Aplysinellamides A-C, bromotyrosine-derived metabolites from an australian aplysinella sp. marine sponge

Article abstract of DOI:10.1021/np500119e

Mass-directed fractionation of an extract from the Australian marine sponge Aplysinella sp., from the Great Barrier Reef, resulted in the isolation of four new bromotyrosine derivatives, aplysinellamides A-C (1-3) and aplysamine-1-N-oxide (4), along with six known compounds (5-10). The structure elucidation of compounds 1-4 was based on their 1D and 2D NMR and MS spectroscopic data. Aplysamine-1 (6) increased the apolipoprotein E secretion from human CCF-STTG1 astrocytoma cells by 2-fold at the concentration of 30 μM.

Full text of DOI:10.1021/np500119e

Article
pubs.acs.org/jnp
Aplysinellamides A−C, Bromotyrosine-Derived Metabolites from an
Australian Aplysinella sp. Marine Sponge
Li-Wen Tian,† Yunjiang Feng,† Yoko Shimizu,‡ Tom Pfeifer,‡ Cheryl Wellington,§ John N. A. Hooper,⊥
and Ronald J Quinn*,†
†Eskitis Institute, Griffith University, Brisbane, QLD 4111, Australia
‡Centre for Drug Research and Development, Vancouver, BC 2405, Canada
§Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC V6T 1Z4, Canada
⊥Queensland Museum, South Brisbane, QLD 4101, Australia
S
* Supporting Information
ABSTRACT: Mass-directed fractionation of an extract from
the Australian marine sponge Aplysinella sp., from the Great
Barrier Reef, resulted in the isolation of four new
bromotyrosine derivatives, aplysinellamides A−C (1−3) and
aplysamine-1-N-oxide (4), along with six known compounds
(5−10). The structure elucidation of compounds 1−4 was
based on their 1D and 2D NMR and MS spectroscopic data.
Aplysamine-1 (6) increased the apolipoprotein E secretion from human CCF-STTG1 astrocytoma cells by 2-fold at the
concentration of 30 μM.
was then extracted by CH2Cl2 and MeOH. Mass-directed
fractionation and purification of the combined CH2Cl2/MeOH
extract led to the isolation of 10 bromotyrosine derivatives,
including four new compounds, aplysinellamides A−C (1−3)
and aplysamine-1-N-oxide (4), together with six known natural
products, suberedamine A (5),7 aplysamine-1 (6),8 purealidine
G (7),9 2,6-dibromo-4-[2-(dimethylamino)ethyl]phenol (8),10
N,N,N-trimethyl-3-bromo-4-methoxytyrosine (9),11 and 3,5-
dibromo-4-methoxybenzoic acid (10).12 Given solvents con-
taining 0.1% TFA were used for the isolation, compounds 1, 2,
4, 5, 6, 7, 8, and 9 were isolated as TFA salts. Herein we report
the isolation and structure elucidation of aplysinellamides A−C
(1−3) and aplysamine-1-N-oxide (4), as well as the ApoE
modulation activities of compounds 1−10.
