Isolation and synthesis of pulmonarins A and B, acetylcholinesterase inhibitors from the colonial ascidian Synoicum pulmonaria

Article abstract of DOI:10.1021/np401002s

Pulmonarins A and B are two new dibrominated marine acetylcholinesterase inhibitors that were isolated and characterized from the sub-Arctic ascidian Synoicum pulmonaria collected off the Norwegian coast. The structures of natural pulmonarins A and B were tentatively elucidated by spectroscopic methods and later verified by comparison with synthetically prepared material. Both pulmonarins A and B displayed reversible, noncompetitive acetylcholinesterase inhibition comparable to several known natural acetylcholinesterase inhibitiors. Pulmonarin B was the strongest inhibitor, with an inhibition constant (K _i) of 20 μM. In addition to reversible, noncompetitive acetylcholinesterase inhibition, the compounds displayed weak antibacterial activity but no cytotoxicity or other investigated bioactivities.

Full text of DOI:10.1021/np401002s

Article
pubs.acs.org/jnp
Isolation and Synthesis of Pulmonarins A and B, Acetylcholinesterase
Inhibitors from the Colonial Ascidian Synoicum pulmonaria
Margey Tadesse,† Johan Svenson,*,‡ Kristina Sepcic,§ Laurent Trembleau,⊥ Magnus Engqvist,‡
̌
́
Jeanette H. Andersen,∥ Marcel Jaspars,# Klara Stensvag,† and Tor Haug*,†
̊
†Norwegian College of Fishery Science, University of Tromsø, Breivika N-9037, Tromsø, Norway
‡Department of Chemistry, University of Tromsø, Breivika N-9037, Tromsø, Norway
§Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecn
̌
a pot 111, 1000 Ljubljana, Slovenia
⊥Department of Chemistry, University of Aberdeen, Old Aberdeen, AB24 3UE, Scotland, U.K.
∥Marbio, University of Tromsø, Breivika, N-9037 Tromsø, Norway
#Marine Biodiscovery Centre, University of Aberdeen, Old Aberdeen, AB24 3UE, Scotland, U.K.
S
* Supporting Information
ABSTRACT: Pulmonarins A and B are two new dibromi-
nated marine acetylcholinesterase inhibitors that were isolated
and characterized from the sub-Arctic ascidian Synoicum
pulmonaria collected off the Norwegian coast. The structures
of natural pulmonarins A and B were tentatively elucidated by
spectroscopic methods and later verified by comparison with
synthetically prepared material. Both pulmonarins A and B
displayed reversible, noncompetitive acetylcholinesterase in-
hibition comparable to several known natural acetylcholines-
terase inhibitiors. Pulmonarin B was the strongest inhibitor, with an inhibition constant (Ki) of 20 μM. In addition to reversible,
noncompetitive acetylcholinesterase inhibition, the compounds displayed weak antibacterial activity but no cytotoxicity or other
investigated bioactivities.
arine invertebrates and their associated bacterial
Msymbionts represent organisms with great potential for
the discovery of novel bioactive compounds and chemical
scaffolds.1−3 The sessile benthic lifestyle promotes the
production of defensive secondary metabolites to provide an
evolutionary advantage. To investigate their potential as sources
for bioactive compounds, several hundred different Arctic
marine invertebrates, algae, and microorganisms have been
collected during a systematic study of Arctic marine organisms
along the Norwegian coast.4 The Arctic waters provide a
significantly less studied biotope in comparison to warmer
waters; however they are still rich in biodiversity.4−6 One such
Arctic organism, the colonial ascidian Synoicum pulmonaria, has
been shown to contain a family of dibrominated bioactive
compounds known as the synoxazolidinones.7−9 In the current
paper, further studies of the extract from S. pulmonaria revealed
additional novel compounds named pulmonarins A (1) and B
(2).
