Antibacterial butenolides from the korean tunicate Pseudodistoma antinboja

Article abstract of DOI:10.1021/np300544a

Six new (1, 2, and 5-8) and three known (3, 4, and 9) butenolide metabolites were isolated from the tunicate Pseudodistoma antinboja by activity-guided fractionations. The structures were elucidated by combined NMR and MS spectroscopic methods. These comp

Full text of DOI:10.1021/np300544a

Article
pubs.acs.org/jnp
Antibacterial Butenolides from the Korean Tunicate Pseudodistoma
antinboja
Weihong Wang,†,‡ Hiyoung Kim,†,‡ Sang-Jip Nam,§ Boon Jo Rho,⊥ and Heonjoong Kang*,†,∥
†Center for Marine Natural Products and Drug Discovery, School of Earth and Environmental Sciences, Seoul National University,
NS-80, Seoul, 151-747, Korea
§Sunchon National University, Jeollanam-do, Korea
⊥Ewha Womans University, Seoul, Korea
∥Research Institute of Oceanography, Seoul National University, Seoul, Korea
S
* Supporting Information
ABSTRACT: Six new (1, 2, and 5−8) and three known (3, 4,
and 9) butenolide metabolites were isolated from the tunicate
Pseudodistoma antinboja by activity-guided fractionations. The
structures were elucidated by combined NMR and MS
spectroscopic methods. These compounds were evaluated for
their antibacterial activity, and most of them exhibited
moderate to significant activity that selectively targeted
Gram-positive strains and did not exhibit cytotoxicity in the
MTT assay at 100 μM. Cadiolides 5−9 in particular exhibited
significant antibacterial activity that was comparable to or even better than those of marketed drugs such as vancomycin and
linezolid against all of the drug-resistant strains tested.
atural products-derived antibiotics have played a pivotal
role in the treatment of bacterial infections and diseases
synthetic pathways. However, more significant than their
interesting structures, tunicate metabolites exhibit a wide
spectrum of pharmacological activities, such as anticancer,
antiviral, and antifungal activities, and antibacterial activity
against Gram-positive bacteria.1−3 Among tunicate metabolites,
ecteinascidin 743 (marketed under the trade name Yondelis)
and dehydrodidemnin B (also known as aplidine) are
noteworthy examples.4
As a part of our ongoing project directed toward the search
for antibacterial metabolites from marine tunicates, we analyzed
the colonial tunicate Pseudodistoma antinboja (order Aplouso-
branchia, family Pseudodistomidae). Bioassay-guided fractiona-
tion using various chromatographic techniques resulted in the
isolation of six new (1, 2, and 5−8) and three known (3, 4, and
9) non-nitrogenous metabolites that are responsible for the
antibacterial properties of the EtOAc extract. These compounds
are structurally related to rubrolides5,6 and cadiolides,7
respectively. Rubrolides A, B, and C have been reported to
possess potent antibiotic activity.5 Cytotoxicity,6 aldose
reductase inhibitory activity,8 inhibitory activity against protein
phosphatases 1 and 2A,5 and anti-inflammatory activity9 have
been observed for other rubrolide compounds, making these
compounds worthwhile targets for synthetic studies.10−15
However, only two compounds with a cadiolide carbon
skeleton have been reported from a natural source, and another
two have been reported as synthetic intermediates.16 There are
N
since their discovery in the early and mid 20th century.
However, with the widespread use of broad-spectrum anti-
biotics, enormous selection pressures have been placed on
bacterial populations, provoking the evolution of resistance
mechanisms and thus causing the emergence of resistant
bacterial strains. The resistance of Gram-positive pathogens to
antibiotics is a great concern because these bacteria pose a
continuous and serious threat to public health. One Gram-
positive pathogen of significance in public health, Staphylococcus
aureus, is currently the most frequent cause of nosocomial
bacteremia and skin/wound infections and the second most
frequent cause of nosocomial lower respiratory infections.
Despite the importance of antibiotics, many pharmaceutical
companies have reduced or completely eliminated their
antibiotics research and development efforts for economic
reasons. Most of the natural product-derived antibacterial
compounds currently undergoing clinical evaluation are based
on well-known antibiotic templates. Therefore, there is an
urgent and continuing need to develop new antibiotics,
preferably ones that are structurally different from extant
ones, to combat the worldwide public health crisis.