lzheimer’s disease (AD) has emerged as the most
Aprevalent form of late-life mental disorder in humans
and is typified by deposition of β-amyloid (Aβ) within the
brain.1 Aβ is a neurotoxic peptide, and the accumulation of Aβ
leads to its deposition into plaques and the launching of a
pathologic cascade that ultimately leads to neuronal death.1 An
impaired ability to clear Aβ peptides from the brain, rather than
increasing production of Aβ peptides, is thought to underlie
most cases of late-onset AD.2
Apolipoprotein E (ApoE) is the major apolipoprotein in the
central nervous system and plays a central role in cholesterol
transport.3 In the brain, ApoE is mostly produced by astrocytes,
and some of this ApoE binds to specific neuronal receptors for
cholesterol uptake.3 A previous study suggested that increased
levels of highly lipidated ApoE could facilitate the clearance of
Aβ peptides,4,5 implying that the interference of ApoE secretion
could potentially lead to the treatment of AD. Bexarotene is the
only reported compound to date that activates ApoE
expression.6
In our ongoing search for new lead compounds for
neurodegenerative disorders, a drug discovery program was
initiated to identify natural products as ApoE modulators. A
high-throughput screening method was developed and used to
screen a prefractionated natural product library of 102 432
fractions for their ability to modulate ApoE secretion from
CCF-STTG1 cells. From 11 fractions derived from an extract of
the Australian sponge Aplysinella sp., a single active fraction was
identified with activity against ApoE. (+)-LRESIMS showed
molecular ions at m/z 204/205/206, 212/213/214, 322/324/
326, and 316/318 within the active fraction, and MS was used
to guide large-scale isolation. Twenty grams of sponge material
RESULTS AND DISCUSSION
■
The freeze-dried and ground marine sponge (20.0 g) was
sequentially extracted with n-hexane, CH2Cl2, and MeOH. The
CH2Cl2 and MeOH extracts were combined and chromato-
graphed using reverse phase C18-bonded silica HPLC (gradient
MeOH/H2O/0.1% TFA) to yield 60 fractions. Fractions 22−
27 contained the ions of interest (see above). Purification of
these fractions using a Luna C18 semipreparative HPLC column
(gradient MeOH/H2O/0.1% TFA) yielded a new compound,
aplysamine-1-N-oxide (4, 0.5 mg), and four known compounds,
aplysamine-1 (6, 0.6 mg), purealidine G (7, 0.3 mg), 2,6-
dibromo-4-[2-(dimethylamino)ethyl]phenol (8, 0.2 mg), and
N,N,N-trimethyl-3-bromo-4-methoxytyrosine (9, 0.5 mg).
Received: February 7, 2014
Published: April 23, 2014
© 2014 American Chemical Society and
American Society of Pharmacognosy
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carbonyl group was linked to the 1,3,4-trisubstituted aromatic
ring, thus forming a cinnamoyl-derived moiety. The lysine
moiety was connected to the substituted cinnamoyl moiety
through an amide bond by the HMBC correlations from the
methylene protons (δH 3.31) in lysine to the carbonyl carbon
C-9 (δC 167.3). Finally the substitution of a methoxy group on
the benzene ring C-4 position was established by the HMBC
correlations from the aromatic protons H-2/H-5/H-6 and the
methoxy protons (δH 3.91) to the aromatic carbon (δC 157.3);
thus the bromine atom was assigned to aromatic carbon C-3.
The absolute configuration of the lysine moiety in
aplysinellamide A (1) was determined by Marfey’s method.13
Acid hydrolysis of 1, followed by derivatization with Marfey’s
reagent and LC-MS analysis, showed a peak with a retention
time of 10.6 min, suggesting a D-lysine in the molecule
(standard D-lysine derivative had a retention time of 10.5 min,
while the L-lysine derivative eluted at 9.8 min). The structure of
aplysinellamide A was therefore established as 1.
Aplysinellamide B (2) exhibited a cluster of isotopic ions at
m/z 463/465/467 (1:2:1) in the (+)-LRESIMS spectrum,
indicating the presence of two bromine atoms in the molecule.
The molecular formula, C16H20Br2N2O4, was deduced by
HRESIMS, with seven degrees of unsaturation. The 1H, HSQC,
and COSY NMR data of compound 2 were very similar to
those of 1, except that compound 2 had a two-proton singlet
(δH 7.79, 2H), representing a symmetrical benzene moiety.
Both the HSQC and HMBC correlations were observed from
the aromatic proton (δH 7.79) to the carbon (δC 131.3),
confirming a 3,5-dibromo-4-methoxybenzene ring system in the
molecule. The structure of aplysinellamide B was therefore
elucidated as 2. Acid hydrolysis followed by derivatization with
Marfey’s reagent was carried out in an attempt to determine the
absolute configuration of the lysine residue in 2. However, LC-
MS failed to detect any lysine derivative, maybe due to the low
quantity of compound 2. We propose that compound 2 has a D-
lysine in the molecule based on its shared biogenesis with 1.