The pulmonarins are structurally related to the synoxazoli-
dinones (3−5), which are heavily modified antimicrobial
dipeptide derivatives displaying a rare 4-oxazolidinone core
linking a dibrominated tyrosine derivative to an arginine/
agmatine side chain. The pulmonarins do not display the 4-
oxazolidinone core or the guanidine group of the synoxazoli-
dinones, but instead they contain quaternary ammonium
groups implying potential acetylcholinesterase (AChE)-inhibit-
ing properties. AChE inhibitors are a class of drugs used for the
treatment of Alzheimer’s disease, glaucoma, myasthenia gravis
(an autoimmune disorder), and the recovery of neuromuscular
block after surgery.10,11 Some well-known AChE inhibitors in
clinical use today include physostigmine (6), a naturally
occurring carbamate from the Calabar bean, used for the
treatment of glaucoma.10,12 Examples of other current AChE-
inhibitor-based drugs are galanthamine (7) and donepezil, both
used in most countries for the treatment of Alzheimer’s disease.
Several toxins such as nerve agents, insecticides, and
antiparasitic chemicals used in agriculture and aquaculture
also act by inhibiting AChE and consist mostly of organo-
phosphorus compounds and carbamates.13,14 At least three
Received: December 3, 2013
Published: February 18, 2014
© 2014 American Chemical Society and
American Society of Pharmacognosy
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Journal of Natural Products
Article
other marine natural products with similar structures to 1 and 2
have been reported in the literature. Compound 8, with
significant structural similarities to 1, was isolated by Fattorusso
et al. from the Caribbean sponge Pseudoceratina crassa (later
reclassified as Aiolochroia crassa) in 1994.15 Compound 8 was
recently studied by Munoz and co-workers and shown to
display weak antiparasitic properties.16 Compound 9 was
isolated from another Caribbean sponge Verongula sp., but no
Table 1. 1D and 2D NMR Data (600/175 MHz DMSO-d6)
of Natural 1 and 2
δH (mult, J in
a
no.
δC, type
Hz)
gCOSY
gHMBC
Pulmonarin A
1
61.2, CH3
158.2, C
3.84, s
2
2
3/3′
118.5, C
biological activity was reported for the compound.17
A
4/4′
5
133.9, CH
b
8.15, s
2, 3/3′, 4/4′, 6
derivative of 9, compound 10, was isolated from the sponge
Verongia fistularis.18
6
163.2, C
7
59.9, CH2
64.2, CH2
53.3, CH3
4.66, m
8
7
8
3.78, t (4.5)
3.14, s
9
8
9/9′/9″
Pulmonarin B
1
60.9, CH3
151.9, C
117.0, C
133.7, CH
136.1, C
41.0, CH2
169.3, C
NH
3.77, s
2
2
3/3′
4/4′
5
7.53, s
3.38, s
2, 3/3′, 4/4′
4/4′, 5, 7
6
7
8
8.19, t (5.5)
3.05, m
1.45, m
1.25, m
1.66, m
3.24, m
3.02, s
9
9
38.9, CH2
29.0, CH2
23.8, CH2
22.3, CH2
65.2, CH2
52.2, CH3
8, 10
9, 11
10, 12
11, 13
12
10
11
12
13
14/14′/14″
13, 14
a
Carbon shifts obtained from gHSQC and gHMBC experiments.
Not observed.
b
ment, while the quaternary carbon signals are derived from
gHMBC data by comparison with the structurally related
synoxazolidinones isolated from the same organism. The
tentative structure of 1 was solved based on the following
correlations: The gHMBC correlation from the H3-1 methoxy
protons to C-2 indicated the position of the methoxy group on
the substituted aromatic ring; the shift value of C-3/3′ (δC
118.5) was indicative of two bromine atoms at these positions;7
and protons H-4/4′ showed a gHMBC correlation to the ester
carbonyl C-6. Furthermore, a gCOSY correlation was observed
between the two adjacent methylene protons H2-7 and H2-8,
and the high chemical shift of H2-7 (δH 4.66) placed it adjacent
to the ester oxygen. The gHMBC correlation from protons H3-
9/9′/9″ to C-8 supported the terminal trimethylated
quaternary ammonium. The shift values of C-9/9′/9″ (δC
53.3) and H3-9/9′/9″ (δH 3.14) are consistent with literature
values.19−21 The assignment of the terminal trimethylated
ammonium was also supported by a loss of 59 mass units from
the parent ion as indicated by a prominent peak at m/z 334 in
the mass spectrum of 1.
