Bromopyrrole alkaloids isolated from the patagonian bryozoan aspidostoma giganteum

Article abstract of DOI:10.1021/np500012y

Nine new bromopyrrole alkaloids, aspidostomides A-H and aspidazide A (1-9), were isolated from the Patagonian bryozoan Aspidostoma giganteum. Aspidostomides A-H have dibromotyrosine- or bromotryptophan-derived moieties forming either linear amides or pyrroloketopiperazine-type lactams with a bromopyrrole carboxylic acid as a common structural motif. On the other hand, aspidazide A is a rare asymmetric acyl azide formed by an N-N link of two different pyrroloketopiperazine lactams and is the first isolated compound of this class from marine invertebrates. This work is the first report of secondary metabolites isolated from a bryozoan from the Patagonian region. The structures of compounds 1-9 were elucidated by spectroscopic methods and chemical transformations. One of these compounds, aspidostomide E (5), was moderately active against the 786-O renal carcinoma cell line.

Full text of DOI:10.1021/np500012y

Article
pubs.acs.org/jnp
Bromopyrrole Alkaloids Isolated from the Patagonian Bryozoan
Aspidostoma giganteum
Laura P. Patino C,† Claudia Muniain,‡ Maria Elena Knott,§ Lydia Puricelli,§ and Jorge A. Palermo*,†
̃
†UMYMFOR, Departamento de Química Organ
́
ica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad
2 (1428), Buenos Aires, Argentina
‡CONICET, Laboratorio de Ecología Química y Biodiversidad Acuat
Universitaria, Pabellon
́
́
́
ica, Instituto de Investigacion e Ingeniería Ambiental,
Universidad de San Martín, Peatonal Belgrano 3563 (1650), San Martín, Buenos Aires, Argentina
§Research Area, “Angel H. Roffo” Institute of Oncology, University of Buenos Aires, Avenida San Martín 5481 (C1417DTB), Buenos
Aires, Argentina
S
* Supporting Information
ABSTRACT: Nine new bromopyrrole alkaloids, aspidosto-
mides A−H and aspidazide A (1−9), were isolated from the
Patagonian bryozoan Aspidostoma giganteum. Aspidostomides
A−H have dibromotyrosine- or bromotryptophan-derived
moieties forming either linear amides or pyrroloketopiper-
azine-type lactams with a bromopyrrole carboxylic acid as a
common structural motif. On the other hand, aspidazide A is a
rare asymmetric acyl azide formed by an N−N link of two
different pyrroloketopiperazine lactams and is the first isolated
compound of this class from marine invertebrates. This work is
the first report of secondary metabolites isolated from a
bryozoan from the Patagonian region. The structures of compounds 1−9 were elucidated by spectroscopic methods and
chemical transformations. One of these compounds, aspidostomide E (5), was moderately active against the 786-O renal
carcinoma cell line.
ryozoans have emerged over the last decades as a source of
derived from either bromotryptophan or bromotyrosine,
Bbiologically active and structurally diverse compounds, aspidostomides A−H (1−8), together with aspidazide A (9),
including a wide array of alkaloids.1−7 Many species belonging
and 9-O-ethyl aspidostomide C (10), which is probably an
artifact, plus three brominated small aromatic compounds that
are biosynthetically related to the above-mentioned substances.
The structures of the isolated compounds were elucidated by
HRMS, NMR techniques, and chemical transformations. One
of these compounds, aspidostomide E (5), was moderately
active against the 786-O renal carcinoma cell line. The present
work is the first report on the chemistry of a bryozoan from the
Patagonian region.
to the order Cheilostomata, especially from the Bugulidae and
Flustridae families, have been the focus of extensive studies by
chemists. These organisms have provided a great variety of
secondary metabolites, among them the large family of the
bryostatins8 from the Bugulidae family and the ubiquitous
brominated physostigmine alkaloids from Flustridae.9−11
However, there are no previous reports of secondary
metabolites from Aspidostomatidae bryozoans. In particular,
the genus Aspidostoma comprises 27 species, and among them,
Aspidostoma giganteum (Busk, 1854) has a wide distribution
along the Magellanic and Antarctic regions, with records in
Patagonia, Tierra del Fuego, Malvinas, and the South Shetland
Islands.12−14
As part of a research program on bioactive secondary
metabolites from South Atlantic marine invertebrates, an
investigation was conducted on the associated epibenthos
that form the by-catch of commercial Patagonian fisheries.
From an associated fauna sample, collected by trawling (60−
100 m) at the fishing grounds of the prawn Pleoticus muelleri
(Bate, 1888) located in the Gulf of San Jorge (Patagonia,
Argentina), two colonies of A. giganteum were obtained. A
detailed analysis of their organic extract resulted in the isolation
and structure elucidation of a series of bromopyrrole alkaloids
RESULTS AND DISCUSSION
■
The ethanolic extract of A. giganteum was fractionated by silica
gel column chromatography, Sephadex LH20 permeation, and
RP-18 HPLC. Further purification of the major components
yielded pure compounds 1−10. Compound 1 was isolated as a
light yellow, amorphous solid. HRESIMS of 1 showed an
isotopic pattern corresponding to four bromine atoms, and a
molecular formula of C13H9Br4N2O3 was established, with eight
degrees of unsaturation. The 1H NMR spectrum of 1 (Table 1)
showed eight signals, while the 13C NMR spectrum had only 11
signals, which could indicate the presence of two pairs of
Received: January 6, 2014
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Chart 1
a
1
Table 1. H NMR Spectroscopic Data (500.13 MHz; CDCl3, CD3COCD3) of Compounds 1−3, 8, and 10
position
NH-1
1 (δH, J in Hz)
9.82, bs
2 (δH, J in Hz)
8.03, bs
3 (δH, J in Hz)
10.10, bs
8 (δH, J in Hz)
10 (δH, J in Hz)
10.50, bs
10.10, bs
2
7.01, d (3.3)
4
6.96, s
6.86, s
NH-7
8
7.29, bt (7.0)
7.24, bt (7.9)
7.25, bt (6.0)
7.31, d (8.7)
6.45, d (8.7)
7.35, bdd (7.0, 3.5)
3.91, ddd (14.0, 7.0, 3.9)
3.34, ddd (14.0, 7.5, 4.0)
4.33, dd (7.5, 3.9)
7.45, s
3.82, ddd (14.0, 7.0, 3.4)
3.54, ddd (14.0, 7.4, 3.5)
4.86, dd (7.4, 3.4)
7.52, s
3.62, ddd (14.0, 7.9, 5.0)
3.43, ddd (14.0, 7.5, 4.8)
4.85, dd (7.5, 5.0)
7.59, d (0.6)
3.87, ddd (13.7, 6.0, 3.6)
3.56, ddd (13.7, 7.7, 3.8)
4.89, dd (7.7, 3.6)
7.55, s
9
11
8.08, dd (8.7, 2.1)
7.00, d (8.7)
14
15
7.52, s
7.59, d (0.6)
5.85, bs
7.55, s
8.31, d (2.1)
7.45, s
OH
5.93, bs
5.94, bs
5.93, bs
C-13 OCH3
C-8 OCH3
O-CH2CH3
O-CH2-CH3
4.02, s
3.51, s
3.50, 3.38 (m)
1.23, t (7.0)
a
Solvent used for spectra of compound 2.