Aplysinellamide C (3) was isolated as brown, amorphous
powder. (+)-LRESIMS showed an isotopic ion cluster at m/z
356/358 (1:1), indicating the presence of one bromine atom in
the molecule. The molecular formula of 3 was determined to be
C15H18BrNO4, on the basis of HRESIMS with seven degrees of
unsaturation. The 1H, HSQC, and COSY NMR data of
compound 3 were also very similar to those of 1; the difference
was the absence of an amino methine in the aliphatic chain.
HMBC correlations were observed from the methylene protons
(δH 1.66 and 2.34) in the aliphatic chain to a carboxyl carbon
(δC 176.0), indicating that the lysine functionality in 1 and 2
was replaced by a 5-aminopentanoic acid in 3. Hence,
aplysinellamide C was assigned to structure 3.
Aplysamine-1-N-oxide (4) was isolated as a pale, amorphous
powder. (+)-LRESIMS showed a cluster of isotopic ions at m/z
423/425/427 (1:2:1) and 212/213/214 (1:2:1), indicating the
presence of two bromine atoms in the molecule. The molecular
formula of 4 was determined to be C15H24Br2N2O2 based on a
HRESIMS measurement, with four degrees of unsaturation. 1H,
COSY, and HSQC NMR data suggested that compound 4 had
similar structural feature to that of the known compound
aplysamine-1 (6),8 including a symmetrical tetrasubstituted
benzene ring, two isolated N-methyl protons (δ 3.44, s, 6H and
δ 2.81, s, 6H), and substituted ethyl and propyl groups. The
obvious differences between 4 and 6 were that the proton
chemical shifts of H-8 (δ 3.81, m, 2H) and one set of N-methyl
groups (δ 3.44, m, 6H) in 4 were downfield shifted in
Mass analysis of other HPLC fractions indicated the presence
of brominated metabolites. Further purifications of these
fractions using a Luna C18 semipreparative HPLC column
(gradient MeOH/H2O/0.1% TFA) yielded three more new
compounds, aplysinellamide A (1, 0.4 mg), aplysinellamide B
(2, 0.3 mg), and aplysinellamide C (3, 0.5 mg), along with two
known compounds, suberedamine A (5, 0.3 mg) and 3,5-
dibromo-4-methoxybenzoic acid (10, 0.8 mg).
Aplysinellamide A (1) was obtained as a pale amorphous
powder. The cluster of two isotopic ions at m/z 385/387 (1:1)
in the (+)-LRESIMS spectrum indicated the presence of one
bromine atom in the molecule. The molecular formula,
C16H21BrN2O4, was deduced on the basis of HRESIMS
1
measurement, with seven degrees of unsaturation. The H
and HSQC NMR data suggested that compound 1 contained
three aromatic protons (δH 7.76, d, J = 2.1 Hz, 7.50, dd, J = 8.6,
2.1 Hz, and 7.05, d, J = 8.6 Hz), one isolated pair of trans
olefins (δH 7.42, d, J = 15.7 Hz, and 6.46, d, J = 15.7 Hz), one
methoxy (δH 3.91, s), one methine (δH 3.91, m), and several
methylenes in the upfield region. The COSY correlation data
established a 1,3,4-trisubstituted benzene moiety and a
substituted aliphatic pentyl group. The proton and carbon
chemical shifts of the methylene (δH 3.31 m, δC 38.5) and the
methine (δH 3.91 m, δC 52.6) in the pentyl subunit suggested
that a nitrogen was attached at the 1- and 5-positions of the
subunit, respectively. HMBC correlations from the methylene
(δH 1.97/1.90, H-4′) and methine (δH 3.91, H-5′) in the pentyl
functionality to a carboxyl carbon (δC 170.6) suggested that the
methine group in the pentyl group was connected to a carboxyl
group. Thus, a lysine moiety was assigned. In addition, HMBC
correlations from the olefinic proton (δH 7.42) to the aromatic
carbons (δC 128.5, 128.6, and 131.6) and the carbonyl carbon
(δC 167.3) (Figure 1) indicated that an α,β-unsaturated
Figure 1. Key COSY and HMBC correlations for compound 1.