The current paper describes the isolation, structural
characterization, and syntheses of 1 and 2 and the evaluation
of their biological acitivities including AChE inhibition
employing electric eel AChE.
RESULTS AND DISCUSSION
■
Stepwise fractionation of an aqueous extract of S. pulmonaria
resulted in the isolation of two compounds. The aqueous
extract was loaded on a solid-phase extraction (SPE) cartridge
and eluted with 40/60 CH3CN/H2O. The eluate was loaded on
a semipreparative RP-HPLC C18 column. MS analysis revealed
compounds with isotope patterns indicating dibrominated
compounds. Compounds 1 and 2 were isolated as yellow gels
with the molecular formula C13H18Br2O3N for 1 and
C17H27Br2O2N2 for 2. Database searching suggested new
compounds, and their tentative structures were solved on the
basis of a number of 1D and 2D NMR experiments and mass
spectometric analyses. Spectroscopic similarities with the
synoxazolidinones were rapidly established.7 Table 1 lists
proton and carbon NMR shift values and both gCOSY and
gHMBC correlations of natural 1 and 2.
Compound 2 displayed signals in the aromatic area of the 1H
and 13C NMR spectra that were nearly identical to 1, suggesting
a similar substructure. The chemical shift of H-4/4′ is
somewhat lower than that of the corresponding protons
found in both 1 and the synoxazolidinones due to the
methylene at C-6. The H2-6 methylene protons displayed a
high shift (δH 3.38) and showed gHMBC correlations to C-4/
4′, C-5, and the amide carbonyl C-7, placing it between the
amide and the aromatic ring. The chemical shifts for H2-6, C-6,
and C-7 correlated well with a similar synthetic methoxy phenyl
An initial inspection of the 2D NMR data revealed similar
motifs in the aromatic region to those found in the
synoxazolidinones.7 The carbon shifts (Table 1) used for the
structure elucidation were obtained from the gHSQC experi-
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acetamide derivative of orthidine F.22 C-9 was placed next to
the amide nitrogen based on gCOSY correlations between
amide protons H-8 and H2-9. This spin system further involved
H2-10 to H2-13, generating a saturated five-carbon segment
between the amide and terminal trimethyl ammonium, which
was further verified by the gHMBC correlations from protons
H3-14/14′/14″ to C-13 in analogy with 1. This spin system
matched well with the chemical shifts of the analogous spin
system reported for clavatadine D, isolated from the Australian
sponge Suberea clavata.23 Figure 1 shows key gHMBC and
gCOSY correlations of natural 1 and 2.
a
Scheme 1. Synthesis of 1 and 2
a
Reagents, conditions, isolated yield: (a) Choline chloride, DMAP,
DCC, CH2Cl2, rt, 6 days, 4% (HPLC yield); (b) Br2/FeBr3,24 CHCl3,
rt, 48h, 21%; (c) HBTU, DIPEA, DMF, rt, 16 h, 57%; (d) K2CO3,
CH2Cl2; 16 h, 13% (HPLC yield).
Figure 1. Key gHMBC (H→C) and gCOSY () correlations of 2.
our screening process.4 Electric eel AChE, sharing a high degree
of sequence homology and having a 3D structure very similar to
human AChE,28 was employed as a model vertebrate AChE
system, and the well-established method of Ellman was
employed to study the inhibition kinetics.29,30 Synthetic 1
induced 50% inhibition of electric eel AChE at 42 μg/mL (105
μM) in the presence of acetylthiocholine chloride as a substrate
(Figure 2). The inhibitory constant (Ki) was determined from
Because the purification led to sample loss and the
incomplete NMR data of the compounds, total synthesis was
required to generate sufficient material for structural verification
and biological testing. Compound 1 was prepared in one step.