Marine tunicates (phylum Chordata) are widely recognized
as a prolific source of structurally diverse nitrogen-containing
secondary metabolites. One major group of tunicate metabo-
lites is peptides that contain phenylalanine- or tyrosine-derived
residues. A second major group of tunicate metabolites is the
alkaloids. Marine tunicates have also yielded a small number of
non-nitrogenous metabolites produced through diverse bio-
Received: August 7, 2012
Published: November 12, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
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Article
no previously published biological data for these compounds. In
this study, metabolites 5−9 of the cadiolide class were found to
exhibit in vitro antibacterial activities that are more potent or
comparable to those of marketed drugs against all of the drug-
resistant Gram-positive strains in our bioassay study. Herein we
report the isolation and structural elucidation of these new
metabolites and their antibacterial activities.
1). To simplify our description, the minor isomer is symbolized
by 1a and the major one by 1b. The 13C NMR spectrum of 1a
contained only 14 resonance signals, indicating that there were
some elements of symmetry present in the molecule. In the 1H
NMR spectrum, only six proton signals were observed. The two
doublet signals at δ 6.65 and 6.96, which were mutually
coupled, arose from two pairs of adjacent aromatic protons on a
para-disubstituted phenyl ring (H-3″/5″ and H-2″/6″). The
three-proton singlet at δ 3.70 can be assigned to the methoxy
group at C-4″. The two-proton singlet at δ 7.23 arose from two
equivalent protons attached to two methine carbons situated
meta to each other (H-2′/6′) on a symmetrically tetrasub-
stituted phenyl ring. On the basis of the comparison of these
NMR data with those of related natural products and with
calculated values, the presence of a 4-methoxyphenyl ring and a
3,5-dibromo-4-hydroxyphenyl ring was established.
The remaining fragment of 1a had to account for an
elemental composition of C5H2O2 and four degrees of
unsaturation. An unsaturated butenolide ring bearing an
exocyclic trisubstituted double bond was evident based on
the NMR data and IR absorptions and supplied the four
degrees of unsaturation required for this fragment. Long-range
correlations between the protons of the phenyl rings and
nearby butenolide carbons in the HMBC experiment located
the 3,5-dibromo-4-hydroxyphenyl residue and the 4-methox-
yphenyl residue at C-3 and C-5, respectively.
A similar set of arguments led to the assignment of the NMR
signals for the major isomer, 1b. Compounds 1a and 1b have
identical atomic connectivities. The difference in the structures
of the two isomers is due to the different configuration at Δ4,5,
which was defined by a series of NOE difference experiments.
Irradiation of H-5 of 1b induced NOEs for the H-2′/6′ and H-
2″/6″ signals, indicating that the Δ4,5 olefin has the Z
configuration in 1b. In contrast, irradiation of H-5 of the E
isomer (1a) resulted in an enhancement of only the H-2″/6″
signal. In addition, irradiation of H-2′/6′ in 1a induced an
enhancement at H-2″/6″, indicating that the two phenyl rings
are much closer to each other in this compound, consistent
RESULTS AND DISCUSSION
■
Rubrolide P (1) was isolated as a yellow, amorphous solid. It
yielded an isotopic cluster of [M + H]+ ion peaks at m/z 451/
453/455 with intensities in a 1:2:1 ratio in the FAB mass
spectrum, which is characteristic of a dibrominated compound.
