Daminin, a bioactive pyrrole alkaloid from the Mediterranean sponge Axinella damicornis

Article abstract of DOI:10.1016/j.tet.2005.05.025

The isolation and characterization of the known pyrrole alkaloid agelongine (6) and of the new natural product daminin (7), the bromine-free analogue of 6, from a specimen of the marine sponge Axinella damicornis is described. Compound 7 showed significant neuroprotective properties. Moreover, for the supply of sufficient material for future medicinal investigations, a short total synthesis of 7 was developed.

Full text of DOI:10.1016/j.tet.2005.05.025

Tetrahedron 61 (2005) 7266–7270
Daminin, a bioactive pyrrole alkaloid from the Mediterranean
sponge Axinella damicornis
a
Anna Aiello,a Monica D’Esposito,a Ernesto Fattorusso,a, Marialuisa Menna,
*
Werner E. G. Mu¨ller,b, Sanja Perovic-Ottstadt, Hideyuki Tsuruta, Tobias A. M. Gulder
b
c
c
*
´
and Gerhard Bringmannc,
*
a
`
Dipartimento di Chimica delle Sostanze Naturali, Universita di Napoli ‘Federico II’, via D. Montesano 49, I-80131 Napoli, Italy
b
¨
Institut fur Physiologische Chemie, Abt. Angewandte Molekularbiologie, Duesbergweg 6, D-55099 Mainz, Germany
c
¨
¨
¨
¨
Institut fur Organische Chemie, Universitat Wurzburg, Am Hubland, D-97070 Wurzburg, Germany
Received 4 March 2005; revised 2 May 2005; accepted 11 May 2005
Available online 13 June 2005
Abstract—The isolation and characterization of the known pyrrole alkaloid agelongine (6) and of the new natural product daminin (7), the
bromine-free analogue of 6, from a specimen of the marine sponge Axinella damicornis is described. Compound 7 showed significant
neuroprotective properties. Moreover, for the supply of sufficient material for future medicinal investigations, a short total synthesis of 7 was
developed.
q 2005 Published by Elsevier Ltd.
1. Introduction
Hymeniacidon sp.15 and agelongine (6), from the sponge
Agelas longissima,16 have extended the structural variety of
the so far known pyrrole alkaloids in having a 3,4,5,6-
tetrahydropyrimidine and a pyridinium ring, respectively,
instead of the commonly found imidazole nucleus, and an
ester linkage that replaces the usual amidic bond in the
central segment. Agelongine (6) exhibits antiserotonergic
activity on rat stomach fundus strip.
Marine sponges are rich sources of pyrrole alkaloids.1 These
secondary metabolites are of interest because of their
structural variety and pharmacological activities. Most of
them are characterized by the presence of a short linear
aliphatic segment connecting a pyrrole-2-carboxylic acid
moiety equipped with different bromine substitution
patterns, with a heterocyclic ring, frequently an amino-
imidazole unit (Fig. 1). The first member of this group to be
discovered, initially in Agelas oroides in 1971 and then in
various other sponges, was the dibrominated compound
oroidin (1).2 This alkaloid 1 shows antimicrobial activity
and interacts with muscarinic acetylcholine receptors
(mAChR) in rat brain membranes.3 In the meantime,
numerous similar alkaloids have been isolated from
Agelasidae, Hymeniacidonidae, and Axinellidae species,4–9
with diverse structures and interesting biological properties
ranging from antiserotonergic and antihistaminic activities
to inhibitory effects against EGF receptor kinase.3,10–14
During oursearchforbioactive substances fromMediterranean
sponges in the frame of the NOMATEC (BIOTECmarin)
project, which aims at the sustainable development of the
Mediterranean resources, we have now chemically investi-
gated the organic extract of a specimen of Axinella
damicornis. This study led to the renewed isolation of
agelongine (6), together with the identification of a new
alkaloid, daminin (7), the first naturally occurring
agelongine analogue. In vitro tests on rat cortical cell
cultures showed that daminin (7) might represent a new
therapeutic tool for the treatment of CNS diseases such as
Parkinson’s and Alzheimer’s diseases.17
More recently, manzacidins A–D (2–5), from
a
2. Results and discussions
Keywords: Marine natural products; Sponges; Pyrrole alkaloids; Total
synthesis.
