Aflaquinolones A-G: Secondary metabolites from marine and fungicolous isolates of aspergillus spp.

Article abstract of DOI:10.1021/np200958r

Seven new compounds (aflaquinolones A-G; 1-7) containing dihydroquinolin-2-one and terpenoid units have been isolated from two different fungal sources. Two of these metabolites (1 and 2) were obtained from a Hawaiian fungicolous isolate of Aspergillus sp. (section Flavipedes; MYC-2048 = NRRL 58570), while the others were obtained from a marine Aspergillus isolate (SF-5044) collected in Korea. The structures of these compounds were determined mainly by analysis of NMR and MS data. Relative and absolute configurations were assigned on the basis of NOESY data and ¹H NMR J-values, comparison of calculated and experimental ECD spectra, and analysis of a Mosher's ester derivative of 2. Several known compounds, including alantrypinone, aspochalasins I and J, methyl 3,4,5-trimethoxy-2((2-((3-pyridinylcarbonyl)amino)benzoyl) amino)benzoate, and trans-dehydrocurvularin were also encountered in the extract of the Hawaiian isolate.

Full text of DOI:10.1021/np200958r

Article
pubs.acs.org/jnp
Aflaquinolones A−G: Secondary Metabolites from Marine and
Fungicolous Isolates of Aspergillus spp.
Scott A. Neff,† Sang Un Lee,‡ Yukihiro Asami,§ Jong Seog Ahn,§ Hyuncheol Oh,‡ Jonas Baltrusaitis,†
James B. Gloer,*,† and Donald T. Wicklow⊥
†Department of Chemistry, University of Iowa, Iowa City, Iowa 52242, United States
‡College of Medical and Life Sciences, Silla University, Busan 617-736, Korea
§Korea Research Institute of Bioscience and Biotechnology (KRIBB), 52 Eoun-dong, Yuseong-gu, Daejeon 305-333,
Republic of Korea
⊥Bacterial Foodborne Pathogens and Mycology Research Unit, Agricultural Research Service, National Center for Agricultural
Utilization Research, USDA, Peoria, Illinois 61604, United States
S
* Supporting Information
ABSTRACT: Seven new compounds (aflaquinolones A−G;
1−7) containing dihydroquinolin-2-one and terpenoid units
have been isolated from two different fungal sources. Two of
these metabolites (1 and 2) were obtained from a Hawaiian
fungicolous isolate of Aspergillus sp. (section Flavipedes; MYC-
2048 = NRRL 58570), while the others were obtained from
a marine Aspergillus isolate (SF-5044) collected in Korea. The
structures of these compounds were determined mainly by
1
analysis of NMR and MS data. Relative and absolute configurations were assigned on the basis of NOESY data and H NMR
J-values, comparison of calculated and experimental ECD spectra, and analysis of a Mosher’s ester derivative of 2. Several known
compounds, including alantrypinone, aspochalasins I and J, methyl 3,4,5-trimethoxy-2((2-((3-pyridinylcarbonyl)amino)benzoyl)-
amino)benzoate, and trans-dehydrocurvularin were also encountered in the extract of the Hawaiian isolate.
ur long-term studies of fungi from a variety of ecological
groups have resulted in the discovery of many new bio-
and 2, as well as the known metabolites alantrypinone,11
aspochalasins I and J,12 methyl 3,4,5-trimethoxy-2((2-((3-
pyridinylcarbonyl)amino)benzoyl)amino)benzoate,13 and trans-
dehydrocurvularin.14 The known compounds were identified by
analysis of MS and NMR spectroscopic data in comparison with
literature values.11−14
Aflaquinolone A (1) was found to have the molecular formula
C26H29NO5 (13 unsaturations) on the basis of HRESITOFMS
and NMR data. The 1H NMR spectrum of 1 (Table 1) exhibited
signals for a phenyl group, an isolated trans olefin unit, a pair of
ortho-coupled aromatic protons indicative of a 1,2,3,4-tetrasub-
stituted benzene ring, an isolated amide NH, and three methyl
groups, including one methoxy group. There were also numerous
signals for diastereotopic methylene protons. 13C NMR data
(Table 1) revealed the presence of two carbonyl carbons (one
ketone and one amide), 14 aromatic/olefinic carbons (corre-
sponding to the two aromatic rings and the trans olefin), and two
quaternary sp3 carbons. Chemical shift data indicated that one
of the aromatic ring carbons is oxygenated (δC 155.0). The
remaining eight carbon signals were located in the aliphatic
region of the spectrum. These units account for 11 degrees of
unsaturation, requiring two additional rings to be present.