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Davisil 40−60 μm Å C18 bonded silica was used for flash
chromatography. A Waters 600 pump equipped with a Waters 996
PDA detector and a Waters 717 autosampler was used for HPLC. A
Thermo Scientific C18 Betasil 5 μm 143 Å column (21.2 mm × 150
mm) and a Phenomenex Luna C18 5 μm 143 Å column (21.2 mm ×
250 mm) were used for semipreparative HPLC separations. All
solvents used for chromatography, UV, and MS were Lab-Scan HPLC
grade, and the H2O was Millipore Milli-Q PF filtered. A BIOLINE
orbital shaker was used for the large-scale extraction of the sponge
material.
Animal Material. The sponge Aplysinella sp. (family Aplysinelli-
dae) was collected from Sykes Reef, Capricorn-Bunker Group, Great
Barrier Reef, Queensland, Australia, latitude −23.41888889°, longitude
152.0505556°, depth 25 m. The sponge was kept frozen prior to
freeze-drying and extraction. A voucher specimen (G307202) has been
lodged at the Queensland Museum, Brisbane, Australia. A description
and images of this species (as Aplysinella sp. OUT QM1194) are
available from the following link (http://www.spongemaps.org/
#!search-for-sponges-public/c16m4) using the search tool for
Mudmap Number inserting the text “QM1194”.
comparison with corresponding signals in aplysamine-1 (6).
These shifts were due to the formation of an N-oxide, thus the
inductive effect of the positively charged nitrogen atom. This
was supported by an uncounted oxygen atom in the molecule
and the HMBC correlation from methyl protons (δ 3.44, s) to
C-8 (δ 68.5). Hence, the structure of aplysamine-1-N-oxide was
assigned as 4.
Six known natural products, suberedamine A (5), aplys-
amine-1 (6), purealidine G (7), 2,6-dibromo-4-[2-
(dimethylamino)ethyl]phenol (8), N,N,N-trimethyl-3-bromo-
4-methoxytyrosine (9), and 3,5-dibromo-4-methoxybenzoic
acid (10), were also isolated from the Australian sponge
1
Aplysinella sp. Their H and 13C NMR data were identical to
those reported in the literature.
Compounds 1−10 were evaluated for their ability to
modulate ApoE secretion. Of the tested compounds, aplys-
amine-1 (6) increased ApoE secretion by 2-fold at the
concentration of 30 μM (Figure 2). All of the other compounds
showed no significant activity at concentrations of up to 40 μM
(data not shown).
Detailed description of the species is as follow: it has a massive,
digitate, encrusting growth form. In life its color is dark gray in the
upper exterior, yellow in the lower exterior and with a yellow interior.
The sponge is aerophobic such that when removed from seawater its
pigments turn black. Oscules are large, on apex of digits, with some
specimens known to have a large stalked central osculum and smaller
oscules on the smaller digits. Sponge texture is firm and difficult to
tear. The surface is conulose, with a tracery of minute conules in
between large surface conules, superficially resembling species of
Dysidea. The ectosome is membranous, heavily collagenous, and
slightly arenaceous externally. The choanosomal skeleton consists of
spongin fibers that are very heavy, concentrically stratified but lightly
pithed, and form very wide meshes producing an irregular reticulation.
The mesohyl collagen is also very heavy. There is no mineral skeleton.
Extraction and Isolation. The freeze-dried and ground sponge
(20.0 g) was poured into a conical flask (1 L), n-hexane (500 mL) was
added, and the flask was shaken at 200 rpm for 2 h. The n-hexane
fraction was filtered under vacuum and then discarded. CH2Cl2/
MeOH (4:1, 500 mL) was added to the defatted marine sample in the
conical flask and shaken at 200 rpm for 2 h. The resulting extract was
filtered under gravity and set aside. MeOH (500 mL) was added, and
the MeOH/sponge mixture was shaken for a further 2 h at 200 rpm.