2.1. Isolation and structural elucidation of daminin (7)
*
Corresponding authors. Tel.: C39 81 678503 (E.F.); tel.: C49 61 31
3925910; fax: C49 61 31 3925243 (W.M.); tel.: C49 931 888 5323;
fax: C49 931 888 4755 (G.B.); e-mail addresses: fattoru@unima.it;
wmueller@uni-mainz.de; bringman@chemie.uni-wuerzburg.de
Specimens of the sponge Axinella damicornis collected in
the Bay of Calvi (Corsica) were extracted with methanol
0040–4020/$ - see front matter q 2005 Published by Elsevier Ltd.
doi:10.1016/j.tet.2005.05.025

A. Aiello et al. / Tetrahedron 61 (2005) 7266–7270
7267
Figure 1. Structures of known pyrrole alkaloids (1–6) isolated from marine sponges and of the new natural product daminin (7).
and, subsequently, with chloroform. The combined extracts
were concentrated in vacuo and partitioned between butanol
and water. The butanol soluble material was initially
subjected to a medium pressure liquid chromatography
(MPLC) using a reversed-phase C-18 column. Further
separation of the alkaloid-containing fractions was achieved
by repeated preparative high performance liquid chroma-
tography (HPLC) on an RP-18 column, thus giving the
known agelongine (6, 3 mg) and the new compound
daminin (7, 4 mg), both in a pure form. Compound 6 was
identified by comparison of its spectral properties with those
reported for agelongine (6) isolated from the sponge Agelas
longissima.16
The 13C NMR spectrum also contained seven sp2 methines
and two sp2 unprotonated carbons, which indicated the
presence of aromatic rings, and furthermore two sp3
methylene signals. The 1H NMR spectrum (DMSO-d6)
exhibited ten well separated signals. The lowfield region of
the spectrum contained seven signals of aromatic protons
and one signal exchangeable with D2O, while two mutually
coupled methylene signals were visible in the central region
of the spectrum (see Table 1). Analysis of the NMR spectra,
including 2D COSY, ROESY, HSQC, and HMBC, clearly
showed 7 to be a close structural analogue of agelongine (6).
In fact, the spectral data obtained for 7 revealed it to contain
the same pyridinium-b-carboxylate moiety as agelongine
(6) linked through an ethylenoxy bridge to a pyrrole-2-
carboxylic acid connected via an ester bond. The carboxyl-
ate portion of the ester corresponds to the chemical structure
of the known betaine alkaloid pyridinebetaine A.18 The
latter heterocyclic moiety, however, was found to lack a
bromine substituent, as clearly indicated by NMR signals of
a complete set of three neighboring aromatic protons [dH
6.78 (t, JZ0.9; 1.9 Hz), H-3; 6.16 (d, JZ1.9 Hz), H-4; 7.05
(d, JZ0.9 Hz), H-5; dC 120.7, C-2; 115.7, C-3; 109.5, C-4;
124.6, C-5], as well as by mass data (see Section 4). Thus,
daminin (7) was concluded to be the non-brominated parent
compound of agelongine (6). The NMR analysis also
allowed us to assign all of the resonances present in 13C and
The ESI (positive ions) mass spectrum of daminin (7, Fig. 2)
exhibited pseudomolecular ion peaks at m/z 261 [MCH]C
and 283 [MCNa]C. The molecular formula C13H12N2O4
was deduced from the HRFABMS (positive ions) of this
compound, measured on the peak at m/z 261.0890 [MCH]C
(calculated value: m/z 261.0875).
The presence of an aromatic ester function and a
carboxylate group was suggested by IR absorptions at
nmax 1710 and 1646 cmK1, respectively, which was
confirmed by the carbonyl resonances in the 13C NMR
spectrum of 7 (DMSO-d6) at d 161.6 and 159.0 (Table 1).
Figure 2. Synthesis of daminin (7) from the building blocks 8 and 1.