A molecular formula of C18H12Br2O4 was indicated by its
HRFABMS data. Compound 1 always exhibited two sets of
NMR signals with an integral ratio of 1:2 due to the rapid
interconversion of its geometrical isomers in solution (Table
1
with the E configuration at C-4/C-5. A comparison of the H
NMR data of 1a with those of 1b showed that H-2′/6′ and H-
a
1
Table 1. H NMR Data for Rubrolides P (1) and Q (2)
b
b
c
c
1 (E)
1 (Z)
2 (E)
2 (Z)
compd
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
δC
δH (J in Hz)
1
167.6
119.7
152.9
145.5
117.0
123.5
132.2
111.1
151.7
111.1
132.2
124.0
131.5
113.4
159.7
55.2
168.2
113.4
155.2
145.3
113.0
124.3
132.4
112.3
152.9
112.3
132.4
125.6
132.4
114.4
160.1
55.3
169.1
120.6
156.1
148.9
117.9
125.4
134.7
110.8
156.5
117.2
130.4
125.9
133.1
114.9
161.5
56.3
169.7
114.2
158.6
148.0
114.2
124.9
134.8
111.6
157.7
118.1
130.9
127.6
133.9
115.8
161.9
56.3
2
6.66, s
6.55, s
6.39, s
6.34, s
3
4
5
7.11, s
7.23, s
6.33, s
7.80, s
7.00, s
6.36, s
1′
2′
3′
4′
5′
6′
1″
2″/6″
3″/5″
4″
7.23, d (2.0)
7.81, d (2.0)
6.83, d (8.3)
7.22, d (8.4)
7.23, s
7.80, s
7.07, dd (8.3, 2.0)
7.53, dd (8.4, 2.0)
6.96, d (8.4)
6.65, d (8.4)
7.82, d (8.9)
7.04, d (8.9)
6.98, d (8.4)
6.65, d (8.4)
7.85, d (8.8)
7.03, d (8.8)
4″-OMe
3.70, s
3.81, s
3.74, s
3.87, s
a
b
c
1
500 MHz for H NMR and 125 MHz for 13C NMR. Spectra were recorded in DMSO-d6. Spectra were recorded in acetone-d6.
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2″/6″ are considerably more shielded in the E isomer (1a) than
in the Z isomer (1b) because the two phenyl rings are in closer
proximity in the E isomer. Conversely, H-5 is less shielded in
the E isomer because H-5 points out into space in the E isomer,
whereas in the Z isomer, H-5 is close to the aromatic ring
attached at C-3. These observations corroborated the assign-
ment of the geometry at C-4/C-5.
Rubrolide Q (2, this compound has been reported from the
South African tunicate Synoicum globosum as 3′-bromorubrolide
F),17 an amorphous yellow solid, gave a [M + H]+ ion cluster at
m/z 373/375 with intensities in a 1:1 ratio in the FABMS
spectrum. Its molecular formula was established as C18H13BrO4
on the basis of the HRFABMS data. Similar to the case of
compound 1, E and Z isomers of this compound exist in
equilibrium, with the Z form predominating. The NMR data
and IR absorptions suggested that 2 is a 5′-debromo derivative
of 1 (Table 1).
On the basis of the combined spectroscopic analyses and the
comparison of the spectroscopic data with those reported in the
literature, compounds 3 and 4 were identified as rubrolides A5
and J,6 respectively. It should be mentioned that the color and
NMR spectra of rubrolides and cadiolides described below vary
with pH of the solvent. The minute amount of residual TFA
from our experimental procedure makes the NMR data slightly
different from those obtained for compounds isolated without
acidic mobile phase additives.
locations of the two phenyl residues at C-3 and C-5 exchanged.
This structure can explain the similar multiplicity patterns and
the different chemical shift values of the H NMR signals of
these two phenyl rings [6: δ 7.70 (s), 7.14 (d, J = 8.5 Hz), 7.84
(dd, J = 8.5, 1.9 Hz), and 8.14 (d, J = 1.9 Hz); 7: δ 8.14 (s),
7.07 (d, J = 8.3 Hz), 7.34 (dd, J = 8.3, 2.0 Hz), and 7.67 (d, J =
2.0 Hz)]. This assignment was also supported by the HMBC
experiment.
1
Cadiolide F (8) was isolated as an amorphous, yellow solid.
It displayed two distinct HPLC peaks. Both peaks showed the
same distinct peaks upon reinjection into the HPLC system as
well as the same molecular formula of C25H14Br4O6 in the
HRFABMS data when these peaks were collected separately.