O
active natural products.1−3 Fungicolous and mycoparasitic
isolates from Hawaii have been a productive source of such
compounds over the past few years,4−6 as have marine fungi.7
Chemical investigations of two isolates of Aspergillus sp.
(Trichocomaceae) obtained from these two different habitats
carried out independently in our two laboratories led to the
isolation of seven new dihydroquinolin-2-one-containing natural
products that we named aflaquinolones A−G (1−7). These
compounds are members of a known general class of fungal
metabolites that includes aspoquinolones and penigequino-
lones.8−10 Details of the isolation and structure elucidation of
these compounds are presented here.
RESULTS AND DISCUSSION
■
A culture of Aspergillus sp. (MYC-2048 = NRRL 58570) was
obtained from a basidioma of Rigidoporus microsporus found
on a dead hardwood branch in a Hawaiian alien wet forest. The
fungus was cultured by solid-substrate fermentation on rice, and
the EtOAc extract of the resulting fermentation mixture showed
antifungal activity against Aspergillus flavus (NRRL 6541) and
Fusarium verticillioides (NRRL 25457) as well as the ability to
reduce the growth rate of Spodoptera frugiperda (fall army-
worm) in a dietary assay. The extract was therefore subjected to
chemical investigation, leading to the isolation of compounds 1
Special Issue: Special Issue in Honor of Gordon M. Cragg
Received: December 9, 2011
Published: February 1, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
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Table 1. NMR Spectroscopic Data for Aflaquinolones A (1)
a
and B (2) in CDCl3
1
2
position
δC
δH, mult (J in Hz)
δC
δH, mult (J in Hz)
7.44, br s
1-NH
2
7.52, br s
165.1
84.0
165.2
84.1
3
3.67, d (1.5)
3.65, d (1.5)
4
79.0
78.9
5
110.9
155.0
122.3
127.2
106.9
134.3
137.2
128.9
126.2
129.2
122.5
135.9
37.3
110.8
154.7
121.2
127.1
106.8
133.9
137.8
128.9
126.3
129.3
123.0
137.4
30.7
6
7
8
7.40, d (8.3)
6.35, d (8.3)
7.35, d (8.3)
6.32, d (8.3)
9
10
11
12/16
13/15
14
17
18
19
20eq
20ax
21
7.26−7.33, m
7.26−7.33, m
7.30, m
7.25−7.32, m
7.25−7.32, m
7.25−7.32, m
6.58, d (17)
6.06, d (17)
6.67, d (17)
6.26, d (17)
47.5
2.09, m
40.0
1.32−1.45, m
1.32−1.45, m
1.55−1.73, m
3.72, br q (2.7)
1.55−1.73, m
1.55−1.73, m
1.55−1.73, m
1.55−1.73, m
1.01, s
1.45, t (13)
41.3
213.7
38.6
2.53, dpent (13, 6.0)
37.1
70.1
32.4
b
22
c
c
The structure of the dihydroquinolone portion of 1 was
established by analysis of 2D NMR data and through com-
parison to a known series of compounds that includes the
aspoquinolines and penigequinolones.7−9 HMBC correlations
from the H-12/H-16 signal for the phenyl group to C-4; from
the isolated oxymethine H-3 to C-2, C-4, C-5, and C-11; and
from the amide NH to C-5 and C-10 were consistent with
the corresponding features of the structure of aspoquinolone C,
albeit without the para-methoxy group on the aromatic ring
substituent.8
23ax
23eq
24eq
24ax
25
2.47, ddd (14, 14, 5.8)
2.22, ddd (14, 4.5, 2.5)
2.13, m
38.4
31.7
1.71, td (14, 4.5)
1.09, s
30.4
14.4
58.9
30.1
18.3
58.9
26
0.96, d (6.4)
3.60, s
0.91, d (6.9)
3.59, s
27
4-OH
6-OH
4.60, s
4.58, s
9.08, s
9.02, s
a
b
Data were collected at 400 MHz (1H) or 100 MHz (13C). A 22-OH
1H NMR signal for 2 was not observed, presumably due to signal
The remaining portion of the structure was significantly
different from those of the aforementioned compounds. HMBC
correlations of H3-25 to C-20 and C-24 and correlations of
H3-26 to C-20 and C-22, with the last remaining methylene unit
(C-23) bridging C-22 and C-24, complete a cyclohexane ring,
accounting for the remaining unit of unsaturation. This ring was
connected to the trans olefin unit at C-18 on the basis of
correlations of H-17 and H-18 to C-19 and of H3-25 to C-18.