equivalent carbons. The presence of a 2H singlet at δH 7.52,
which correlated (DEPT-HSQC) to an intense carbon signal at
δC 129.6, strongly favored this assumption and suggested a
possible symmetric aromatic ring. Nine of the 13C signals
corresponded to sp2 carbons, one of them (δC 160.2) probably
an amide carbonyl, while the two remaining signals belonged to
an oxo-methine and a methylene. The presence of a second
aromatic system was suggested by a doublet at 7.01 ppm (J =
3.3 Hz, 1H), which had a different set of HMBC correlations
than the protons at δH 7.52. The DEPT-HSQC spectrum
indicated the presence of three exchangeable protons: two
broad singlets at 9.82 and 5.93 ppm and a broad triplet at 7.29
ppm. COSY correlations established a two-carbon side chain
formed by the methylene (3.82 and 3.54 ppm) and the oxo-
methine at 4.86 ppm (δC 72.2), which ended in an amide (δH
7.29, bt). HMBC correlations between the methylene protons
and the amide carbonyl (δC 160.2) confirmed this substructure.
The equivalent aromatic protons at δH 7.52 had HMBC
correlations with three quaternary aromatic carbons at 149.1,
136.2, and 110.1 ppm. The relative intensity of the last signal
suggested that this was the other duplicated carbon, while the
signal at δC 149.1 was consistent with a phenolic moiety, thus
accounting for the third oxygen atom required by the molecular
formula. Taking into consideration the number of bromine
atoms, this symmetric benzene ring is presumably derived from
3,5-dibromotyrosine, a frequent structural motif in many
B
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marine natural products.15 Comparison of the NMR values
with literature data confirmed this assumption.16 An additional
HMBC correlation of the aromatic protons at δH 7.52 with the
oxo-methine indicated that the aliphatic side chain was linked
to C-1 of the aromatic ring (C-10). Up to this point, the
remaining portion of the molecule to be assigned was an
additional aromatic C4H2Br2N system with three unsaturations,
and a dibromopyrrole unit was the obvious choice. A COSY
correlation between the pyrrole aromatic proton (7.01 ppm, J =
3.3 Hz) and the exchangeable proton at 9.82 ppm established
that the aromatic proton was vicinal to the pyrrole nitrogen,
while an HMBC correlation between δH 9.82 and δC 160.2
linked the carbonyl to C-5 of the pyrrole, and in this way C-3
and C-4 were assigned as the brominated positions on the
pyrrole. The absolute configuration at the carbinolic position
was determined as R by the modified Mosher’s method.17 In
this way the structure of compound 1 was established as a 3,5-
dibromooctopamine derivative, for which the name aspidosto-
mide A was coined. The numbering system adopted for this
and the following compounds was based on the related
bromopyrrole alkaloid oroidin.
typical AMX aromatic system was evident (δH 7.23, d, J = 8.6
Hz, δH 7.12, dd, J = 8.6, 1.6 Hz, and δH 6.91, bs), while the
characteristic aliphatic system of compounds 1−3 was replaced
1
by two broad H singlets at 5.68 and 5.22 ppm (Table 2).
1
Table 2. H NMR Spectroscopic Data (500.13 MHz;
CD3COCD3) of Compounds 4−7
4 (δH, J in
5 (δH, J in
position
Hz)
Hz)
6 (δH, J in Hz) 7 (δH, J in Hz)
NH-1 4
NH-7
8
11.5, bs 6.68, s
7.68, d (4.5)
8.18, d (5.1)
10.1, s
7.48, s
5.22, bd (4.5) 4.96, dd (5.1, 6.79, bs
1.5)
3.50, dd (13.5,
6.7)
9
5.68, bs
5.83, bd (1.5)
11.50, bs
2.94, t (6.7)
10.55, bs
NH-12
14
11.5, bs
11.6, bs
7.23, d (8.6)
7.40, d (8.7)
7.45, d (8.70) 6.90, d (1.5)
15
7.12, dd (8.6, 7.28, dd (8.7, 7.35, dd
1.6)
1.8)
(8.70,1.8)
16
6.53, d (1.5)
9.11, bs
17
6.91, bs
7.01, d (1.8)
3.49, s
7.70, d (1.75)
OH
OCH3
These two protons had HSQC correlations with 13C signals at
59.8 and 78.1 ppm, respectively, clearly indicating that the latter
corresponded to an oxidized position. On the other hand, the
deshielding of the signal at 5.68 ppm was probably explained by
an N-substitution in combination with anisotropic effects. Two
exchangeable signals were also observed at 7.68 (d, J = 4.5 Hz)
and 11.5 ppm (bs), and all these data gave the idea of a
different core for compound 4 from that of 1−3. Sequential
1H−1H couplings were observed in the COSY spectrum
between δH 5.68, δH 5.22, and the exchangeable signal at δH
7.68, which was assigned to an amide NH. An HMBC
correlation between δH 5.22 and an amide carbonyl at δC 156.8
confirmed this substructure (Figure 2).
Figure 1. COSY () and HMBC correlations of 1 and NOESY
correlations of 2.
Aspidostomide B (2) had the same molecular formula
(C13H10Br4N2O3) as aspidostomide A (1), and the only
observable difference in the 1H and 13C NMR spectra
corresponded to the pyrrole proton (δH 6.96, s, δC 113.2),
which in this case was a singlet. This multiplicity, together with
the chemical shifts, suggested that this proton was located at
either C-3 or C-4 of the pyrrole ring. An HMBC correlation
with the amide carbonyl, as well as a NOESY correlation with
the amide proton, placed the pyrrole proton at C-4. The
remaining 2D NMR correlations confirmed that the backbones
of compounds 1 and 2 were identical.
Figure 2. COSY () and HMBC correlations of 4 and NOESY
correlations of 4a.
By analysis of the HMBC correlations of the three aromatic
protons in the AMX system, it was observed that all three
correlated with the same two quaternary carbons (135.2 and
113.6 ppm). The proton corresponding to the ortho doublet at
7.23 ppm had two additional correlations to other quaternary
carbons (126.9 and 108.0 ppm). In particular the quaternary
carbons at 126.9 and 135.2 ppm had the typical chemical shifts
of C-3a and C-7a positions in a C-3-substituted indole
system.18 The exchangeable proton at 11.5 ppm resembled a
typical indole NH, reinforcing this idea; however, no HMBC
correlations were observed for this broad signal, which could
confirm this hypothesis. As positions C-11 and C-16 (which
correspond to C-2 and C-5 of the probable indole core) had to
be substituted, two bromine atoms were assigned, out of the
five bromines required by the molecular formula. In turn, the
The molecular formula C13H9Br5N2O3 was obtained by
HRESIMS for aspidostomide C (3), and in this case an isotope
pattern for five bromines was clearly observed. The absence in
1
the H NMR spectrum of the signal corresponding to the
pyrrole proton, together with the replacement of a hydrogen
atom by a bromine in the molecular formula compared with
compounds 1 and 2, suggested the presence of a fully
brominated pyrrole ring in 3, which was confirmed by analysis
of the 2D NMR spectra. The absolute configurations of
compounds 2 and 3 were assumed to be the same as that of 1.