Dicyclohexylcarbodiimide (DCC) was used as coupling agent
to react 3,5-dibromo-4-methoxybenzoic acid and choline
chloride in the presence of a catalytic amount of 4-
(Dimethylamino)pyridine (DMAP) in CH2Cl2. The reaction
was slow, and sufficient amounts of 1 could only be obtained
after an extended reaction time followed by HPLC purification.
Several different solvent systems were evaluated, but only
CH2Cl2 generated 1 due to solubility issues. Compound 2 was
synthesized in three steps from 4-methoxyphenylacetic acid,
which was initially dibrominated with Br2/FeBr3 to produce
3,5-(dibromo-4-methoxyphenyl)acetic acid (2a) according to
Weller et al.24 The bromination reaction was followed by MS,
and a complete conversion to the desired dibrominated product
required additional Br2/FeBr3 after 24 h. Compound 2a was
then coupled with 5-(dimethylamino)amylamine using 2-(1H-
Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-
phosphate (HBTU) as coupling agent to generate 2-(3,5-
dibromo-4-methoxyphenyl)-N-(5-(dimethylamino)pentyl)-
acetamide (2b). N-Methylation of 2b with CH3I completed the
synthesis of 2. The synthetic routes to 1 and 2 are displayed in
Scheme 1.
1
Both H and 13C NMR shifts of the natural and synthetic
compounds are analogous, thus validating their proposed
structures (Table S3). This is further supported by comparing
the 2D NMR data.
Figure 2. Inhibition of vertebrate AChE by 1 and 2. 50% inhibition
was observed at 42 and 16 μg/mL (105 and 36 μM), respectively.
The para-methoxy dibrominated phenyl group moiety is a
common structural motif among marine secondary metabolites
and particularly those that are tyrosine derivatives.25 The
isolated compounds bear structural resemblances with not only
the synoxazolidinones7,8 and 8, 9, and 1015,17,18 but also
ianthelline26 and subereamine B27 among others.
As both pulmonarins are terminally trimethylated ammo-
nium compounds, they were evaluated as AChE inhibitors in
addition to the standard screens routinely performed during
the Dixon plot31 and revealed 1 to be a reversible,
noncompetitive inhibitor with a Ki of 36 μg/mL (90 μM)
(Figure 3). Compound 2 showed a stronger reversible,
noncompetitive inhibition, and 50% inhibition was obtained
at 16 μg/mL (36 μM) with a Ki of 9 μg/mL (20 μM) (Figures
2 and 3).
A Ki of 9 μg/mL (20 μM) for 2 indicates moderate binding
affinities for vertebrate AChE, similar to that of the FDA-
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Figure 3. Determination of the type of inhibition and the inhibition constant Ki for 1 (left graph) and 2 (right graph) by Dixon plot analysis. The
□
■
●
concentrations of the substrate acetylthiocholine were 0.25 ( ), 0.5 ( ), and 1 mM ( ). Ki was determined to 36 μg/mL (90 μM) for 1 and 9 μg/
mL (20 μM) for 2.
approved drug galanthamine.32 The affinity of 1 for AChE is 4
times weaker. AChE inhibitors with significantly varying
binding affinities are used in approved medications. Phys-
ostigmine is a strong AChE inhibitor with an IC50 value of
approximately 30 nM,33 while galanthamine, used to combat
Alzheimer’s disease, displays a moderate Ki of 2−20 μM against
AChE from different vertebrate species.32 The Ki values
obtained here are therefore within the pharmaceutically
interesting regime. For competitive inhibitors of AChE, studies
based on molecular modeling and quantitative structure−
activity relationship (QSAR) analysis have shown that a degree
of hydrophobicity and the presence of an ionizable nitrogen are
prerequisites for the inhibitor−enzyme interactions.10 Consid-
ering this, it was expected that the more hydrophobic 1, as
determined by HPLC retention times, would be more active. As
the observed AChE inhibition contradicts this, it is clear that
further investigations are required for a mechanistic explan-
ation.