Following filtration the sponge sample was extracted with another
volume of MeOH (500 mL), while being shaken at 200 rpm for
another 2 h. All CH2Cl2/MeOH extractions were combined and dried
under vacuum to yield a dark brown solid (3.02 g). This material was
preabsorbed onto C18-bonded silica and then packed into a HPLC
stainless steel guard cartridge (10 × 30 mm) that was subsequently
attached to a C18 semipreparative HPLC column. A gradient HPLC
condition from 90% H2O (0.1% TFA)/10% MeOH (0.1% TFA) to
MeOH (0.1% TFA) was run over 40 min, followed by isocratic
conditions of 100% MeOH (0.1% TFA) for a further 10 min, all at a
flow rate of 9.0 mL/min. Sixty fractions (60 × 1 min) were collected
and then analyzed by LC-MS. Fractions 22/23 [(+)-LRESIMS, m/z
204/205/206], 24/25 [(+)-LRESIMS, m/z 212/213/214, m/z 322/
324/326], and 26/27 [(+)-LRESIMS, m/z 316/318] contained the
ions of interest. Further purification of these fractions with a Luna C18
semipreparative HPLC column yielded compounds 4 (0.5 mg,
0.0025% dry wt), 6 (0.6 mg, 0.003% dry wt), 7 (0.3 mg, 0.0015%
dry wt), 8 (0.2 mg, 0.001% dry wt), and 9 (0.5 mg, 0.0025% dry wt).
Further purification of the fractions 33, 34, 39, 40, 42, and 43 with a
Luna C18 semipreparative column yielded compounds 1 (0.4 mg,
0.002% dry wt), 2 (0.3 mg, 0.0015% dry wt), 3 (0.5 mg, 0.0025% dry
wt), 5 (0.3 mg, 0.0015% dry wt), and 10 (0.8 mg, 0.004% dry wt).
Aplysinellamide A (1): pale, amorphous powder; [α]2D3 −3.1 (c
0.007, MeOH); UV (MeOH), λmax (log ε) 290 (3.76), and 218 (3.78)
nm; IR (KBr) νmax 2924, 2853, 1652, 1596, 1498, 1261, and 1052
cm−1; 1H NMR (CD3OD, 600 MHz), see Table 1; 13C NMR
(CD3OD, 150 MHz), see Table 2; (+)-LRESIMS m/z 385 (100%),
Figure 2. Activity of aplysamine-1 (6) in the ApoE modulation assay.
Marine sponges of the order Verongida have proven to be a
remarkable source of chemically diverse bromotyrosine
derivatives.14 The chemical diversity of this structural class
arises from the degree of bromination of the tyrosine moiety, as
well as the subsequent oxidation, reduction, ring-opening, and
rearrangement. Four sponge families from the order of
Verongida, Aplysinidae, Aplysinellidae, Ianthellidae, and
Pseudoceratinidae have been frequently reported to contain
bromotyrosine derivatives.15 Our study together with literature
data suggests that this unique structural feature could be
considered as a marker for chemotaxonomic identification of
the Verongida sponges.
In conclusion, 10 bromotyrosine derivatives (1−10) were
isolated from the Australian marine sponge Aplysinella sp., from
the southern Great Barrier Reef, including the new compounds
aplysinellamides A−C (1−3) and aplysamine-1-N-oxide (4).
Aplysamine-1 (6) showed ApoE-modulating activity, increasing
ApoE secretion from human astrocytoma cells by 2-fold at the
concentration of 30 μM.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured with a HORIBA SEPA-300 high-sensitivity polarimeter.