Aspidostomides A−C were C13 compounds; however, the
molecular formula C15H8Br5N3O2 was obtained for compound
4, indicative of 11 degrees of unsaturation. Another distinctive
1
feature of 4 was a different pattern in the H NMR signals: a
C
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methine at δH 5.68 correlated in the HMBC spectrum with two
quaternary carbons of the indole (C-11 and C-18) plus two
additional quaternary carbons (δC 123.6 and 107.9), which
probably belonged to another aromatic system. A C4Br3N
fragment and four unsaturations still needed to be assigned. On
the basis of the previously isolated aspidostomides, a
tribromopyrrole structure was highly possible. The absence of
the pyrrole NH, together with the chemical shift of C-9 and H-
9 (δC 59.8 and δH 5.68), strongly favored the closure of an
additional ring between C-9 and the pyrrole nitrogen, forming a
bicyclic tribromopyrroloketopiperazine core, bound to C-10
(which corresponds to position 3 of an indole core), thus
establishing the structure of compound 4, which was named
aspidostomide D. Diagnostic NOESY correlations of H-17 with
H-8 and H-9 supported the proposed structure.
HMBC correlation of this proton with the amide carbonyl. The
structure of compound 6 was thus confirmed as the dehydro
derivative of aspidostomide D (4).
A molecular formula of C15H11Br4N3O2 was obtained for
1
aspidostomide G (7). The H NMR spectrum showed nine
signals, four of them corresponding to exchangeable protons at
7.48, 9.11, 10.55, and 11.50 ppm as observed in the HSQC
spectrum. The five remaining signals belonged to a pair of
methylene groups (2.94 and 3.50 ppm) and three aromatic
protons, two of which were meta-coupled [δH 6.53 (d, J = 1.5
Hz), δH 6.68 (bs), and δH 6.90 (d, J = 1.5 Hz)]. Both meta-
coupled protons correlated in the HMBC spectrum with the
same three quaternary carbons (151.2, 116.2, and 114.9 ppm)
(Figure 3). The signal at δH 6.53 showed an NOE correlation
The structure of compound 4 was finally confirmed by
chemical transformations. As no HMBC correlations were
observed for the indole NH, a methylation reaction was
performed (NaH and MeI in DMSO), to obtain the methylated
derivative 4a.19 In the H NMR spectrum of 4a, two N-Me
1
singlets (δH 3.50, δC 35.0; δH 3.89, δC 32.3) were observed,
indicating that both the lactam and the indole nitrogens were
methylated. The absence of both H-8 and H-9 signals was also
evident, being replaced by the formation of a new trisubstituted
double bond. Analysis of the 2D NMR spectra of 4a confirmed
the formation of an enamide moiety by elimination of the C-8
hydroxy and H-9. The N-methyl group at δH 3.50 showed an
HMBC correlation to the enamide carbonyl (δC 154.0) and a
NOE correlation with the enamide proton (δH 6.41), while an
additional NOE correlation was observed between the enamide
proton and H-17. On the other hand, the N-methyl at δH 3.89
had an HMBC correlation with C-14 (position 7 of the indole
core) and a NOESY correlation with the ortho-coupled doublet
at 7.28 ppm (H-14), thus confirming the substitution by
bromine at C-16. A detailed analysis of the ms2 spectrum on the
quasimolecular ion m/z 659.6496 (Supporting Information)
also confirmed the proposed structure, since a main fragment
ion was observed at m/z 342.7742, which may arise from the
cleavage of the C-8−NH and C-9−N-1 bonds.
Figure 3. HMBC and NOESY correlations of compounds 7 and 8.
with δH 9.11, while δH 6.90 had a NOE with δH 10.55. These
correlations were indicative of an indole ring with a hydroxy
group at C-4. The methylene at 2.94 ppm showed correlations
in the HMBC spectrum not only with the other methylene
(3.50 ppm) but also with C-3, C-2, and C-3a of the indole core.
All of these data indicated substitution by the methylene chain
at C-10 (C-3 of the indole core). Once again, the presence of a
carbonyl carbon at 159.1 ppm and the NH signal at 7.48 ppm
were typical of an amide group. This amide was N-bonded to
the alkyl chain connected to the indole group and also to a
dibromo pyrrole ring. The presence of a singlet at δH 6.68 that
had HMBC correlations with C-5 and C-3 and an NOE
correlation with the amide NH clearly identified the pyrrole
proton as H-4.
The absolute configuration of 4 was determined by the
modified Mosher’s method with negative Δδ (δS − δR) values
for H-14, H-15, and H-17 and a positive Δδ value for the amide
N−H. In this way, the absolute configuration of C-8 was
established as R, while an S configuration was deduced for C-9
based on the NOE correlation and small coupling constant
between H-8 and H-9, which were cofacial on the lactam ring.