Both of these peaks exhibited two sets of NMR signals in an
integral ratio of 1:2. These phenomena can be interpreted as
reflective of the rapid interconversion of the E/Z geometrical
isomers in solution. Compound 8 was obtained as a mixture of
1
E/Z isomers, with the Z form predominating. The H and 13C
NMR data, including those for a methoxy group of the Z
isomer, showed that this compound is a monomethyl ether of
cadiolide C (5). The location of the methoxy group at C-4″ was
determined by the ROESY correlation between the methoxy
protons (δ 3.99) and H-5″ (δ 7.24). Two other ROESY
correlations, from H-5 to H-2′/6′ and H-2″/6″, established the
Z configuration at Δ4,5. The E isomer showed a series of 1H and
13C NMR signals with the same splitting pattern but different
chemical shifts compared with those of the Z isomer. In
particular, H-5 is less shielded in the E isomer than it is in the Z
isomer, and conversely, H-2′/6′ and H-2″/6″ are much more
shielded in the E isomer than they are in the Z isomer. These
shielding patterns are consistent with the geometrical assign-
ment established by the ROESY experiments. Compound 9 was
determined to be cadiolide B by comparison of its
spectroscopic data with those reported previously.7
The compounds were evaluated for their antibacterial activity
by the 2-fold microtiter broth dilution method.19 Most of these
compounds displayed moderate to significant antibacterial
activity against all of the four Gram-positive strains tested
(Staphylococcus aureus ATCC 6538, Kocuria rhizophila (Micro-
coccus luteus) ATCC 9341, Staph. epidermidis ATCC12228, and
Bacillus subtilis ATCC 6633) (Table 4). However, they did not
show inhibitory activity toward the Gram-negative bacteria
including Escherichia coli ATCC 11775, Salmonella typhimurium
Cadiolide C (5) was isolated as an amorphous, yellow-orange
solid. The molecular formula was established as C24H12Br4O6
1
on the basis of the HRFABMS data. The H NMR spectrum
contained two spin systems of three signals each at δ 7.07 (d, J
= 8.3 Hz), 7.34 (dd, J = 8.3, 1.9 Hz), and 7.67 (d, J = 1.9 Hz)
and at δ 7.13 (d, J = 8.5 Hz), 7.82 (dd, J = 8.5, 1.9 Hz), and
8.13 (d, J = 1.9 Hz), respectively, reminiscent of two 1,2,4-
trisubstituted phenyl rings. The two-proton singlet at δ 8.04 is
reminiscent of a symmetrically tetrasubstituted phenyl ring.
Analysis of the 13C NMR and HMBC data revealed the
presence of two 3-bromo-4-hydroxy phenyl residues and a 3,5-
dibromo-4-hydroxyphenyl residue. The IR absorption (1754
cm−1) and the 13C NMR signals at δ 166.4, 157.3, 147.3, 123.3,
and 116.3 suggested that compound 5 contained, similarly to
rubrolide P (1), a butenolide ring bearing an exocyclic
trisubstituted double bond. The carbon signal at δ 186.4 and
the carbonyl stretching band at 1705 cm−1 in the IR spectrum
were indicative of the presence of a conjugated keto carbonyl
group, which is attached to C-2. The carbon signals at δ 157.3
and 123.3 could be assigned to the olefinic carbons positioned
β and α to this carbonyl group, respectively. The locations of
the three phenyl rings were assigned based on HMBC
correlations. The Z geometry at C-4/C-5 was deduced from
a 2D NOESY experiment, which showed correlations from H-5
(δ 6.42) to H-2′ (δ 7.67) and H-6′ (δ 7.34) in addition to
correlations from H-5 to H-2″ (δ 8.13) and H-6″ (δ 7.82).
The molecular formula of cadiolide D (6), an amorphous,
orange solid, was determined to be C24H11Br5O6 based on the
HRFABMS data. The 1H NMR data revealed that compound 6
differed from 5 only in the replacement of the 3-bromo-4-
hydroxyphenyl residue attached at C-3 in 5 with a 3,5-dibromo-
4-hydroxyphenyl residue in 6. The 13C NMR, COSY, HSQC,
and HMBC data for 6 were in complete agreement with this
assignment.