Finally, the two main structural units of 1 were linked on the
basis of HMBC correlations of both trans olefinic protons to C-7
to complete the gross structure as shown. Additional HMBC
data were fully consistent with this conclusion. The differences
in structures relative to those of the aspoquinolones led to the
proposal of a distinct name for 1 (aflaquinolone A). However,
the numbering system shown is consistent with that of the
aspoquinolones.7
c
broadness. These assignments may be interchanged.
a long-range “w-coupling”. NOESY results for this structural
unit were also analogous to those previously reported.
Key NOESY data included correlations of H-3 with signals for
both the phenyl group and the 4-OH (requiring H-3 to be
pseudoequatorial), as well as a correlation from the methoxy
group (C-27) to the 4-OH, all of which are consistent with
literature observations for molecules having the relative config-
uration shown for 1 at C-3 and C-4.10 The relative config-
uration of the left-hand portion of 1 was assigned on the basis
of NMR J-values and NOESY data (Table 1 and Figure 1).
H-21 was assigned an axial (or pseudoaxial) orientation by virtue
of a large trans-diaxial coupling (J = 13 Hz) with Hax-20. NOESY
correlations of H-21 with both Hax-23 and olefinic proton H-17
supported this assignment and placed H-21 and the trans olefin
unit on the same face of the cyclohexanone ring. Further NOESY
correlations shown in Figure 1 were consistent with these con-
clusions. On the basis of these data, the cyclohexanone unit of
1 was assigned the relative configuration shown. However, no
NOESY correlations were observed that enabled relative stereo-
chemical correlation of the cyclohexanone and dihydroquinolone
portions of the molecule.
1
Analysis of NOESY data and H NMR J-values enabled
assignment of the relative configuration of each half of 1. The
dihydroquinolone unit of 1 exhibited NMR shifts and J-values
virtually identical to those of the corresponding unit in the
aspoquinolones. In addition, the appearance of the H-3 signal
as a doublet long-range-coupled to the NH (1.5 Hz) matched a
characteristic signal and J-value described for these compounds
in the literature.8 Observation of such a J-value would be
consistent with the near coplanarity of the H-3 and NH bonds
reflected in the energy-minimized molecular model of 1, which
approximates the kind of arrangement that sometimes results in
ECD (electronic circular dichroism) data were collected for 1
and matched closely with the spectrum of the literature com-
pound peniprequinolone,10 a member of this class having a
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Figure 1. Key NOESY correlations for the terpene-derived portions of
aflaquinolones A (1) and B (2).
simple prenyl substituent at C-7 and a para-methoxyphenyl
group in place of the phenyl group of 1. A simpler, co-occurring
metabolite in the same report lacking the prenyl group also
afforded an ECD curve of similar shape.10 However, these data
were provided to help characterize the compounds and were
not used to propose the absolute configuration. The similarity
in ECD data among these compounds suggested that the shape
of the ECD curve is dictated largely by the configuration of the
dihydroquinoline unit and that these three compounds all share
the same absolute configuration in that portion of the molecule.
However, the literature does not offer definitive assignment
of absolute configuration for a member of this class, and the
structure does not lend itself well to stereochemical analysis by
standard empirical methods. Ultimately, TDDFT computa-
tional methods proved to be helpful in making a stereochemical
assignment.
Figure 2. Experimental ECD spectrum (top) and TDDFT-calculated
ECD spectrum with energy-minimized model (bottom) for aflaquino-
lone A (1).
After geometry optimization of each possible isomer of 1 to
obtain minimum energy conformers, TDDFT-calculated,
smoothed ECD spectra were generated for each and compared
with the experimental data. Comparison of the experimental
and calculated spectra for 1 showed excellent agreement for the
3S, 4S-absolute configuration at C-3 and C-4 in 1 (Figure 2),
regardless of the choice of configuration for the cyclohexanone
portion of the molecule. Both the calculated and experimental
data spectra showed a pair of positive Cotton effects (CE)
above 275 nm, a negative CE near 250−260 nm, and a positive
CE below 220 nm. These close similarities enabled assignment
of the absolute configuration for the dihydroquinoline unit of 1
as shown. However, those of the remote terpenoid-derived
portion could not be assigned by these methods. This issue is
addressed further below.
equatorial position and indicating that the new C-22 hydroxy
group adopts an axial orientation. A NOESY correlation
between the axial H-21 and H-17 of the disubstituted olefin
unit required these two units to be on the same face of the
molecule, thereby setting the relative configuration at C-19.