Compounds 5 and 6 were closely related to aspidostomide
D. Compound 5, aspidostomide E, had a molecular formula of
C16H9Br5N3O2 obtained by HRESIMS. The major differences
Compound 8 (aspidostomide H) had a molecular formula of
C15H13Br3N2O4, indicative of nine degrees of unsaturation. The
13C NMR spectrum of 8 showed signals corresponding to two
carbonyl carbons: an amide (160.7 ppm) and a conjugated
ketone (189.4 ppm). This was the first compound of the series
1
found in the H and 13C NMR spectra of compound 5 with
1
to present a ketone in its structure. In the H NMR spectrum
respect to aspidostomide D (4) were the presence of signals
corresponding to a methoxy group at δH 3.49 and δC 54.2,
which correlated in the NOESY spectrum with H-8, H-9 (4.96
and 5.83 ppm) and the amide-NH (8.18 ppm), thus confirming
that 5 was the O-methyl derivative of aspidostomide D. On the
other hand, aspidostomide F (6) had a molecular formula of
some characteristic fragments could be identified: an AMX
aromatic system [δH 8.31 (d, J = 2.1 Hz), δH 8.08 (dd, J = 8.7,
2.1 Hz), and δH 7.00 (d, J = 8.7 Hz)], two methoxy groups at
3.51 and 4.02 ppm, together with two exchangeable protons
(δH 7.31, probably an amide, and δH 10.10 ppm). Two
additional methine signals were also observed: one of them (δH
6.45, δC 77.6) was clearly an oxomethine, while the other (δH
6.86, δC 113.7) probably belonged to a bromopyrrole. Analysis
of the COSY spectrum showed a correlation between the amide
proton at 7.31 ppm and the oxomethine at 6.45 ppm. This in
turn had HMBC correlations with the two carbonyls and with a
methoxy group (δH 3.51). In this way, it was clear that the
oxomethine (C-8) had a methoxy substituent and was located
1
C15H5Br5N3O according to the HRESIMS. The H and 13C
NMR spectra showed a signal corresponding to a vinyl proton
at δH 6.79, δC 119.6 and that the signals that originally
corresponded to H-8 and H-9 were no longer present. The
chemical shifts of the vinyl proton and its associated carbon
closely resembled those of the enamide obtained in derivative
4a. The presence of this enamide moiety was confirmed by an
D
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Table 3. 13C NMR Spectroscopic Data (125.13 MHz; CDCl3) of Compounds 1−3, 8, and 10
position
1 (δC)
2 (δC)
3 (δC)
8 (δC)
10 (δC)
2
3
4
5
6
8
9
121.3, CH
100.6, C
101.5, C
122.9, C
160.2, C
47.5, CH2
72.2, CH
136.2, C
129.6, CH
110.1, C
149.1, C
110.1, C
129.6, CH
104.0, C
98.6, C
105.6, C
100.5, C
104.1, C
124.2, C
159.3, C
47.3, CH2
72.0, CH
136.0, C
129.5, CH
110.0, C
149.1, C
110.0, C
129.5, CH
106.8, C
100.4, C
113.7, CH
125.9, C
159.6, C
77.9, CH
189.4, C
127.4, C
134.9, CH
112.4, C
160.7, C
111.3, CH
130.9, CH
56.6, CH3
55.3, CH3
105.4, C
100.1, C
103.8, C
124.6, C
158.2, C
45.7, CH2
78.8, CH
134.2, C
130.1, CH
110.2, C
149.2, C
110.2, C
130.1, CH
113.2, CH
128.3, C
160.1, C
47.4, CH2
70.9, CH
137.9, C
130.0, CH
110.4, C
149.5, C
110.4, C
130.0, CH
10
11
12
13
14
15
C-13 OCH3
C-8 OCH3
O-CH2CH3
O-CH2-CH3
65.0, CH2
15.3, CH3
Table 4. 13C NMR Spectroscopic Data (125.13 MHz;
CD3COCD3) of Compounds 4−7
between the ketone (C-9) and amide groups, which explained
its high δH. As for the aromatic AMX system, two of these
protons were heavily deshielded and showed HMBC
correlations with the ketone group. This could be explained if
the ketone was conjugated with a 3,4-disubstituted aromatic
ring. Both deshielded protons showed HMBC correlations with
a quaternary carbon at δC 160.7, which in turn correlated with
the additional methoxy group (δH 4.02). These data
determined the nature of the substituents on the benzene
ring: a methoxy at position 4 and a bromine at position 3 of the
aromatic ring (C-13 and C-12, respectively). NOE correlations
between the methoxy at δH 4.02 and the aromatic proton at δH
7.00 (H-14), H-8 and H-11, and H-8 and H-15 confirmed this
partial substructure. As in the previous compounds, the portion
of the molecule that remained to be identified corresponded to
a dibromopyrrole carboxylic acid. The chemical shifts of the
pyrrole proton and its associated carbon (δH 6.86; δC 113.7), its
multiplicity (singlet), and a diagnostic NOE with the amide
NH (δH 7.31) placed the pyrrole proton at C-4, finally
establishing the gross structure of aspidostomide H (8). The
absolute configuration at C-8 could not be determined in this
case due to a lack of sample.
position
4 (δC)
5 (δC)
6 (δC)
7 (δC)
2
107.9, C
104.5, C
102.7, C
123.6, C
156.8, C
78.1, CH
59.8, CH
108.0, C
110.7, C
135.2, C
113.1, CH
125.2, CH
113.6, C
119.7, CH
126.9, C
108.0, C
104.5, C
103.0, C
123.4, C
156.6, C
85.0, CH
58.0, CH
107.5, C
111.0, C
135.3, C
113.1, CH
125.3, CH
113.7, C
119.7, CH
126.8, C
54.2, CH3
109.5, C
101.1, C
100.9, C
123.2, C
150.4, C
119.6, CH
123.8, C
106.4, C
110.6, C
134.8, C
113.0, CH
125.4, CH
113.8, C
121.1, CH
131.9, C
103.9, C
98.5, C
3
4
112.0, CH
128.7, C
159.1, C
40.2, CH2
25.8, CH2
112.4, C
107.7, C
138.9, C
105.8, CH
114.9, C
107.9, CH
151.2, C
116.2, C
5
6
8
9
10
11
13
14
15
16
17
18
O-CH3
clearly resembled the characteristic 3,5-dibromo-4-hydroxy-
phenyl moieties typical of compounds 1−3. The singlet at δH
7.61 (H-11/H-15) correlated to C-13 (δC 151.6), C-9 (δC
125.6), C-10 (δC 117.3), and C-12/C-14 (δC 109.6), while the
signal at δH 7.60 (H-11′/H-15′) showed correlations with C-
13′ (δC 151.7), C-10′ (δC 117.0), C-9′ (δC 125.8), and C-12′/
C-14′ (δC 109.7). A similar trend was evident with the protons
at δH 6.47 and 6.53, which also had similar HMBC subspectra.
Both protons correlated to the corresponding C-10 of their
respective substructures and with an amide carbonyl. These
correlations resembled those observed for the enamide moiety
in aspidostomide F (6), except that in compound 6 the
pyrroloketopiperazine ring was connected to an indole, and in
this case each enamide seemed to be connected to a different
3,5-dibromo-4-hydroxyphenyl group. The two remaining
proton singlets at δH 7.12 and 6.58 showed HMBC correlations
consistent with bromopyrrole substructures. The difference in
chemical shift could be explained in terms of different
substitution patterns on the pyrrole rings. An intriguing fact
was that, although two distinct amide carbonyls were present in
the molecule, no amide protons could be detected. In the
The last new compound of this series (9) showed two
symmetric clusters in the negative mode HRESIMS spectrum,
which corresponded to polybrominated ions (m/z 1072.4232
[M − H]− and m/z 1094.4100 [M − 2H + Na]−), giving rise to
a molecular formula of C26H10Br8N4O4 with 20 degrees of
unsaturation. This molecular formula was especially interesting,
as aspidostomides A−C were C13 compounds, suggesting that
compound 9 could be a dimeric structure formed by the union
of two C13H5Br4N2O2 monomers. In the negative mode mass
spectrum, an additional ion was observed at m/z 536.7130,
1
which corresponded to the formula C13H5Br4N2O2. The H
NMR spectrum of 9 showed only six downfield singlets [δH
7.61 (s, 2H), 7.60 (s, 2H), 7.12 (s, 1H), 6.58 (s, 1H), 6.53 (s,
1H), and 6.47 (s, 1H)], while, in the 2D NMR spectra, two sets
of paired signals could be clearly detected. Inspection of the 13C
NMR spectrum confirmed the presence of paired signals of
similar chemical shifts, indicative of either a dimeric structure or
the presence of two unresolved very similar compounds (Table
5). In particular the proton signals at 7.61 and 7.60 ppm (2H
each) had similar sets of COSY and HMBC correlations, which
E
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6.53. On the other hand, the aromatic protons at δH 7.61
correlated with both pyrrole protons. From the evidence
gathered so far, especially the NOE correlations between
protons of the different parts of the molecule, it was clear that
there was some kind of symmetry in the dimeric structure,
which was only broken by the different substitution in the
pyrrole rings. At this point, the possibility of a mixture of
unresolved isomers was less probable.