Table 2. 1H NMR Data for Cadiolides C−F (5−8) (acetone-
a
d6, 500 MHz)
position
5
6
7
8 (E)
7.16, s
8 (Z)
6.47, s
5
6.42, s
6.50, s
7.70, s
6.45, s
2′
7.67, d
(1.9)
7.67, d
(2.0)
7.20, d
(2.0)
7.66, d
(1.9)
5′
7.07, d
(8.3)
7.07, d
(8.3)
6.78, d
(8.3)
7.14, d
(8.4)
6′
7.34, dd
(8.3, 1.9)
7.70, s
7.34, dd
(8.3, 2.0)
7.00, dd
(8.3, 2.0)
7.34, dd
(8.4, 1.9)
2″
8.13, d
(1.9)
8.14, d
(1.9)
8.14, s
7.19, d
(1.9)
8.20, d
(1.9)
5″
7.13, d
(8.5)
7.14, d
(8.5)
6.82, d
(8.5)
7.24, d
(8.6)
6″
7.82, dd
7.84, dd
8.14, s
8.05, s
7.11, dd
7.95, dd
(8.5, 1.9)
(8.5, 1.9)
(8.5, 1.9)
(8.6, 1.9)
4″-OMe
2′″/6′″ 8.04, s
3.84, s
8.03, s
3.99, s
8.05, s
Cadiolide E18 (7) was isolated as an amorphous, brown solid,
and a formula of C24H11Br5O6 was indicated by the HRFABMS
data. This compound is a constitutional isomer of 6, with the
8.06, s
a
Multiplicities and coupling constants are in parentheses.
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Table 3. 13C NMR Data for Cadiolides C−F (5−8) (acetone-
d6, 125 MHz)
activity. The analysis of the structure−activity relationships of
these compounds may provide useful information on the
structural requirements for antibacterial activity against Gram-
positive bacteria, including drug-resistant strains. In addition, it
should be noted that none of these compounds showed
significant cytotoxicity in the MTT assay at 100 μM. Therefore,
cadiolides could serve as new lead compounds for the
development of antibiotics for the treatment of bacterial
infections caused by Gram-positive bacteria such as Staph-
ylococcus aureus. The cadiolides may also provide a tool for
identifying new drug templates, perhaps with a novel
mechanism of action, which would be significant for the
development of novel antibiotics because the cadiolides possess
a totally different scaffold from the benchmark antibiotics.
position
5
6
7
8 (E)
8 (Z)
1
166.4
123.3
157.3
147.3
116.3
186.4
122.7
135.0
110.9
157.2
117.5
131.2
127.7
136.9
110.9
156.7
117.8
133.0
166.2
124.1
156.7
147.0
117.0
186.1
123.7
134.2
111.7
153.4
111.7
134.2
127.6
137.0
111.0
156.8
117.8
133.1
166.1
123.8
157.5
148.2
114.5
186.4
122.4
135.1
110.9
157.2
117.5
131.3
129.0
135.7
111.9
152.8
111.9
135.7
166.6
123.3
153.9
148.9
119.0
186.4
123.3
134.5
110.7
156.7
117.0
130.4
126.7
135.6
111.7
157.3
112.6
131.9
56.9
166.5
123.3
157.3
147.9
115.7
186.4
122.6
135.0
110.0
157.5
117.6
131.2
128.4
136.4
112.6
158.1
113.5
133.2
57.0
2
3
4
5
6
1′
2′
3′
4′
5′
6′
EXPERIMENTAL SECTION
1″
2″
3″
4″
5″
6″
4″-OMe
1″′
2″′
3″′
4″′
5″′
6″′
■
General Experimental Procedures. UV spectra were obtained in
MeOH using a Scinco UVS-2100 spectrophotometer. IR spectra were
measured by a Thermo Electron Corp. Nicolet 570 spectrometer. H
1
and 13C NMR spectra were recorded on a Bruker Avance DPX-500
instrument. Chemical shifts were reported with reference to the
respective solvent peaks and residual solvent peaks (δH 2.05, δC 29.9
and 206.7 for acetone-d6). FABMS spectra were measured on a JEOL
JMS-AX505WA mass spectrometer. HPLC was performed with a
Synersi Fusion-RP column (250 × 10 mm, 4 μm, 80 Å) using a
Younglin M 720 UV detector.