Other NOESY data (Figure 1) were consistent with these
relative stereochemical assignments. The data also showed
correlations for the dihydroquinolone moiety that matched
those observed for 1, enabling assignment of the analogous
relative configuration at C-3 and C-4.
The ECD spectrum collected for 2 was virtually identical to
that of 1, enabling assignment of the analogous 3S, 4S-absolute
configuration to the dihydroquinolinone portion of the
molecule. Unlike compound 1, the presence of the secondary
alcohol moiety on the terpenoid-derived unit of 2 suggested
that the absolute configuration of this portion of the molecule
could be assigned through the use of Mosher’s method.15
Treatment of 2 with R-(−)-MTPA-Cl or S-(+)-MTPA-Cl in
pyridine-d6 and CDCl3 afforded the S-MTPA ester (2a) or
R-MTPA ester (2b), respectively. Formation of the esters was
confirmed by the significant downfield shift of the signal for
H-22 in each case and the appearance of the expected new
Compound 2 was assigned the molecular formula
C26H31NO5 (12 unsaturations) on the basis of HRESITOFMS
and NMR data. The structure of 2 was nearly identical to that
of 1. The main difference was evident in the absence of a
1
ketone signal in the 13C NMR spectrum, the appearance of H
and 13C NMR signals for an oxymethine unit (CH-22), and
associated changes in shifts and multiplicities of nearby
diastereotopic protons (Table 1). These observations indicated
that 2 differs from 1 by reduction of the C-22 ketone unit to a
1
1
aromatic and methoxy signals in the H NMR spectra. The
secondary alcohol moiety. Some of the key H NMR J-values
analysis was complicated somewhat by the formation of minor
products arising from a second acylation at the phenolic OH
group. However, assignment of H NMR signals for relevant
were difficult to measure due to overlap in the upfield region,
but a spectrum in acetone-d6 afforded better resolution of these
signals. The resulting data indicated that the axial proton at
C-20 showed a large trans-diaxial-type coupling (12.5 Hz) to
H-21, indicating that H-21 must be axially oriented, and placing
the C-21 methyl group in an equatorial position. The oxymethine
signal (H-22) shows only small couplings, with a large trans-
diaxial coupling clearly absent, thereby placing H-22 in an
1
portions of 2a and 2b was accomplished by comparison with
1
the data for 2 and verified by H−1H decoupling experiments.
The resulting Δδ values observed for key signals of 2a and 2b
(Figure 3) were consistent with the S-configuration at C-22,
leading to assignment of the overall absolute configuration
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Isolation of reduced analogue 2 from the same source as 1 and
the assignment of its absolute configuration by Mosher’s method
as noted above led us to propose that 1 has the absolute config-
uration analogous to that of 2, thereby enabling the proposal
of the opposite cyclohexanone unit absolute configuration for 3,
as shown.
1
The H NMR spectrum of aflaquinolone D (4), another
isomer of 1 and 3 as established by HRESIMS data, was almost
identical to those of 1 and 3 except for some chemical shift
variations of signals corresponding to the C-17 to C-26 portion
of the molecule (Table 1). Therefore, aflaquinolone D was
presumed to be another stereoisomer of compounds 1 and 3.
Figure 3. Observed chemical shift differences (Δδ = δS − δR, ppm;
400 MHz) for the R- and S-MTPA esters of aflaquinolone B (2).
shown for 2. The absolute configuration for the cyclo-
hexanone unit of 1 was then proposed as shown by analogy to
2. The name aflaquinolone B is proposed for compound 2.
In an effort to chemically correlate 1 and 2 to further support
the above conclusions, attempts were made to oxidize 2 to 1
under mild conditions using TPAP/NMO. Unfortunately, the
approaches employed led to decomposition and did not succeed
in providing detectable product. More extensive efforts toward this
end were hampered by sample limitations. However, treatment
of 1 with NaBH4 afforded a mixture of alcohol products, one of
which gave NMR signals and chromatographic properties identical
to those of 2.