Table 5. NMR Spectroscopic Data of Aspidazide A (9)
1
(500.13 MHz for H, 125.13 MHz for 13C; CD3COCD3)
position
δH
δC, type
2
119.9, CH
100.0, C
105.7, C
123.3, C
154.3, C
116.9, CH
125.6, C
117.3, C
135.4, CH
109.6, C
151.6, C
109.6, C
135.4, CH
99.4, C
6.58, s
3
4
5
6
All the facts gathered so far suggested a possible N−N link
between the two lactams, giving rise to an asymmetrical
diacylazide, a functional group that had been reported
previously in a few natural products, but is extremely rare.
Because no suitable crystals for X-ray diffraction could be
obtained, the final confirmation of the proposed structure was
obtained by chemical transformations (Figure 4). Methylation
of compound 9 under basic conditions (NaH, MeI in DMSO)
yielded two products, 9a and 9b, which were purified by
8
6.53, s
7.61, s
9
10
11
12
13
14
15
2′
3′
4′
5′
6′
7.61, s
7.12, s
6.47, s
7.60, s
1
preparative TLC. The H NMR spectrum of compound 9a
101.5, C
112.6, CH
127.5, C
153.7, C
117.4, CH
125.8, C
117.0, C
135.3, CH
109.7, C
151.7, C
109.7, C
135.3, CH
showed five singlets [δH 7.83 (H-11 and H-15), δH 6.80 (H-8),
δH 6.74 (H-2), δH 3.95 (δC 60.4, O-CH3), and δH 3.43 (δC 33.5,
N-CH3)], while compound 9b had a similar set of signals [δH
7.85 (H-11′ and H-15′), δH 6.85 (H-8′), δH 7.24 (H-4′), δH
3.96 (δC 60.4, O-CH3), and δH 3.49 (δC 33.8, N-CH3)]. It was
evident that the diacylazide bond was cleaved by a hydride
reduction, followed by N-methylation, together with O-
methylation of the phenolic groups. This N−N cleavage was
no surprise, since it is well known that hydrazo and azo
compounds are easily cleaved by a variety of reducing agents.20
NaH is not frequently used as a reducing agent due to its
basicity; however, it has been reported that it can easily reduce
S−S, Si−Si, and Si−Cl bonds.21 In fact, a well-known method
for the reduction of disulfide bridges in proteins makes use of
NaH in DMSO.22 This dual character of NaH as a base or
hydride donor has been studied.23a,b In this case, the cleavage of
the easily reduced N−N bond was a bonus that helped with the
structure elucidation. Correlations observed in the NOESY
spectra confirmed the assignment of the methyl groups and the
substructures that formed compound 9: in the case of 9a, NOE
correlations were observed between the N-Me (δH 3.43) and
the enamide double-bond proton (δH 6.80), between the latter
and the phenyl ring protons (δH 7.83), and between the phenyl
ring protons and the pyrrole proton (δH 6.74). These data
confirmed that in this substructure the pyrrole proton was
8′
9′
10′
11′
12′
13′
14′
15′
7.60, s
previously isolated compounds, the amide NH protons were
always clearly observed. The fact that both enamide vinylic
protons were singlets was consistent with the absence of both
amide protons. The only major difference in the 2D NMR
spectra of the two substructures was observed precisely in the
NOESY correlations of the enamide sp2 protons at 6.53 and
6.47 ppm. Each enamide proton correlated to its respective
aromatic two-proton singlet of the corresponding 3,5-dibromo-
4-hydroxyphenyl group (δH 7.61; 6.53; δH 7.60; 6.47), while δH
6.47 had an additional NOE with the pyrrole proton at δH 6.58.
In turn, both sp2 enamide protons correlated with each other,
and no additional NOESY correlations were observed for δH
Figure 4. Chemical transformations of compound 9.
F
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vicinal to the nitrogen. On the other hand, compound 9b
displayed NOE correlations only between δH 3.49 and 6.85 and
between δH 6.85 and 7.85. In order to rule out definitively the
possibility of two unresolved compounds for 9, the previous
reaction was repeated without addition of MeI. In the case of
two unresolved compounds no change would be expected.
However, once again two compounds were formed, in this case
9c and 9d, which were purified by preparative TLC and
characterized by NMR and HRMS. Both products preserved
Compounds 1−10 were tested for their cytotoxic activity
against the 786-O cell line (human renal carcinoma). The only
active compound (IC50 < 10 μM) was aspidostomide E (5),
with an IC50 value of 7.8 μM. The IC50 values for all
compounds are shown in Table S1, Supporting Information.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a PerkinElmer 343 polarimeter. UV spectra were
recorded in MeOH on a Hewlett-Packard 8452 spectrometer. Infrared
spectra were obtained (film on KBr) on a Nicolet-Magna 550 FT-IR
spectrometer. All NMR spectra were recorded in (CD3)2CO or CDCl3
using the signals of residual nondeuterated solvents as internal
reference on a Bruker Avance II 500 MHz spectrometer operating at
500.13 MHz for 1H and 125.13 MHz for 13C. All 2D NMR
experiments (COSY, DEPT-HSQC, HMBC, NOESY) were per-
formed using standard pulse sequences. High-resolution mass spectra
were recorded on a Bruker microTOf-Q instrument. Column
chromatography was performed using Merck silica gel 60, 230−400
mesh. Merck silica gel 60 PF254 plates were used for preparative TLC.
Gel permeation chromatography was performed in MeOH, using
Sephadex LH20 (Pharma Inc.). HPLC separations were performed
using a Thermo Separations SpectraSeries P100 pump and a Thermo
Separations Refractomonitor IV RI detector connected to a Thermo
Separations SpectraSeries UV 100 detector, with simultaneous UV
(220 nm) and RI detection. A YMC RP-18 (5 μm, 20 mm × 250 mm)
column working at a flow rate of 5 mL/min was used for HPLC
separations. All solvents were HPLC grade.
1
the H NMR signals corresponding to their respective portion
of compound 9, but in this case, the NH protons of the lactams
were clearly observable, coupled to their corresponding
enamide sp2 protons. This was the definitive proof of the
asymmetrical diacylazide structure of compound 9, which was
named aspidazide A.