As an allosteric inhibitor of AChE, the presence of a
quaternary nitrogen in both compounds suggests binding
through electrostatic interactions at the peripheral anionic site
(PAS) of the enzyme, which is located on the surface of the
protein at the entrance of the active site cleft. This site is
implicated in a number of nonclassical functions such as
amyloid deposition, cell adhesion, and neurite outgrowth.34
The PAS is thus a potential target in rational drug design for
the development of novel and improved inhibitors and
therapeutics for the treatment of neural cancers, nerve
regeneration, and neurodegenerative disorders such as
Alzheimer’s disease.11,34 QSAR studies of 1 and 2 could
potentially contribute to future development of new drug
candidates in various therapeutic fields.
effect in the active fraction. Compound 2 was slightly more
active against C. glutamicum, displaying a minimal inhibitory
concentration of 250 μg/mL. No activities were seen in the
other bacterial screens in our panel (test strains used were S.
aureus (ATTC 9144), E. coli (ATCC 25922), P. aeruginosa
(ATTC 27853), and C. glutamicum (ATTC 13032)) (data not
included). As well as testing negative against other bacteria,
both 1 and 2 also tested negative for cytotoxic activities against
three adherent cancer cell lines (A2058 melanoma, HT29 colon
carcinoma, and MCF7 breast carcinoma cells) and against
adherent, nonmalignant lung fibroblasts (MRC-5 cells) (data
not included). They were also inactive in an anti-inflammatory
assay targeted against TNF-α and in a biofilm formation
inhibition assay using S. epidermidis (ATCC 35984) (data not
included). Finally 1 and 2 were shown to be inactive as cellular
antioxidants (data not included). Compounds 1 and 2 thus
appear to possess a highly specific action as reversible
noncompetitive AChE inhibitors displaying no apparent cellular
toxicity. As reported for the synoxazolidinones (3−5), it was
not possible to determine whether symbiotic microorganisms
are responsible for the biosynthesis of 1 and 2, which is a
common case for marine natural products.1 On the basis of the
key structural differences between the synoxazolidinones and
the compounds in the present study, it seems unlikely that they
share a common biosynthetic route in the organism apart from
being tyrosine derivatives. Compound 1 can be considered as a
dibrominated version of p-hydroxybenzoylcholine, which has
been isolated from the seeds of Sinapis albae, and it was
proposed to originate biosynthetically from a p-hydroxy benzyl
alcohol derivative.36,37 An analogous biosynthetic route toward
1 is therefore plausible. The proposed biosynthesis of 2 is
similar (see Supporting Information) and is suggested to be
derived from amide formation between methylated 2-(4-
hydroxy-3,5-dibromophenyl)acetic acid and ascophylline, both
previously described in extracts of marine organisms.38,39
We have previously reported that the 40/60 (v/v) CH3CN/
H2O SPE fraction of S. pulmonaria exhibited antibacterial
activity against a number of bacteria, showing an activity at 160
μg/mL against Corynebacterium glutamicum and at 80 μg/mL
against Staphylococcus aureus.35 These compounds were there-
fore also included in the extensive screening performed at
MabCent to investigate other potential bioactivities.4 A lack of
activity at concentrations above 100 μg/mL classified the
compounds as “inactive” in all assays apart from the
antibacterial screen. Compound 1 displayed only very mild
activity against one bacterial strain, C. glutamicum, at a
concentration of 500 μg/mL, indicating a possible synergistic
CONCLUSIONS
■
Two new marine bioactive compounds have been isolated and
characterized from S. pulmonaria. Curtailed by the lack of the
natural compounds, total synthesis provided enough material
for structure verification and biological testing. Both com-
pounds turned out to be nontoxic, reversible, noncompetitive
inhibitors of AChE. The isolation of 1 and 2 yet again
demonstrates the chemical diversity of the cold-water marine
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3,5-(Dibromo-4-methoxyphenyl)acetic Acid (2a). Compound 2a
was prepared in 21% yield according to the method described by
Weller et al.24 Spectral data matched those previously reported.