In the course of this work, it came to our attention that
colleagues at another institution (coauthors on this manuscript)
had independently encountered and identified members of the
same class of compounds from a marine isolate of Aspergillus sp.
(SF-5044), including one metabolite that initially appeared to
be identical to 1. Because of this overlap, a decision was made
to pool our efforts and compile a joint report. Earlier studies
of this marine isolate led to isolation of an unrelated set of
compounds.7
1
Detailed analysis of H NMR and COSY data supported the
conclusion that 4 has the same planar structure as 1 and 3, and
the NOESY correlation of H-3 with the aromatic protons of the
phenyl group, along with ECD analysis, indicated that the
quinolin-2-one unit in 4 has the same absolute configuration as
in 1−3. However, comparisons of the NOESY data and J-values
of 4 with those of 1 and 3 revealed a difference in relative
configuration in the cyclohexanone unit A trans-diaxial J-value
(13 Hz) for H-21 and Hax-20 again indicated an axial orien-
tation for H-21 and an equatorial orientation for CH3-26.
However, in this instance, the H-21 signal showed a NOESY
correlation with H3-25, rather than H-17 as in 1−3, placing
CH3-25 in an axial orientation cis to H-21 and trans to CH3-26.
Correlations of Hax-20 with H-18 and H3-26 were consistent
with the location of all of these protons on the same face of the
cyclohexanone ring. Therefore, aflaquinolone D (4) was
identified as a diastereomer of 1 and 3 possessing an inverted
configuration at one of the centers in the cyclohexanone ring.
The structure shown for 4 displays an inverted orientation at C-
21 (α to the ketone carbonyl) relative to that of 3, but the other
possible diastereomer (i.e., with inversion at C-19 instead)
could not be ruled out. Unlike the case for 1 and 3, an isomer
with the same relative configuration in the cyclohexanone unit that
would have enabled an analogous comparison was not obtained
from the fungicolous fungal culture, and sample limitations
precluded further study of the issue for this compound.
The molecular formula of aflaquinolone E (5) was esta-
blished as C16H15NO4 on the basis of HRESIMS analysis and
NMR data, making it significantly smaller than 1−4. Analysis
of the 1H and 13C NMR data for 5 and comparison with those
of 1−4 suggested that 5 retains the phenylquinolin-2-one
skeleton, but lacks the terpenoid side-chain appended to the
aromatic ring in 1−4. The planar structure of 5 was readily
determined by independent analysis of 1D- and 2D-NMR data
(Table 2). NOESY correlation of H-3 with the aromatic
protons of the phenyl group again indicated that 5 possesses a
relative configuration analogous to those found in the quinolin-
2-one moieties of 1−4, and the ECD spectrum was again very
similar to those of 1−4, aside from a modest blue shift in
wavelength values, indicating analogous absolute configurations
as well. The observed shift to lower wavelength in the ECD
curve of 5 might be expected given the change in the chro-
mophore associated with the absence of the olefin unit, but the
overall shape was relatively unaffected. In fact, the similarity in
ECD spectral shape among all five metabolites 1−5 (Figure 4)
strongly supports the conclusion that the quinolin-2-one moiety
is dominant in dictating the shape of the ECD curve in this class
of compounds, regardless of the presence or absence of side-
chains like those found in 1−4 and any stereocenters that might
be associated with them.