Aspidostomides A−C are structurally related to the
dibromotyrosine-derived compounds that are typical in sponges
of the order Verongida, such as the agelasins and fistularins, and
especially to hemifistularin-3, which is also a dibromooctop-
amine derivative.24 However, to the best of our knowledge,
compounds of this type have not been previously isolated from
bryozoans. The same can be said for aspidostomides D−F,
which are brominated pyrroloketopiperazine alkaloids very
similar to a series of compounds typically isolated from sponges
of the Agelasidae and Axinellidae families, such as the
longamides,25 agelastatins,26a,b mukanadin C,27 hanishin,28
cyclooroidin,29 and the agesamides.30 As in other cases in
which compounds of great structural similarity have been
isolated from taxonomically unrelated marine invertebrates
belonging to different phyla, there is always the possibility of
common microbial symbionts as the true producers of the
isolated substances. This theory will need further investigation.
On the other hand, aspidazide A is the first natural product
isolated directly from a marine invertebrate that has a
diacylazide functional group. Although there are several
examples of synthetic hydrazides and diacylazides, some of
which are in clinical use, only recently has this functional group
been detected in natural products. There are only a few
examples to date, from a variety of sources, mostly from
microorganisms,31−33 but also from plant seeds or bulbs.34,35 In
the marine environment, diacylazides have been isolated from
the brown alga Sargassum vachellianum36 and from a symbiotic
dinoflagellate isolated from a sponge.37 These previous reports
point toward a possible microbial origin of aspidazide A,
another point that will merit further investigation.
Animal Material. Samples of Aspidostoma giganteum (Busk, 1854)
were collected by trawling at a depth of 85 m during the prawn P.
muelleri fishing season at the Gulf of San Jorge (45°09′ S, 65°35′ W),
Patagonia, Argentina, and identified by one of us (C.M). The
biological material (160 g) was frozen on board (−20 °C), transported
to the laboratory, and kept frozen until processed. A voucher specimen
(MACN-in: 39252) is deposited at the “Bernardino Rivadavia”
Museum of Natural Sciences, Buenos Aires, Argentina.
Extraction and Isolation. Frozen samples of A. giganteum (160 g)
were triturated and extracted three times with EtOH. The combined
extracts were evaporated under reduced pressure to obtain 7.10 g of a
brown syrup. The extract was permeated on Sephadex LH20 (4 × 120
cm, MeOH), to afford 12 fractions (LH1−LH12). Fraction LH5 (29.0
mg) was subjected to HPLC (YMC Rp18, 20 × 250 mm) with
MeOH/H2O (65:35), as eluent, yielding compounds 1 (12.0 mg
0.024% dry wt) and 2 (2.2 mg, 0.0037% dry wt). Fraction LH6 (63.0
mg) was purified by preparative HPLC, employing MeOH/H2O
(70:30) as eluent, yielding compounds 3 (3.2 mg, 0.0053% dry wt), 4
(16.0 mg, 0.027% dry wt), 5 (2.0 mg, 0.0033% dry wt), and 6 (2.2 mg,
0.0037% dry wt). Purification of fraction LH7 (180.7 mg) by column
chromatography using a cyclohexane/EtOAc/MeOH gradient fol-
lowed by HPLC purification (MeOH/H2O, 1:1) afforded compound
9 (23.5 mg, 0.039% dry wt). Fractions LH8 and LH9 were pooled and
subjected to preparative TLC (cyclohexane/EtOAc, 55:45), to yield
compound 7 (32.1 mg, 0.054% dry wt). Finally, fraction LH10 was
further fractionated by column chromatography on silica gel, eluting
with a cyclohexane/EtOAc gradient, and after final purification by
TLC (cyclohexane/EtOAc, 1:1) provided compound 8 (2.0 mg,
0.0033% dry wt).
Besides the above-mentioned new compounds, the 9-O-ethyl
ether of aspidostomide C (10) was also isolated. Compound 10
had a molecular formula of C15H13Br5N2O3, and its mass
spectrum also showed a fragment at m/z 616.6441, which
corresponded to a loss of EtOH. The presence of an ethoxy
1
group was clearly evident in the H NMR spectrum by the
Aspidostomide A (1): amorphous, yellow solid; [α]25 −26.5 (c
presence of an extra methylene signal (δH 3.50 and 3.38)
coupled to a methyl group at δH 1.23. HMBC correlations of
this group with H-9 and C-9 confirmed that compound 10 was
the ethyl ether of aspidostomide C. Because EtOH was used for
the extractions, it is highly probable that compound 10 is an
artifact. Three additional compounds were also isolated in this
work: 3,4-dibromopyrrole-2-carboxamide, which has been
previously isolated from several sponges, 3-bromo-4-methox-
ybenzoic acid, and methyl-3-bromo-4-methoxybenzoate.38a,c
These compounds are probably biosynthetically related to the
aspidostomides or their precursors.
D
0.60, MeOH); UV (MeOH) λmax (log ε) 255 (4.51) nm; IR (film
1
KBr) νmax 3389, 2925, 1707, 1635, 1546, 1227, 1144, 736 cm−1; H
and 13C NMR, see Tables 1 and 3; HRESIMS m/z 556.7332 [M −
H]− (calcd for C13H979Br4N2O3, 556.7352; Δ −2.0 mmu).
Aspidostomide B (2): off-white, amorphous solid; [α]25 −28.8 (c
D
0.50, MeOH); UV (MeOH) λmax (log ε) 237 (6.43), 273 (5.68) nm;
IR (film KBr) νmax 3403, 2967, 1726, 1635, 1553, 1227, 1201, 734
cm−1; 1H and 13C NMR, see Tables 1 and 3; HRESIMS m/z 556.7305
[M − H]− (calcd for C13H979Br4N2O3, 556.7352; Δ 4.7 mmu).
Aspidostomide C (3): off-white, amorphous solid; [α]25 −31.2 (c
D
0.65, MeOH); UV (MeOH) λmax (log ε) 224 (1.97) nm; IR (film
G
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1
KBr) νmax 3394, 2920, 1704, 1635, 1557, 1230, 1085, 736 cm−1; H
and 13C NMR, see Tables 1 and 3; HRESIMS m/z 634.6463 [M −
H]− (calcd for C13H879Br5N2O3, 634.6457; Δ −0.6 mmu).
was extracted with EtOAc (10 mL × 3). The organic phase was
concentrated and purified by TLC on silica gel, using cyclohexane/
EtOAc (1:1), to yield 7N,12N-dimethyldehydroaspidostomide D (4a)
(0.6 mg, 60% yield).
Aspidostomide D (4): off-white, amorphous solid; [α]25 −2.68 (c
D
1
15.3, MeOH); UV (MeOH) λmax (log ε) 228 (0.83) nm; IR (film
KBr) νmax 3276, 2923, 1696, 1668, 1535, 1435, 1318, 1044, 799 cm−1;
1H and 13C NMR, see Tables 2 and 4; HRESIMS m/z 655.6496 [M −
H]− (calcd for C15H779Br5N3O2, 655.6461; Δ −3.5 mmu).