UV spectra were recorded on a Jasco V650 UV/vis spectropho-
tometer. NMR spectra were recorded at 30 °C on a Varian 600 MHz
Unity INOVA spectrometer equipped with a triple resonance
1
cryoprobe. The H and 13C NMR chemical shifts were referenced to
the solvent peak for CD3OD at δH 3.31 and δC 49.5 or DMSO-d6 at δH
2.50 and δC 39.5. LRESIMS spectra were recorded on a Waters ZQ
mass spectrometer. HRESIMS spectra were recorded on a Bruker
Daltronics Apex III 4.7e Fourier-transform mass spectrometer. Alltech
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387 (100%); (+)-HRESIMS m/z 385.0755 [M + H]+ (calcd for
Aplysamine-1-N-oxide (4): pale, amorphous powder; UV
(MeOH), λmax (log ε) 283 (1.97), and 217 (3.11) nm; IR (KBr)
C16H2279BrN2O4, 385.0765).
1
νmax 3038, 2965, 1676, 1384, 1200, 1130, and 906 cm−1; H NMR
(DMSO-d6, 600 MHz), see Table 1; 13C NMR (DMSO-d6, 150
MHz), see Table 2; (+)-LRESIMS m/z 423 (50%), 425 (100%), 427
(50%); (+)-HRESIMS m/z 445.0097 [M + Na]+ (calcd for
C15H2479Br2N2O2Na, 445.0077).
1
Table 1. H NMR Data (600 MHz) for 1−4
δH (J in Hz)
a
a
a
b
position
1
2
3
4
Marfey’s Derivatization and LC-MS Analysis of 1. Compound
1 (0.3 mg) was hydrolyzed with 6 N HCl (1 mL) at 110 °C for 20 h.
After cooling, the reaction mixture was concentrated under the
vacuum to dryness. The hydrolysate was added to 40 μL of 1 M
NaHCO3 and 200 μL of 1% 1-fluoro-2,4-dinitrophenyl-5-alaninamide
in acetone. The solution was reacted at 40 °C for 1 h and subsequently
analyzed by LC-MS [Waters ZQ-LCMS, Phenomenex Luna C18 (50 ×
4.6 mm), 3.0 μm, gradient MeOH/H2O/0.1% TFA; flow rate 1.0 mL/
min]. LC-MS analysis showed a peak with a retention time of 10.6
min, while the standard D-lysine derivative had a retention time of 10.5
min. The L-lysine derivative had a retention time of 9.8 min.
ApoE Modulation Activity. The ApoE modulation assay was
carried out on fractions and compounds as previously described.16
Briefly, fractions or compounds were incubated with human CCF-
STTG1 astrocytoma cells for 96 h. Supernatant samples were assayed
for secreted ApoE using an ELISA assay detecting ApoE. The fold
change over control cells was reported.
2
7.76, d (2.1)
7.05, d (8.6)
7.79, s
7.79, s
7.77, d (2.2)
7.05, d (8.6)
7.66, s
7.66, s
5
6
7.50, dd (8.6,
2.1)
7.50, dd (8.6,
2.1)
7
7.42, d (15.7)
6.46, d (15.7)
3.31, m
7.38, d
(15.7)
7.41, d (15.7)
6.47, d (15.7)
3.33, m
3.10, t
(8.4)
8
6.55, d
(15.7)
3.81, t
(8.4)
1′
3.32, m
3.98, t
(5.8)
2′
3′
1.62, m
1.54, m
1.49, m
1.97, m
1.90, m
3.91, m
3.91, s
1.62, m
1.50, m
1.60, m
1.66, m
2.14, m
3.31, m
4′
1.94, m
2.34, t (7.1)
3.92, s
1.87, m
5′
3.72, t (6.2)
3.88, s
OCH3
8-NCH3
3′-NCH3
ASSOCIATED CONTENT
3.44, s
2.81, s
■
S
* Supporting Information
1H, COSY, HSQC, and HMBC NMR spectra for aplysinella-
mide A−C (1−3) and aplysamine 1-N-oxide (4). LC-MS trace
for Marfey’s derivative of 1 and the two standards. This material
is available free of charge via the Internet at http://pubs.acs.org.
a
b
In CD3OD. In DMSO-d6.