The first of these compounds, aflaquinolone C (3), was
assigned the same molecular formula as 1 on the basis of
HRESIMS data and was initially thought to be identical to 1, as
the structure and relative configuration of the two stereo-
chemically insulated portions of the molecule were found to be
identical to those of 1 by independent analysis of COSY, HMBC,
1
and NOESY data, as well as H NMR J-values. Moreover,
the NMR shifts for the two compounds (as measured for
both samples in acetone-d6) were virtually identical by direct
comparison. However, despite the nearly identical ECD spectra
obtained for the two samples, the specific rotation values
recorded under matching conditions were of somewhat
different magnitudes and, more importantly, of opposite
sign. These observations led to the hypothesis that 1 and 3
have the same dihydroquinolinone unit absolute configuration
and the same relative configuration in the cyclohexanone ring,
but have opposite absolute configurations at both stereo-
centers in the latter unit. This would make the two com-
pounds diastereomers of one another, but because the two
stereogenic features in the structures are well insulated from
one another, they might be expected to possess virtually
identical NMR data. Their specific rotations might well differ,
even though their ECD curves might match. Indeed, a mixed
sample of 1 and 3 gave a single 1H NMR spectrum, as well as a
single peak by HPLC analysis on three different stationary
phases. While an inability to resolve the two stereoisomers
was somewhat unexpected, the reproducible difference in the
sign (and magnitude) of specific rotation for 1 and 3, together
with the accompanying virtually identical ECD spectra,
was consistent with this hypothesis. It has been noted that
diastereomers having stereochemical differences in regions
remote from one another can give identical NMR spectra.16
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a
Table 2. NMR Spectroscopic Data for Aflaquinolones E−G (5−7) in CD3OD
5
6
7
position
δC
δH, mult (J in Hz)
δC
δH, mult (J in Hz)
δC
δH, mult (J in Hz)
2
169.1
86.3
172.7
75.8
172.6
76.6
3
3.64, s
4.76, s
4.58, s
4
79.9
78.7
78.2
5
113.0
159.1
108.1
131.0
113.2
138.1
140.9
127.6
129.6
129.8
59.2
130.1
130.1
124.0
130.6
117.0
138.4
143.2
128.3
129.0
128.4
132.4
127.6
124.8
130.1
116.8
136.7
141.5
128.6
128.7
128.7
6
6.71, dd (7.8, 2.1)
6.90, t (7.8)
7.53, dd (7.7, 1.2)
7.11, t (7.7)
7
6.45, d (8.0)
7.14, t (8.0)
6.53, d (8.0)
8
7.25, dt (7.8, 1.4)
6.95, d (7.8)
7.29, dt (7.7, 1.2)
6.95, d (7.7)
9
10
11
12/16
13/15
14
7.24−7.29, m
7.26−7.31, m
7.26−7.31, m
3.52, s
7.51, d (7.5)
7.39, d (7.5)
7.32, t (7.5)
7.34, d (8.0)
7.19−7.25, m
7.19−7.25, m
17
a
Data were collected at 400 MHz (1H) or 100 MHz (13C).
Figure 4. ECD spectra of aflaquinolones A−E (1−5).
HRESIMS data established the molecular formula
consistent with the absence of a NOESY correlation between
H-3 and the protons of the phenyl group in the data for 7.
A literature search revealed that similar spectroscopic patterns
were observed for the two diastereomers of 3,4-dihydroxy-4-
(4′-methoxyphenyl)-3,4-dihydro-2(1H)-quinolinone.17
1
C15H13NO3 for both aflaquinolones F (6) and G (7). H and
13C NMR data of 6 and 7 showed almost identical patterns with
some minor chemical shift variations. Comparison of these data
1
with the H and 13C NMR data for 5 indicated replacement of
Comparison of the ECD spectra of 6 and 7 with that of 5,
the closest relative among the other metabolites encountered,
showed some differences in both cases. However, as might be
expected given the relative configuration assignments for 6 and
7, the data for 6 more closely resembled those of 5, as both
compounds show a positive CE at approximately 270 nm,
a negative CE near 250 nm, and a strongly positive CE below
230 nm. By contrast, the ECD data for 7 included a negative
CE at 286 nm not present in any of the spectra for compounds
1−6, although other ECD features retained some resemblance
with those of 1−6. Thus, while the absolute configuration of
6 was assigned to match those of 1−5, that of 7 (the only
metabolite encountered in this study with a trans orientation of
the phenolic OH group with an additional aromatic proton and
also revealed the absence of the methoxy group (Table 2).
These observations led to the proposal that 6 and 7 are dia-
stereomers of one another, each having a 3,4-dihydroxy-4-
phenyl-3,4-dihydro-2(1H)-quinolinone structure. Detailed anal-
ysis of 2D-NMR data confirmed this proposal. The relative
configurations of 6 and 7 were determined by analysis of
NOESY data. NOESY correlation of H-3 with aromatic protons
of the phenyl group in 6 indicated a cis arrangement of the two
hydroxy groups in 6, thus leading to assignment of the same
relative configuration found in the quinolin-2-one moieties of
1−5. In order for 7 to be a diastereomer of 6, a trans orientation
of the two hydroxy groups is required, and this assignment is
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the oxygen substituents at C-3 and C-4) could not be assigned
with confidence by comparison of the experimental ECD
curves. In an effort to resolve this ambiguity, we again
employed computational methods to calculate ECD spectra
for the two possible enantiomers of 7. Although the calculated
absorption wavelengths (Figure 5) did not match with the
are remote from this unit remains a complicated issue and would
still require independent analysis on a case-by-case basis.