2-(3,5-Dibromo-4-methoxyphenyl)-N-(5-(dimethylamino)pentyl)-
acetamide (2b). A 0.25 mL amount of 5-(dimethylamino)amylamine
(1.61 mmol, 1.05 equiv) was added to a stirred solution of 2a (494.0
mg, 1.53 mmol) and HBTU (580 mg, 1.53 mmol, 1.0 equiv) in 5 mL
of DMF and 1.06 mL of DIPEA (6.12 mmol, 4 equiv). The reaction
mixture was stirred overnight at rt before being diluted with 20 mL of
EtOAc and washed with 2 × 25 mL of 10% citric acid, 25 mL of 10%
NaHCO3, and 25 mL of saturated brine. The EtOAc extract was dried
over Na2SO4, filtered, and concentrated in vacuo to yield a yellow oil
organism S. pulmonaria and the potential of the species for the
discovery of new structural motifs for further lead compound
development in a number of therapeutic areas.
EXPERIMENTAL SECTION
■
General Experimental Procedures. UV spectra were determined
on a Lambda 25 spectrometer from Perkin-Elmer Instruments.
Infrared spectra were recorded on an Avatar 320 FT-IR spectrometer
from Nicolet. 1D and 2D NMR spectra of natural products were
recorded on a Varian VNMRS 600 MHz spectrometer in DMSO-d6.
Carbon resonances were derived from gHSQC and gHMBC
experiments. NMR spectra of synthetic compounds were recorded
on a Varian 7000e 400 MHz spectrometer in DMSO-d6. Chemical
shifts were referenced to the residual solvent peaks, δH 2.50 and δC
39.5. Mass spectra were acquired on an LTQ Orbitrap XL Hybrid
Fourier Transform mass spectrometer from Thermo Scientific and the
Thermo Scientific Accela HPLC-LTQ Ion Trap-Orbitrap Discovery
system. HPLC of synthetic compounds was performed on a 1200
series instrument with a binary pump and a photodiode array detector
from Agilent Technologies. All starting materials were purchased from
Sigma-Aldrich apart from 5-(dimethylamino)amylamine, which was
provided by Matrix Scientific. Chemicals were used without further
purification except for methyl iodide, which was distilled and stored
over Cu(s) prior to use. Electric eel AChE (6.25 U/mL) was provided
by Sigma-Aldrich.
1
(57% yield). No further purification was performed. H NMR (400
MHz, CDCl3) δ 7.36 (s, 2H), 3.79 (s, 3H), 3.34 (s, 2H), 3.14 (q, J =
6.0 Hz, 2H), 2.28 (m, 2H), 2.24 (s, 6H), 1.47 (m, 2H), 1.40 (m, 2H),
1.24 (m, 2H) ppm; 13C NMR (100 MHz, CDCl3) δ 170.1 (C), 153.0
(C), 134.2 (C), 133.5 (CH), 118.2 (C), 60.7 (CH3), 59.1 (CH2), 45.1
(CH3), 41.8 (CH2), 39.7 (CH2), 29.1 (CH2), 26.8 (CH2), 24.3 (CH2)
ppm; HRESIMS m/z 435.0279 [M + H]+ (calcd for C16H2579Br2O2N2,
435.0283).