130.9
134.9
111.8
157.4
111.8
134.9
131.6
134.9
111.4
156.3
111.4
134.9
131.4
134.9
111.5
156.4
111.5
134.9
130.8
134.9
111.8
157.6
111.8
134.9
130.9
134.9
111.8
157.7
111.8
134.9
Animal Material. The tunicate was collected with the aid of scuba
gear at a depth of 10−15 m off the shore of Tong-Yeong City, South
Sea, Korea, in July 2005. The fresh specimens were immediately frozen
and kept at −24 °C until chemically investigated. The specimen was
taxonomically identified as Pseudodistoma antinboja (order Aplouso-
branchia, family Pseudodistomidae) by one of the authors (B.J.R.). A
voucher specimen (BYD-25) was deposited in the Center for Marine
Natural Products and Drug Discovery, Seoul National University,
Seoul, Korea.
ATCC 14028, and Klebsiella pneumoniae ATCC 4352.
Cadiolides 5−9 exhibited significant antibacterial activity
against all of the drug-resistant Gram-positive strains (Table 5).
It seems that the compounds of the cadiolide family present a
new class of antibiotics active against Gram-positive bacteria.
Although both cadiolides and rubrolides share the same 3-aryl-
4-arylmethylenefuranone unit, the former is distinguished by an
additional 2-ketoaryl substituent and a different carbon
skeleton. It is obvious that, generally, all the cadiolides
exhibited activities better than or comparable to (especially
against drug-resistant strains) compound 3, which was reported
to have antibiotic activity (the antibacterial activity of
compound 4 has not been reported previously). These data
suggest that the benzoyl substituent at C-2 may be beneficial
for stable and potent antibacterial activity. The most potent
compound in the cadiolide series was 5, with MICs against
Gram-positive bacteria ranging from 0.2 to 3.1 μg/mL and with
MICs against drug-resistant Gram-positive strains ranging from
0.13 to 0.5 μg/mL. The positions of the bromines appear to be
important. The phenolic group is also likely responsible for the
good activity because methylation of the phenolic group led to
a reduction in the overall activity, as exemplified by compound
8, which is less active than 5, and by compounds 1 and 2, which
displayed only weak to modest antibacterial activity. The
4′,4″,4″′-trimethylation of 5 caused the complete loss of its
Extraction and Isolation. The lyophilized specimens (dry wt 336
g) were cut into small pieces and extracted three times with 50%
MeOH in CH2Cl2 (700 mL) at room temperature. These extracts
were combined and partitioned three times between n-hexane (500
mL) and MeOH (500 mL). Then, the MeOH-soluble layer was
further partitioned between EtOAc (500 mL) and H2O (500 mL)
three times. The EtOAc extract (1.15 g) was active in the antibacterial
assay and was subjected to silica flash column chromatography, eluting
with a step gradient of EtOAc and n-hexane, to afford 10 fractions.
Active fractions were further separated by reversed-phase HPLC
(Synersi Fusion-RP, 250 × 10 mm, 4 μm, 80 Å, 210 nm), eluting with
75% CH3CN in H2O (0.0025% TFA), to afford compounds 1−9 as
amorphous solids. The overall purified metabolites were isolated in the
following amounts: 2.0, 2.3, 1.9, 2.6, 20.6, 17.0, 19.4, 3.3, and 22.3 mg
for 1−9, respectively.
Rubrolide P (1): yellow, amorphous solid; UV (MeOH) λmax (log
ε) 254 (3.89), 360 (4.00) nm; IR (film) νmax 1736, 1602, 1510, 1468,
1
1255, 1175 cm−1; H NMR data, see Table 1; 13C NMR data, see
Table 1; LRFABMS m/z 451/453/455 [M + H]+; HRFABMS m/z
450.9185 (calcd for C18H1379Br2O4, 450.9181).