Table 2. 13C NMR Data (150 MHz) for 1−4
δC, mult.
a
a
a
b
AUTHOR INFORMATION
Corresponding Author
*Tel: + 61-7-37356000. Fax: + 61-7-37356001. E-mail: r.
quinn@griffith.edu.au.
Notes
position
1
2
3
4
■
1
2
3
4
5
6
7
8
9
128.6, C
133.9, C
128.8, C
136.6, C
131.6, CH
111.5, C
131.3, CH
118.0, C
131.8, CH
111.6, C
133.9, CH
118.0, C
157.3, C
154.9, C
151.7, C
151.5, C
111.9, CH
128.5, CH
138.7, CH
119.2, CH
167.3, C
118.0, C
111.7, CH
128.5, CH
138.5, CH
119.3, CH
167.0, C
118.0, C
The authors declare no competing financial interest.
131.3, CH
136.5, CH
122.7, CH
166.6, C
133.9, CH
27.4, CH2
68.5, CH2
ACKNOWLEDGMENTS
■
The authors thank H. Vu from Griffith Uninversity for
acquiring the HRESIMS measurement. This work was
supported by the Queensland Government Smart Futures
NIRAP Program, the Australian Research Council for NMR
and MS equipment (LE0668477 and LE0237908), in part by
an operating grant from the Alzheimer’s Drug Discovery
Foundation to C.W., and Innovation Funds from the Centre for
Drug Research and Development (Vancouver, Canada).
1′
2′
3′
4′
5′
6′
38.5, CH2
28.6, CH2
21.9, CH2
29.8, CH2
52.6, CH
170.6, C
38.7, CH2
28.7, CH2
22.0, CH2
30.1, CH2
53.6, CH
171.6, C
38.6, CH2
28.6, CH2
21.9, CH2
33.0, CH2
176.0, C
70.7, CH2
25.3, CH2
54.8, CH2
4-OCHH3
8-NCH3
3′-NCH3
55.4, CH3
59.8, CH3
55.3, CH3
56.2, CH3
42.9, CH3
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a
b
CD3OD. DMSO-d6.
Aplysinellamide B (2): brown, amorphous powder; [α]2D3 +0.29 (c
0.017, MeOH); UV (MeOH), λmax (log ε) 280 (3.96), 224 (4.13), and
206 (4.18) nm; IR (KBr) νmax 2925, 2854, 1635, 1420, 1261, and 1203
cm−1; 1H NMR (CD3OD, 600 MHz), see Table 1; 13C NMR
(CD3OD, 150 MHz), see Table 2; (+)-LRESIMS m/z 463 (50%), 465
(100%), 467 (50%); (+)-HRESIMS m/z 484.9682 [M + Na]+ (calcd
for C16H2079BrN2O4Na, 484.9679).
(4) Cramer, P. E.; Cirrito, J. R.; Wesson, D. W.; Lee, C. Y. D.; Karlo,
J. C.; Zinn, A. E.; Casali, B. T.; Restivo, J. L.; Goebel, W. D.; James, M.
J.; Brunden, K. R.; Wilson, D. A.; Landreth, G. E. Science 2012, 335,
1503−1506.
(5) Poirier, J.; Bertrand, P.; Kogan, S.; Gauthier, S.; Davignon, J.;
Bouthillier, D. Lancet 1993, 342, 697−699.
Aplysinellamide C (3): brown, amorphous powder; UV (MeOH),
λmax (log ε) 290 (4.12), and 219 (4.09) nm; IR (KBr) νmax 3422, 2938,
1
2880, 1649, 1497, 1293, and 1206 cm−1; H NMR (CD3OD, 600
MHz), see Table 1; 13C NMR (CD3OD, 150 MHz), see Table 2;
(+)-LRESIMS m/z 356 (100%), 358 (100%); (+)-HRESIMS m/z
378.0322 [M + Na]+ (calcd for C15H1879BrNO4Na, 378.0319).
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Journal of Natural Products
Article
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