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A. Aiello et al. / Tetrahedron 61 (2005) 7266–7270
Table 1. 1H and 13C NMR data (DMSO-d6) of compound 7
No.
dH (mult., J Hz)
dC
HMBC
COSY
1
2
3
4
5
6
7
8
12.3 (s)
—
6.78 (t, 0.9, 1.9)
6.16 (d, 1.9)
7.05 (d, 0.9)
—
4.65 (t, 3.7)
5.01 (t, 3.7)
9.4 (s)
—
120.7
115.7
109.5
124.6
159.0
61.8
2
4, 5
3, 5
3, 4
2, 5
2, 3, 4
8
7
8
7
58.9
9
145.7
140.9
144.7
126.6
144.0
161.6
8, 10, 11, 14 11, 13
10
11
12
13
14
—
8.78 (d, 7.4)
8.06 (t, 6.5, 7.4)
9.04 (d, 6.5)
—
13, 14
10, 11, 13
11, 12
9, 12
11, 13
9, 12
Figure 3. Incubation of the neurons with L-Glu and Ca2C in the absence
(B) or in the presence of 0.5 (:), 1 (,), or 3 (>) mg/mL of daminin (7).
Daminin (7) was added at time 5 min and remained in the cultures during
the entire incubation period. At time 10 min, neurons were stimulated with
L-Glu and CaCl2 as described in Experimental. The results are expressed as
mean value (nZ35) plus standard deviation (GSE).
1H NMR spectra, which are reported in Table 1;
particularly, the assignments of the pyrrole moiety were in
good agreement with those reported for 2-substituted
pyrroles.8,19
on the [Ca2C]i was measured (Fig. 3). In a parallel series of
experiments it could be shown that daminin (7) caused no
effect on the [Ca2C]i level (not presented).
2.2. Synthesis of daminin (7)
A synthetic access (Fig. 2) to the new compound daminin
(7) was realized by esterification of 1-(2-hydroxyethyl)-3-
methoxycarbonyl-pyridinium bromide (10) with pyrrole-2-
carboxylic acid chloride (12). N-alkylation of nicotinic acid
methyl ester (8) using 2-bromoethanol (9) delivered the
pyridinium salt 10 in 88% yield. The acid chloride 12 was
prepared from pyrrole-2-carboxylic acid (11) and oxalyl
chloride under basic conditions. In situ reaction of 12 with
10 and selective cleavage of the more reactive methyl ester
on the electron-poor pyridinium portion of the crude
intermediate 13 using lithium iodide gave daminin (7) in
48% yield, identical in all chromatographic and spectro-
scopic respects with the natural product from A. damicornis.
2.3.3. Modulating effect of daminin (7) on the NMDA-
caused [Ca2C]i level. Incubation of neurons with 200 mM
of NMDA (n-methyl-D-aspartate) and 2.4 mM CaCl2 (here
added at time 10 min) resulted in a strong increase in
[Ca2C]i; the 340/380 nm ratio value increased by 1.117
(235%). This strong rise was statistically reduced to 63.5%
(controls were set to 100) if the cells were pre-incubated
with 1 mg/mL of daminin (7); Figure 4.
2.3. Bioactivities of daminin (7)
2.3.1. Cytostatic/cytotoxic activity of daminin (7).
Daminin (7) was tested for its cytotostatic/cytotoxic activity
against several tumor cell lines, namely murine leukemic
lymphoblasts L5178y, rat adrenal pheochromocytoma PC12
cells, and human cervix carcinoma HeLa S3 cells. It
displayed no cytotoxic activity against all tested cell lines.
In all cases, the IC50 was O40 mg/mL.
2.3.2. Influence of L-Glu and CaCl2 on the [Ca2C]i level
in neuronal cells after pre-incubation with daminin (7).
Incubation of neurons with 200 mM of L-Glu (glutamic acid)
and 2.4 mM CaCl2 resulted in a strong rise in [Ca2C]i. If
these components were added 10 min after starting the
experiment, the 340/380 nm ratio value increased by 1.537
(305%, Fig. 3). However, if the neurons were pre-incubated
for 5 min with 0.5, 1.0 or 3.0 mg/mL of daminin (7) a
significant decrease of the [Ca2C]i levels was recorded; after
Figure 4. Incubation of the neurons with 200 mM of NMDA and 2.4 mM
Ca2C in the absence (%) or in the presence of 1 (,) mg/mL of 7. Daminin
(7) was added at time 5 min and remained in the cultures, while NMDA/
Ca2C was added at time 10 min.