Rubrolide Q (3′-bromorubrolide F, 2): yellow, amorphous solid;
UV (MeOH) λmax (log ε) 253 (3.87), 344 (4.04) nm; IR (film) νmax
1736, 1602, 1510, 1301, 1255, 1175 cm−1; 1H NMR data, see Table 1;
Table 4. Antibacterial Activities (MIC μg/mL) against Non-Drug-Resistant Strains of Compounds 1−9
tested strain
gentamycin
1
2
3
4
5
6
7
8
9
Staphylococcus aureus ATCC 6538
Staphylococcus epidermidis ATCC12228
Kocuria rhizophila ATCC 9341
Bacillus subtilis ATCC 6633
<0.02
<0.02
1.6
>50
50
>50
25
3.1
1.6
1.6
1.6
3.1
0.8
6.3
1.6
0.4
0.4
0.2
3.1
6.3
0.8
1.6
6.3
3.1
6.3
0.2
3.1
12.5
6.3
3.1
3.1
3.1
3.1
6.3
50
3.1
3.1
0.05
50
12.5
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Table 5. Antibacterial Activities (MIC μg/mL) against MSSA and MRSA Strains of Compounds 1−9
microorganisms
platensimycin
linezolid
vancomycin
daptomycin
1
2
3
4
5
6
7
8
9
a
CCARM 0027
4
4
4
8
8
8
8
8
4
2
2
4
2
4
4
4
0.5
0.25
<0.13
0.25
1
16
4
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
4
4
4
8
8
8
8
8
2
2
1
2
4
2
2
2
0.5
0.25
0.25
<0.13
0.5
0.5
2
2
4
4
2
1
1
2
1
2
1
2
a
CCARM 0204
0.5
1
a
CCARM 0205
2
0.25
0.25
0.5
2
a
CCARM 3640
16
0.25
1
1
b
CCARM 3089
>32
>32
>32
>32
1
b
CCARM 3090
1
0.25
0.5
0.25
1
1
1
b
CCARM 3634
0.5
2
1
b
CCARM 3635
1
<0.13
0.25
0.5
0.5
a
b
Methicillin-sensitive Staphylococcus aureus (MSSA). Methicillin-resistant Staphylococcus aureus (MRSA).
13C NMR data, see Table 1; LRFABMS m/z 373/375 [M + H]+;
HRFABMS m/z 373.0071 (calcd for C18H1479BrO4, 373.0075).
Cadiolide C (5): yellow-orange, amorphous solid; UV (MeOH)
λmax (log ε) 254 (4.36), 358 (4.33), 421 (4.04) nm; IR (film) vmax
CCARM 3090, Staph. aureus CCARM 3634, and Staph. aureus
CCARM 3635. The antibacterial activity was determined by the 2-fold
microtiter broth dilution method.17 Dilutions of the test compounds
dissolved in DMSO were added to each well of a 96-well microtiter
plate containing a fixed volume of Mueller Hinton broth (Difco) (final
0.64% DMSO). Each well was inoculated with an overnight culture of
bacteria (5 × 105 cfu/mL), and the plate was incubated at 37 °C for 24
h. The minimum inhibitory concentration (MIC) was taken as the
concentration at which no growth was observed.
1
1754, 1705, 1644, 1579, 1298 cm−1; H NMR data, see Table 2; 13C
NMR data, see Table 3; LRFABMS m/z 464/466/468, 634/636/638/
640, 713/715/717/719/721 [M + H]+, 735/737/739/741/743 [M +
Na]+; HRFABMS m/z 712.7447 (calcd for C24H1379Br4O6, 712.7446).
Cadiolide D (6): orange, amorphous solid; UV (MeOH) λmax (log
ε) 218 (4.00), 256 (3.79), 385 (3.93) nm; IR (film) νmax 1742, 1707,
1
1660, 1567, 1382 cm−1; H NMR data, see Table 2; 13C NMR data,
ASSOCIATED CONTENT
* Supporting Information
■
see Table 3; LRFABMS m/z 541/543/545/547, 790/792/794/796/
798/800 [M + H]+, 812/814/816/818/820/822 [M + Na]+;
HRFABMS m/z 790.6533 (calcd for C24H1279Br5O6, 790.6551).
Cadiolide E (7): brown, amorphous solid; UV (MeOH) λmax (log
ε) 231 (4.23), 258 (4.23), 363 (4.38), 486 (3.76) nm; IR (film) νmax
S
Full characterization details for the compounds 3, 4, and 9 and
NMR spectra for compounds 1−9. This material is available
free of charge via the Internet at http://pubs.acs.org.
