5 min). After 10 min 5-HT/CaCl2,
NMDA/CaCl2 or L-Glu/CaCl2 was added to the
neurons.
1
1
1
6. Romeo, J. T.; Swain, L. A.; Bleecker, A. B. Phytochem-
istry 1983, 22, 1615.
7. Vanderhaeghe, H.; Janssen, G.; Compernolle, F. Tetrahe-
dron Lett. 1971, 28, 268.
8. (a) Fujita, Y.; Kollonitsch, J.; Witkop, B. . J. Am. Chem.
Soc. 1965, 87:9, 2030; (b) Davis, F. A.; Fang, T.; Chao, B.;
Burns, D. M. Synthesis 2000, 14, 2106.
In the next set of experiments PC12 and HEK cells
were first preincubated with 1 lg/ml of compound 1
or 2 (5 min) and after 10 min 5-HT/2.5 mM CaCl2-
was added to the cells. In the case of sponge cells,
the preincubation of the cells was done with 0.1
and 1 lg/ml of compound 1 (5 min) and after
1
9. Copeland, T. D.; Wondrak, E. M.; Toszer, J.; Roberts, M.
M.; Oraszlan, S. Biochem. Biophys. Res. Comm. 1990, 169,
310.
1
0 min 5-HT/10 mM CaCl was added to the cells.
2
As a control, cells treated with 0.1% (v/v) DMSO
20. Dragovich, P. S.; Parker, J. E.; French, J.; Incacuan, M.;
Kalish, V. J.; Kissinger, C. R.; Knighton, D. R.; Lewis, C.
T.; Moomaw, E. W.; Parge, H. E.; Pelltier, L. A. K.;
Prince, T. J.; Showalter, R. E.; Tatlock, J. H.; Tucker, K.
D.; Villafranca, J. E. J. Med. Chem. 1996, 39, 1872.
and 200 lM 5-HT with 2.5 or 10 mM CaCl were
2
2
+
used. In all set of experiments the [Ca ]i level was
measured during the whole incubation period (for
at least 20 min).
2
1. Gillard, J.; Abraham, A.; Anderson, P. C.; Beaulieu, P. L.;
Bogri, T.; Bousquet, Y.; Grenier, L.; Guse, Y.; Lavell e´ e, P.
J. Org. Chem. 1996, 61, 2226.
Acknowledgments
2
2. Skiles, J. W.; Giannousis, P. P.; Fales, K. R. Bioorg. Med.
Chem. Lett. 1996, 6, 963.
This work was supported by a grant from the European
Commission (project NOMATEC). Mass and NMR
spectra were recorded at the ‘Centro Interdipartimentale
di Analisi Strumentale’, Universit a` di Napoli ‘Federico
II’.
23. Foley, L. H.; Habgood, G. J.; Gallagher, K. S. Mag. Res.
Chem. 1988, 26, 1037.
2
2
2
2
4. Agami, C.; Couty, F.; Poursoulis, M.; Vaissermann, J.
Tetrahedron 1992, 48, 431.
5. Jones, G. P.; Naidu, B. P.; Paleg, L. G.; Tienik, E. R. T.
Acta Cryst. 1988, C44, 2208.
6. Jones, G. P.; Naidu, B. P.; Paleg, L. G.; Tienik, E. R. T.;
Snow, M. R. Phytochemistry 1987, 26, 3343.
7. (a) Kueng, A.; Trier, G. Z. Physiol. Chem. 1913, 85, 209;
Supplementary data
(
79, 185.
b) Patchett, A. A.; Witkop, B. J. Am. Chem. Soc. 1957,
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.2007.
28. Bringmann, G.; Lang, G.; M u¨ hlbacher, J.; Schaumann,
K.; Steffens, S.; Rytik, P. G.; Hentschel, U.; Morschh a¨ -
0
5.074.

A. Aiello et al. / Bioorg. Med. Chem. 15 (2007) 5877–5887
5887
user, J.; Brun, R.; M u¨ ller, W. E. G. In Marine Molecular
Biotechnology; M u¨ ller, W. E. G., Ed.; Springer-Press:
Berlin, 2003; p 231.
42. Stys, P. K. Curr. Mol. Med. 2004, 4, 113.
43. Scudiero, D. A.; Shoemaker, R. H.; Paull, K. D.; Monks,
A.; Tierney, S.; Nofziger, T. H.; Currens, M. J.; Seniff, D.;
Boyd, M. R. Cancer Res. 1988, 48, 4827.
44. Bickmeyer, U.; Drechsler, C.; Kock, M.; Assmann, M.
Toxicon 2004, 44, 45.
2
9. Kobayashi, J.; Kanda, F.; Ishibashi, M.; Shigemori, H.
J. Org. Chem. 1991, 56, 4574.
0. European Patent Application No. 94302770.6.
1. Johnson, K. W.; Phebus, L. A.; Cohen, M. L. Prog. Drug
Res. 1998, 51, 219.
3
3
45. Kobayashi, J.; Ohizumi, Y.; Nakamura, H.; Hirata, Y.
Experientia 1986, 42, 1176.
3
2. Goodnick, P. J.; Goldstein, B. J. J. Psychopharmacol.
1
46. Rosa, R.; Silva, W.; de Motta, G. E.; Rodr ´ı guez, A. D.;
Morales, J. J.; Ortiz, M. Experientia 1992, 48, 885.
47. Esterle, T. M.; Sanders-Bush, E. J. Neurosci. 1992, 12,
4775.
998, 12(Suppl. B), 5.
33. Hollister, L. E. J. Clin. Psychopharmacol. 1994, 14, 50.
4. Love, R. C.; Nelson, M. W. Expert. Opin. Pharmacother.
3
2
000, 1, 1441.
48. Freshney, R. I. In Culture of Animal Cells. A Manual of
Basic Technique; Freshney, R. I., Ed.; Alan R. Liss Inc.:
New York, 1987; p 257.
3
5. Ikeguchi, K.; Kuroda, A. Eur. Arch. Psychiatry Clin.
Neurosci. 1995, 244, 320.
3
6. Boess, F. G.; Martin, I. L. Neuropharmacology 1994, 33,
49. Perovic, S.; Schleger, C.; Pergande, G.; Iskric, S.; Ushij-
ima, H.; Rytik, P.; M u¨ ller, W. E. G. Eur. J. Pharmacol.
(Mol. Pharmacol. Sec.) 1994, 288, 27.
2
7. Zifa, E.; Fillion, G. Pharmacol. Rev. 1992, 44, 401.
75.
3
3
8. Passchier, J.; van Waarde, A. Eur. J. Nucl. Med. 2001, 28,
50. M u¨ ller, W. E. G.; Wiens, M.; Batel, R.; Steffen, R.;
Borojevic, R.; Custodio, M. R. Marine Ecol. Progr. Ser.
1999, 178, 205.
51. Rottmann, M.; Schr o¨ der, H. C.; Gramzow, M.; Rennei-
sen, K.; Kurelec, B.; Dorn, A.; Friese, U.; M u¨ ller, W. E.
G. EMBO J. 1987, 6, 3939.
1
13.
9. Lindley, C.; Blower, P. Am. J. Health Syst. Pharm. 2000,
7, 1685.
3
4
4
5
0. Homma, K.; Kitamura, Y.; Ogawa, H.; Oka, K.
J. Neurosci. Res. 2006, 84, 316.
1. Takenouchi, T.; Munekata, E. Neurosci. Lett. 1998, 246,
52. Grynkiewicz, G.; Poenie, M.; Tsien, R. Y. J. Biol. Chem.
1985, 260, 3440.
1
41.