3. Conclusion
addition of L-Glu and CaCl2 at time 10 min, the [Ca2C
]
i
Daminin (7) is one of the relatively few non-brominated
pyrrole alkaloids of marine origin. The promising neuro-
protective activities of this natural product combined with
its very low cytotoxicity and its easy synthetic access
presented in this paper make further investigations con-
cerning possible medicinal uses potentially promising.
level dropped to 58.1% (0.5 mg/mL of 7), to 65.4% (1 mg/
mL of 7) or to 25.1% (3 mg/mL of 7, Fig. 3). Daminin (7)
was present from time 5 min up to the end of the
experiments (30 min). In the pre-incubation set of experi-
ments neurons were incubated 5–10 min only in the
presence of 7 and in the absence of L-Glu/CaCl2. No effect

A. Aiello et al. / Tetrahedron 61 (2005) 7266–7270
7269
4. Experimental
4.1. General aspects
reduced pressure and the residue was washed 3 times with
15 mL diethyl ether. The solid was recrystallized from
acetonitrile–diethyl ether (1:2) to give 1.17 g (6.41 mmol,
88%) pure 10 as colorless crystals.
ESI mass spectra (positive ions) were performed on an API
2000 mass spectrometer. High resolution FAB mass spectra
(glycerol matrix) were taken on a VG Prospec (FISONS)
mass spectrometer. NMR experiments were done on a
Bruker AMX-500 and AV-400 spectrometers; chemical
shifts are referred to the residual solvent signal (DMSO:
dHZ2.49 ppm, dCZ39.5 ppm). Homonuclear (1H–1H) and
heteronuclear (1H–13C) connectivities were determined by
COSY and HSQC experiments, respectively. Two- and
three-bond 1H–13C connectivities were investigated by
HMBC experiments optimized for a 2,3J of 10 Hz.
Separations by medium-pressure liquid chromatography
(MPLC) were performed on a Bu¨chi 861 apparatus with
SiO2 (230–400 mesh) packed columns. High performance
liquid chromatography (HPLC) separations were achieved
on a Waters 501 apparatus equipped with an RI detector. IR
(KBr) spectra were recorded on a Bruker model IFS-48
spectrophotometer.
Mp 108–109 8C (acetonitrile–diethyl ether); 1H NMR
(400 MHz, CD3OD) d 4.07 (t, JZ5.0 Hz, 2H), 4.09 (s,
3H), 4.89 (t, JZ5.0 Hz, 2H), 8.31 (dd, JZ8.2, 6.1 Hz, 1H),
9.12 (ddd, JZ8.2, 1.4, 1.4 Hz, 1H), 9.25 (ddd, JZ6.1, 1.4,
1.4 Hz, 1H), 9.57 (bs, 1H); 13C NMR (100 MHz, CD3OD) d
57.0, 64.3, 68.2, 132.1, 134.7, 149.6, 150.4, 152.2, 166.1.
FABMS m/z 182.1 [MCH-Br]C, 445.1 [2MCH-Br]C.
4.3.2. Synthesis of daminin (7). A suspension of 230 mg
(2.07 mmol) pyrrole-2-carboxylic acid potassium salt (11)
(Aldrich, Germany) and 0.25 mL (2.98 mmol) oxalyl
chloride in 5 mL CH2Cl2 was stirred at room temperature
under N2. After 4 h the solvent was removed under reduced
pressure and the residue was re-dissolved in 5 mL CH2Cl2.
262 mg (1.43 mmol) of 10 were added together with a
catalytic amount of 4-N,N-dimethylaminopyridine and
the mixture was stirred over night at room temperature.
The solvent was removed under reduced pressure and the
remaining solid washed with diethyl ether and then
dissolved in 4 mL of pyridine. After addition of 900 mg
(6.72 mmol) of lithium iodide, the mixture was stirred at
100 8C for 3 h. The solvent was evaporated and the
remaining solid was purified by chromatography on a silica
gel column with ethyl acetate–MeOH (2:8) as the eluent.
After recrystallization of the crude product from EtOH–
diethylether (2:1), 177 mg (0.68 mmol, 48%) of pure
daminin (7) were obtained.