1
1744, 1703, 1564, 1294 cm−1; H NMR data, see Table 2; 13C NMR
AUTHOR INFORMATION
Corresponding Author
*Tel: 82-2-880-5730. Fax: 82-2-883-9289. E-mail: hjkang@snu.
ac.kr.
data, see Table 3; LRFABMS m/z 541/543/545/547, 790/792/794/
796/798/800 [M + H]+, 812/814/816/818/820/822 [M + Na]+;
HRFABMS m/z 790.6530 (calcd for C24H1279Br5O6, 790.6551).
Cadiolide F (8): yellow, amorphous solid; UV (MeOH) λmax (log
ε) 222 (4.08), 255 (4.01), 352 (4.18) nm; IR (film) νmax 1753, 1680,
■
Author Contributions
1
1640, 1599, 1270 cm−1; H NMR data, see Table 2; 13C NMR data,
‡These authors contributed equally to the work.
see Table 3; LRFABMS m/z 727/729/731/733/735 [M + H]+, 749/
751/753/755/757 [M + Na]+; HRFABMS m/z 726.7618 (calcd for
C25H1579Br4O6, 726.7602).
Notes
The authors declare no competing financial interest.
Preparation of 4′,4″,4″′-Trimethylated Cadiolide C. Dimethyl
sulfate (4 mL) was added to the acetone solution (5 mL) containing
compound 5 (5 mg) and anhydrous K2CO3 (10 mg). The resulting
solution was refluxed at 50 °C for 24 h. After cooling to room
temperature, the solvent was removed in vacuo. The residue was
dissolved in distilled water (3 mL) and diethyl ether (3 mL), and the
aqueous layer was further extracted twice with diethyl ether. After
evaporation of the solvent under reduced pressure, the residue was
chromatographed over silica gel (hexane−EtOAc = 9.5:0.5) to give the
desired product (4.2 mg). 1H NMR (500 MHz, CDCl3) δ 8.32 (2H, s,
H-2′″/6′″), 8.12 (1H, d, J = 2.1 Hz, H-2″), 7.88 (1H, dd, J = 8.2, 2.1
Hz, H-6″), 7.86 (1H, d, J = 2.0 Hz, H-2′), 7.55 (1H, dd, J = 8.4, 2.0
Hz, H-6′), 6.97 (1H, d, J = 8.2 Hz, H-5″), 6.90 (d, 1H, J = 8.4 Hz, H-
5′), 6.85 (1H, s, H-6), 3.99 (3H, s, 4″-OMe), 3.98 (3H, s, 4′″-OMe),
3.93 (3H, s, 4′-OMe); 13C NMR (75 MHz, CDCl3) δ 187.3, 166.4,
159.4, 158.2, 157.7, 157.2, 147.9, 143.3, 136.4, 135.4, 134.7, 133.2,
130.8, 130.7, 130.2, 127.8, 126.6, 125.6, 118.7, 112.1, 111.9, 111.1,
60.8, 56.5, 56.3.
Bioassay Procedures. The following seven microorganisms were
obtained from the stock culture collection at the American Type
Culture Collection (Maryland): Staphylococcus aureus ATCC 6538,
Kocuria rhizophila (Micrococcus luteus) ATCC 9341, Staph. epidermidis
ATCC12228, Bacillus subtilis ATCC 6633, Escherichia coli ATCC
11775, Salmonella typhimurium ATCC 14028, and Klebsiella pneumo-
niae ATCC 4352. The following eight drug-resistant strains were
obtained from the stock Culture Collection of Antimicrobial Resistant
Microorganisms (Seoul Women’s University): Staph. aureus CCARM
0027, Staph. aureus CCARM 0204, Staph. aureus CCARM 0205, Staph.
aureus CCARM 3640, Staph. aureus CCARM 3089, Staph. aureus
ACKNOWLEDGMENTS
■
H.K. was in part supported by the BK21 Program, Ministry of
Education, Science and Technology. This work was supported
by the Marine Biotechnology Program, the Ministry of Land,
Transport and Maritime Affairs, Korea.
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