7N,12N-Dimethyldehydroaspidostomide D (4a): colorless oil; H
NMR (CDCl3, 500 MHz) δ 7.49 (d, J = 1.9 Hz, 1H, H-17), 7.40 (dd, J
= 8.7, 1.9 Hz, 1H, H-15), 7.28 (d, J = 8.7 Hz, 1H, H-14), 6.41 (s, 1H,
H-8), 3.89 (s, 3H, N-12 Me), 3.50 (s, 3H, N-7 Me); 13C NMR
(CDCl3, 125 MHz) δ 154.0 (C-6), 135.0 (C-13), 131.1 (C-18), 128.8
(C-5), 125.8 (C-15), 123.0 (C-8), 121.2 (C-17), 114.9 (C-16), 111.3
(C-14), 111.0 (C-10), 108.7 (C-2), 105.6 (C-4), 105.2 (C-11), 103.7
(C-9), 98.2 (C-3), 35.0 (N-7 Me), 32.3 (N-12 Me); APPI-MS m/z
667.6804 [M + H]+ (calcd for C17H1179Br5N3O, 667.6814; Δ −0.1
mmu).
Aspidostomide E (5): pale yellow, amorphous solid; [α]25D −78.0 (c
1.10, MeOH); UV (MeOH) λmax (log ε) 262 (19.8), 293 (15.1) nm;
IR (film KBr) νmax 3409, 2923, 1674, 1527, 1432, 1318, 1080, 669
cm−1; 1H and 13C NMR, see Tables 2 and 4; HRESIMS m/z 669.6616
[M − H]− (calcd for C16H979Br5N3O2, 669.6617; Δ 0.1 mmu)..
Aspidostomide F (6): pale yellow, amorphous solid; UV (MeOH)
λmax (log ε) 253 (14.5), 293 (11.5) nm; IR (film KBr) νmax 3284, 2912,
Preparation of Compounds 9a and 9b. To a solution of
aspidazide A (1 mg, 0.92 μmol) in dry DMSO (1 mL) was added 2
equiv of NaH, and the mixture was stirred at rt for 20 min until the
solution turned dark green. Then, a solution of CH3I (1.2 equiv) in
DMSO was added dropwise. The reaction mixture was then stirred
overnight. A few drops of MeOH were added to remove the excess
NaH; then H2O was added (5 mL), and the mixture was extracted
with EtOAc. The organic phase was concentrated and purified by TLC
on silica gel, using cyclohexane/EtOAc (1:1), after which compounds
9a and 9b were obtained in a combined 77% yield.
1
1713, 1646, 1377, 797 cm−1; H and 13C NMR, see Tables 2 and 4
HRESIMS m/z 637.6346 [M − H]− (calcd for C15H579Br5N3O,
637.6355; Δ 0.9 mmu).
Aspidostomide G (7): white, amorphous solid; UV (MeOH) λmax
(log ε) 255 (13.6), 293 (12.1) nm; IR (film KBr) νmax 3200, 2928,
1699, 1621, 1568, 1421, 1224, 1086, 677 cm−1; 1H and 13C NMR, see
Tables 2 and 4; HRESIMS m/z 579.7494 [M − H]− (calcd for
C15H1079Br4N3O2, 579.7512; Δ 1.8 mmu).
Aspidostomide H (8): white, amorphous solid; [α]25 −25.0 (c
D
1
Compound 9a: white, amorphous solid; H NMR (CD3COCD3,
0.40, MeOH); UV (MeOH) λmax (log ε) 228 (9.5) nm; IR (film KBr)
1
νmax 3733, 1643, 1546, 1394, 1116, 666 cm−1; H and 13C NMR, see
500 MHz) δ 7.83 (bs, 2H, H-11/H-15), 6.80 (s, 1H, H-8), 6.74 (s, 1H,
H-2), 3.95 (s, 3H, O-Me), 3.43 (s, 3H, N-Me); 13C NMR
(CD3COCD3, 125 MHz) δ 154.9 (C-13), 153.8 (C-6), 135.5 (C-
11/C-15), 130.1 (C-5), 127.0 (C-9), 122.5 (C-8), 120.2 (C-2), 117.1
(C-10), 109.9 (C-12/C-14), 101.5 (C-4), 95.4 (C-3), 60.4 (O-Me),
33.5 (N-Me); HRESIMS m/z 564.7407 [M − H]− (calcd for
C15H979Br4N2O2, 564.7403; Δ −0.4 mmu).
Tables 1 and 3; HRESIMS m/z 520.8315 [M − H]− (calcd for
C15H1079Br3N2O4, 520.8425; Δ 11.0 mmu).
Aspidazide A (9): yellow, amorphous solid; UV (MeOH) λmax (log
ε) 295 (12.5) nm; IR (film KBr) νmax 3433, 1646, 1541, 1207, 1052,
872, 669 cm−1; 1H and 13C NMR, see Table 5; HRESIMS m/z
1072.4232 [M − H]− (calcd for C26H979Br8N4O4, 1072.4180; Δ −0.52
mmu).
1
Compound 9b: white, amorphous solid; H NMR (CD3COCD3,
500 MHz) δ 7.85 (bs, 2H, H-11/H-15), 7.24 (s, 1H, H-4), 6.85 (s, 1H,
H-8), 3.96 (s, 3H, O-Me), 3.49 (s, 3H, N-Me); 13C NMR
(CD3COCD3, 125 MHz) δ 155.1 (C-13), 153.5 (C-6), 135.7 (C-
11/C-15), 129.9 (C-5), 127.1 (C-9), 122.1 (C-8), 117.1 (C-10), 112.3
(C-4), 109.4 (C-12/C-14), 105.9 (C-2), 100.0 (C-3), 60.4 (O-Me),
33.8 (N-Me); HRESIMS m/z 564.7404 [M − H]− (calcd for
C15H979Br4N2O2, 564.7403; Δ −0.1 mmu).
Preparation of MTPA Esters of Compound 1. To a solution of
compound 1 (1.0 mg, 1.78 μmol) in dry pyridine (300 μL) was added
(R)-MTPA-Cl (5 μL, 26.7 μmol). After 1 h at rt, the reaction mixture
was diluted with EtOAc, extracted three times with HCl, and then
washed with H2O. The organic layer was taken to dryness, and the
product was purified by TLC using cyclohexane/EtOAc (6:4) to yield
0.7 mg of the (S)-MTPA ester of 1. Treatment of 1 (1.0 mg) with (S)-
MTPA-Cl in a similar way yielded the corresponding (R)-MTPA ester
of 1 (0.6 mg).
Preparation of Compounds 9c and 9d. To a solution of
aspidazide A (1 mg, 0.92 μmol) in dry DMSO (1 mL) was added 2
equiv of NaH, and the mixture was stirred at rt for 30 min until the
solution turned dark green. The reaction was monitored by TLC and
was quenched after 30 min. A few drops of MeOH were added to
remove the excess NaH; then H2O was added, and the mixture was
extracted with EtOAc. The organic phase was concentrated and
purified by TLC on silica gel, using cyclohexane/EtOAc (4:6), after
which compounds 9c and 9d were obtained in a combined 80% yield.