5-(2-(3,5-Dibromo-4-methoxyphenyl)acetamido)-N,N,N-trime-
thylpentan-1-aminium Iodide (2). A 380 mg amount of 2b (0.87
mmol) was dissolved in 5 mL of CH2Cl2, and 5 mL of a 1 M K2CO3
solution was added. The resulting mixture was stirred at room
temperature for 15 min before 0.41 mL of methyl iodide (8.70 mmol,
10 equiv) was added and the reaction was stirred overnight. The pH of
the reaction was adjusted to 7 with 2 M HCl. All volatiles were
evaporated, and the crude solid material (537 mg) was dissolved in
CH2Cl2. Silica was added, and the crude material was adsorbed onto
the silica under vacuum. Silica column purification was performed
using a mixture of HOAc/H2O/MeOH/EtOAc (3:2:3:3) and EtOAc
1:9 as eluent. A final purification of the isolated 237 mg of 2 with
semipreparative reversed-phase HPLC was necessary. The isolated 2
was dissolved in 5% CH3CN to a concentration of 25 mg/mL, and
HPLC purification was performed employing a Waters Sunfire Prep C-
18 column (10 μm, 90 Å, 10 × 250 mm) using a gradient of 16−40%
CH3CN in H2O over 40 min until 50 mg (13% yield) of 2 was
collected. IR νmax 3036, 1688, 1473, 1200, 1125 cm−1; UV (MeOH)
λmax 220 nm; 1H NMR and 13C NMR, see Table S3 in the Supporting
Information; HRESIMS m/z 449.0439 [M]+ (calcd for
C17H2779Br2O2N2, 449.0440).
Acetylcholinesterase Inhibition Assay. AChE activity was
measured by Ellman’s method29,30 using acetylthiocholine chloride
(0.25, 0.5, or 1 mM) as a substrate in 100 mM potassium phosphate
buffer pH 7.4 at 25 °C and electric eel AChE as a source of enzyme
(final concentration in the test 0.0075 U/mL). Hydrolysis of
acetylthiocholine chloride was followed on a kinetic microplate reader
(Dynex Technologies) at 405 nm. Stock solutions (2 mg/mL) of 1
and 2 were prepared in deionized H2O and DMSO, respectively.
AChE inhibition by 1 and 2, progressively diluted in H2O, was
monitored for 5 min. All readings were corrected for their appropriate
blanks, and a run with only acetylthiocholine chloride served as assay
positive control. In the case of 2, the blank reactions without the
inhibitor were run in the presence of the appropriate dilution of
DMSO. Every measurement was repeated at least three times.
Antibacterial Assay. Test strains used were Staphylococcus aureus
(ATTC 9144), Escherichia coli (ATCC 25922), Pseudomonas
aeruginosa (ATTC 27853), and Corynebacterium glutamicum (ATTC
13032). All isolates were grown at 37 °C in Mueller Hinton broth
(Difco Laboratories). Bacterial growth was continuously monitored
with an Envision plate reader (Perkin-Elmer). The test was performed
in 96-well Nunc microtiter plates, in which 50 μL of test fractions
dissolved in H2O was incubated with 50 μL of a suspension of an
actively growing (log phase) culture of bacteria diluted to a starting
concentration of approximately 5 × 105 cells per well. The
antimicrobial peptide cecropin B (25 μM) was used as a positive
control. The minimum inhibitory concentration was defined as the
minimum concentration resulting in no change in optical density after
incubation for 24 h at 37 °C. Compounds were tested at
concentrations ranging from 8 to 500 μg/mL.
Extraction and Purification of Pulmonarins A and B.
Specimens of Synoicum pulmonaria (Ellis and Solander, 1786) were
collected off the coast of Tromsø in northern Norway and identified
by Professor Bjørn Gulliksen (Department of Arctic and Marine
Biology, University of Tromsø, Tromsø, Norway). The organisms are
stored at the Norwegian College of Fishery Science, University of
Tromsø, Norway. Specimens (80 g, wet weight) of the organism were
pooled, lyophilized, and extracted with 10 volumes (v/w) of 60/40
CH3CN/H2O containing 0.1% TFA, at 4 °C. The supernatant was
removed after 24 h, and the extraction procedure was repeated. The
combined supernatants were then placed in a −20 °C freezer for 2 h,
resulting in phase separation between a CH3CN-rich organic phase
and an aqueous phase. The aqueous phase was loaded on a C18 solid
phase extraction cartridge and eluted with 10%, 40%, and 80%
CH3CN. The 40% CH3CN SPE eluate was subsequently loaded on a
semipreparative HPLC (column; Waters, Sunfire C18, 250 × 10 mm)
with photodiode array (PDA) detection. Compounds 1 (3.0 mg) and
2 (3.9 mg) were eluted after 41.9 and 38.4, min respectively, using a
gradient of 0% to 40% CH3CN/0.05% TFA in H2O.