4.2. Collection, extraction, and isolation
Specimens of the sponge Axinella damicornis were
collected in November 2001 in the Bay of Calvi (Corsica)
and kept frozen until use. For the extraction, the fresh
thawed sponge (75.2 g dry weight after extraction) was
homogenized and treated at room temperature with
methanol (3!600 mL) and, subsequently, with chloroform
(3!600 mL). The extracts were concentrated in vacuo and
the water-insoluble lipid fraction obtained was treated with
BuOH. The BuOH soluble portion was subjected to MPLC
on a reversed-phase column eluting with H2O/MeOH/
CHCl3. Two main alkaloid-containing fractions, eluted with
MeOH/H2O 3:7 (fraction A) and MeOH/H2O 2:8 (fraction
B), were obtained. The more polar fraction A was separated
by HPLC on an RP-18 column (Luna, 5 mm, 250!4.6 mm)
using MeOH/H2O 3:7 as the eluent and further purified by
HPLC on an C18 column (AQUA, 5 mm, 250!4.6 mm)
thus providing 7 (4 mg) in a pure form. Fraction B was
separated and purified by consecutive HPLC runs on RP-18
columns (Luna, 5 mm, 250!4.6 mm; Luna, 3 mm, 250!
4.6 mm) with MeOH/H2O 1:1 as the eluent, giving rise to
pure 6 (3 mg).
Mp 193 8C (decomposition); all spectral data identical with
those of the natural product 7 (see above).
4.4. Cell lines
L5178y cells (ATTCC CRL-1722) were grown in
RPMI1640 supplemented with 10 mM Hepes, 10% (v/v)
¨
fetal calf serum (FCS) (PAA, Colbe, Germany) and 0.1%
(w/v) Gentamycin. PC12 cells (ATCC CRL-1573) were
kept in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% (v/v) horse serum, 5% (v/v) FCS
and 0.1% (w/v) of gentamycin. HeLa S3 cells (ATCC CCL-
2.2) were cultured in DMEM supplemented with 10% (v/v)
FCS. All cells were routinely passaged once or twice
weekly.
4.2.1. Daminin (7). Amorphous white solid. IR (KBr): nmax
1710, 1646 cmK1. HRFABMS m/z [MCH]C261.0890
(Calcd for C13H12N2O4, 261.0875). 1H (500 MHz,
DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) data:
Table 1.
4.5. MTT assay
The cell viability was determined using MTT assay.20 The
evaluation was performed in 96-well plates at 595 nm using
an ELISA plate reader.
4.2.2. Agelongine (6). The data fully matched those
reported in the literature.16
4.6. Calcium measurement on primary neurons
4.3. Total synthesis of daminin (7)
The primary cortical cell culture was prepared from 17 to 18
days old rat embryos following the modified procedure.21–23
Daminin (7) was dissolved in sterile water (stock concen-
tration of 10 mg/mL) and stored at 4 8C. Neurons were
exposed to 200 mM of L-glutamic acid (L-Glu) or N-methyl-
D-aspartic acid (NMDA), both compounds together with
4.3.1. Synthesis of 1-(2-hydroxyethyl)-3-methoxycar-
bonylpyridinium bromide (10). A solution of 1.00 g
(7.29 mmol) nicotinic acid methyl ester (8) and 5 mL
(70.82 mmol) 2-bromoethanol (9) in 7 mL of toluene was
stirred at 120 8C for 2.5 h. The solvent was removed under

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9. Uemoto, H.; Tsuda, M.; Kobayashi, J. J. Nat. Prod. 1999, 62,
1581–1583.
2.4 mM CaCl2, 10 min after the beginning of the recording.
The [Ca2C]i level was measured for 20 (experiment with
NMDA) or 30 min (L-Glu). Compound 7 was added to the
cultures 5 min prior to the stimulation with L-Glu/CaCl2 or
NMDA/CaCl2.
10. Nakamura, H.; Ohizumi, Y.; Kobayashi, J.; Kasei, M.
Tetrahedron Lett. 1984, 25, 2475–2478.
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¨
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This work was supported by the German Bundes-
ministerium fu¨r Bildung, Wissenschaft und Forschung
(BMBF, project BIOTECmarin nos. 03F0345E) and by
the Fonds der Chemischen Industrie.
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