1
(S)-MTPA ester of 1: white, amorphous solid; H NMR (CDCl3,
500 MHz) δ 9.48 (bs, 1H); 6.98 (d, J = 3.1 Hz, 1H); 6.93 (bt, J = 5.6
Hz, 1H); 3.87, 3.72 (dd, J = 5.6, 4.0 Hz, 2H); 7.55 (bs, 2H).
1
(R)-MTPA ester of 1: white, amorphous solid; H NMR (CDCl3,
500 MHz) δ 9.54 (bs, 1H); 7.00 (d, J = 3.5 Hz, 1H); 7.07 (bt, J = 5.8
Hz, 1H); 3.90, 3.72 (dd, J = 5.8, 4.0 Hz, 2H); 7.34 (s, 2H).
Preparation of MTPA Esters of Compound 4. To a solution of
compound 4 (1.0 mg, 1.51 μmol) in dry pyridine (300 μL) was added
(R)-MTPA-Cl (0.30 μL, 1.51 μmol). After 1 h at rt, the reaction
mixture was diluted with EtOAc, extracted three times with 10% HCl,
and then washed with H2O. The organic layer was taken to dryness,
and the product was purified by TLC using cyclohexane/EtOAc (1:1)
to yield the (S)-MTPA ester of 4 (0.7 mg). Treatment of 4 (1.0 mg)
with (S)-MTPA-Cl in a similar way yielded the corresponding (R)-
MTPA ester of 4 (0.8 mg).
1
Compound 9c: white, amorphous solid; H NMR (CD3COCD3,
500 MHz) δ 9.81 (bd, 1H, NH amide), 7.73 (s, 2H, H-11/H-15), 6.60
(d, J = 5.0 Hz, 1H, H-8), 6.72 (s, 1H, H-2), 5.64 (s, 1H, OH);
HRESIMS m/z 535.7062 [M − H]− (calcd for C13H579Br4N2O2,
564.7090; Δ 2.8 mmu).
1
Compound 9d: white, amorphous solid; H NMR (CD3COCD3,
500 MHz) δ 10.0 (bd, 1H, NH amide), 7.74 (s, 2H, H-11/H-15), 7.25
(s, 1H, H-4), 6.66 (d, J = 5.0 Hz, 1H, H-8), 5.64 (s, 1H, OH);
HRESIMS m/z 535.7066 [M − H]− (calcd for C13H579Br4N2O2,
564.7090; Δ 2.4 mmu).
1
(S)-MTPA ester of 4: white, amorphous solid; H NMR (CDCl3,
500 MHz) δ 6.78 (d, J = 4.5 Hz, 1H); 7.18 (d, J = 8.5 Hz, 1H); 7.29
(dd, J = 8.5, 2.0 Hz, 1H); 7.15 (bs, 1H), δ 8.40 (bs, 1H).
9-O-Ethylaspidostomide C (10): yellow, amorphous solid; [α]25
D
1
5.8 (c 1.00, MeOH); UV (MeOH) λmax (log ε) 257 (14.3); IR (film
KBr) νmax 3400, 3145, 2931, 1713, 1634, 1552, 1230, 1107, 736 cm−1;
1H and 13C NMR, see Tables 1 and 3; HRESIMS m/z 662.6841 [M −
H]− (calcd for C15H1279Br5N2O3, 662.6842; Δ 0.1 mmu).
Cytotoxicity Evaluation. The effects of the different compounds
on cell growth were assayed on log phase unsynchronized monolayers
of the 786-O (human clear cell renal cell carcinoma) cell line. The cells
were cultured at 37 °C in plastic flasks in RPMI medium (Gibco;
Invitrogen Corp.) in a humidified air atmosphere with 5% CO2. Serial
passages were made by treatment of confluent monolayers with 0.25%
(R)-MTPA ester of 4: white, amorphous solid; H NMR (CDCl3,
500 MHz) δ 6.57 (d, J = 4.5 Hz, 1H); 7.19 (d, J = 9.0 Hz, 1H); 7.30
(dd, J = 9.0, 1.5 Hz, 1H); 7.16 (bs, 1H), δ 8.40 (bs, 1H).
Preparation of Compound 4a. NaH (2 equiv) was added to a
solution of 4 (1 mg, 1.51 μmol) in dry DMSO (1 mL), and the
mixture was stirred at rt for 20 min until the solution turned dark
green. Then, a solution of CH3I (230 μL, 1.2 equivs) in DMSO was
added dropwise. The reaction was monitored by TLC and was
quenched after 1 h. A few drops of MeOH were added to remove the
excess sodium hydride; then H2O was added (5 mL), and the mixture
H
dx.doi.org/10.1021/np500012y | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
trypsin and 0.02% EDTA in Ca2+- and Mg2+-free PBS. Cells were
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method.39 The bioassays were carried out as follows: 2 × 103 cells/
well in complete medium were seeded in 96-multiwell plates. After 24
h, the cells received serial doses of the compounds (0.05−100 μM) or
vehicle (DMSO) in medium plus 2% FBS (fetal bovine serum) for 3
days. As control, the cytotoxic activity of doxorubicin (IC50: 0.13 μM)
was also tested. Medium with freshly added compounds was changed
every 2 days. Viability was assessed by reduction of the tetrazolium salt
(MTS) to the formazan product in viable cells (Cell Titer 96, Promega
Corp.) as calculated by the 492/620 nm absorbance ratio. The IC
(inhibitory concentration)50 was defined as the concentration of the
compound required for 50% cell growth inhibition.
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ASSOCIATED CONTENT
* Supporting Information
■
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S
Full sets of 1D and 2D NMR spectra of compounds 1−10. 1H
NMR spectra of MTPA esters of compounds 1 and 4. 1H NMR
spectra of 3,4-dibromopyrrole-2-carboxamide, 3-bromo-4-me-
thoxybenzoic acid, and methyl-3-bromo-4-methoxybenzoate.
Table with IC50 values for all compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel/Fax: 5411-4576-3385. E-mail: palermo@qo.fcen.uba.ar.
Notes
■
The authors declare no competing financial interest.
́
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Pusset, J.; Leroy, S.; Pietra, F. J. Chem. Soc., Chem. Commun. 1993,
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ACKNOWLEDGMENTS
■
Research at the University of Buenos Aires was supported by
grants from CONICET (PIP 2010-2012 No. 516), UBA
(Programation 2014-2017 No. 704), and ANPCyT (PICT
2010 No. 1808). We thank Dr. G. Cabrera (UMYMFOR−
CONICET) for recording the mass spectra and J. Gallardo and
G. Eskuche (UMYMFOR−CONICET) for the NMR spectra.
L.P. thanks SENACYT-IFARHU (Panama) for a doctoral
́
fellowship. The authors are grateful to L. Jerez for his help with
sample and data collection.
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