Pulmonarin A (1): yellow gel; UV (MeOH) λma1x (log ε) 221 nm
(4.4); IR νmax 3036, 1684, 1474, 1275, 1123 cm−1; H and 13C NMR
data, see Table 1; HRESIMS m/z 393.9650 [M]+ (calcd for
C13H1879Br2O3N, 393.9654).
Pulmonarin B (2): yellow gel; UV (MeOH) λma1x (log ε) 220 nm
(4.5); IR νmax 3036, 1688, 1473, 1200, 1125 cm−1; H and 13C NMR
data, see Table 1; HREIMS m/z 449.0439 for [M]+ (calcd for
C17H27O2N2Br2, 449.0440).
Syntheses. 2-((3,5-Dibromo-4-methoxybenzoyl)oxy)-N,N,N-tri-
methylethanaminium Chloride (1). DCC (1.12 g, 3 equiv, 5.40
mmol), 1.68 g of 3,5-dibromo-4-methoxybenzoic acid (1.5 equiv, 5.40
mmol), and 0.16 g of DMAP (0.2 equiv, 1.28 mmol) were added to 2-
hydroxy-N,N,N-trimethylethanaminium chloride (0.67 g, 4.80 mmol)
in 10 mL of CH2Cl2. The mixture was stirred at room temperature (rt)
for 6 days. A white solid was obtained, which was extracted with H2O
(3 × 20 mL), filtered, and dried. An aliquot of the dried filtrate (713
mg) was dissolved in 5% CH3CN to a concentration of 25 mg/mL and
purified by HPLC using a gradient of 5−100% CH3CN over 45 min,
employing a Waters Sunfire Prep C-18 column (10 μm, 90 Å, 10 ×
250 mm) until 70 mg of the sample was collected for biological activity
screening. The isolated compound (4% yield) was a yellow, viscous
1
gel. IR νmax 3036, 1684, 1474, 1275, 1123 cm−1; H NMR and 13C
NMR, see Table S3 in the Supporting Information; HRESIMS m/z
393.9650 [M]+ (calcd for C13H1879Br2O3N, 393.9654).
368
dx.doi.org/10.1021/np401002s | J. Nat. Prod. 2014, 77, 364−369

Journal of Natural Products
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ASSOCIATED CONTENT
* Supporting Information
1D and 2D NMR and HREIMS data of both natural and
synthetic 1 and 2. This material is available free of charge via
the Internet at http://pubs.acs.org.
■
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AUTHOR INFORMATION
Corresponding Authors
*(J. Svenson) Tel: +47 776 45505. Fax: +47 77 64 47 65. E-
mail: Johan.svenson@uit.no.
*(T. Haug) Tel: +47 776 46071. Fax: +47 77 64 51 10. E-mail:
Tor.haug@uit.no.
Author Contributions
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The manuscript was written through contributions of all
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This work was supported with grants from the Marine
Biotechnology Programme in Tromsø (MABIT, BS0058),
Norway, the Research Council of Norway (184688/S40),
MabCent-SFI, and the University of Tromsø (UiT), Tromsø,
Norway. The authors would like to thank Prof. B. Gulliksen
(Department of Arctic and Marine Biology, UiT, Norway) for
sample collection, taxonomic identification, and organism
image and Dr. J. Isaksson (Department of Chemistry, UiT,
Norway) for operating the 600 MHz NMR.
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