The fungicolous Aspergillus isolate from Hawaii (MYC-2048 =
NRRL 58570) was initially suggested to be Aspergillus flavipes
Section Flavipedes on the basis of morphological characteristics.
However, DNA sequence information and subsequent BLAST
queries of GenBank produced a 100% match with an undescribed
species of Aspergillus sp. NRRL 32683 (= MYC-1580 = NRRL
58569), another isolate from our own collection that was
shown to be most closely related to Aspergillus aureofulgens
Section Flavipedes (NRRL 6326).18 The NRRL 6326 isolate
was originally described from truffle soil in France. Its
occurrence in truffle soil is intriguing because this environment
is also suggestive of a possible fungicolous origin.
Neither compound 1 nor 2 showed bioactivity that would
account for the initial activity seen in the extract of the
Hawaiian isolate. However, numerous known compounds were
also encountered in the extract, including alantrypinone,11
aspochalasins I and J,12 methyl 3,4,5-trimethoxy-2((2-((3-
pyridinylcarbonyl)amino)benzoyl)amino)benzoate,13 and trans-
dehydrocurvularin.14 The abundance of these compounds in the
extract, particularly the aspochalasin and curvularin analogues,
which are known to display various bioactivities, likely explains
the effects originally observed. All of the aflaquinolones except
for 4 were evaluated for growth inhibitory activity against chronic
myelogenous leukemia cells (K562), murine melanoma cells
(B16F10), human acute promyelocytic leukemia cells (HL-60),
human breast cancer adenocarcinoma cells (MDA-MB-231),
and hepatocellular carcinoma cells (Hep3B) using in vitro cell
viability assays. While some of the compounds showed effects
at high concentrations, all were effectively inactive, with IC50
values > 80 μM.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured with a Rudolph automatic polarimeter, model APIII. UV
data were recorded with a Varian Cary III UV−visible spectropho-
tometer, and ECD data were collected using an Olis Cary-17
1
instrument (0.1 cm cell). H NMR, 13C NMR, and NOESY spectra
were recorded using Bruker AVANCE-400, DRX-400, or AVANCE-
600 or JEOL JNM ECP-400 spectrometers. Chemical shift values were
referenced to residual solvent signals for CDCl3 (δH/δC, 7.24/77.0),
acetone-d6 (δH/δC, 2.05/29.8), or CD3OD (δH/δC, 3.31/49.0). HMQC
and HMBC data were recorded using the Bruker AVANCE-600
instrument. All 13C NMR multiplicities were determined by analysis
of DEPT and/or HMQC data and are consistent with the position
assignments. HRESITOFMS data were obtained using a Waters Q-ToF
Premier mass spectrometer (or a similar instrument at Korea University)
or a hybrid ion-trap time-of flight mass spectrometer (Shimadzu)
at KBSI. HPLC separations involving 1 and 2 were performed using a
Beckman System Gold instrument with a model 166P variable-
wavelength UV detector connected to a 127P solvent module. The
reversed-phase HPLC method used to isolate metabolites from the
MYC-2048 cultures employed a program of 25% MeCN/H2O to
100% MeCN over 25 min and 100% MeCN for 5 min using an
Alltech HyperPrep 8 μm particle size C18 column (10 × 250 mm) at
a flow rate of 2 mL/min with UV detection at 244 nm. Other HPLC
separations were performed on an Agilent prep-C18 column (21.2 ×
150 mm; 5 μm; 5 mL/min; UV detection at 210 nm). Flash column
chromatography was carried out using YMC octadecyl-functionalized
silica gel (C18).
Figure 5. Experimental ECD spectrum (top) and TDDFT-calculated
ECD spectrum (bottom) for aflaquinolone G (7).
experimental peak positions as well as in the cases above, the
sequence and signs of the key higher wavelength peaks calculated
for structure 7 (the C-3 epimer of 6) showed a much better
match with the experimental data than the inverted spectrum
calculated for its enantiomer, resulting in assignment of the
3R,4S-configuration shown for 7.
Biogenetically, compounds 1−4 appear likely to be derived
from one unit each of anthranilic acid and phenylalanine, along
with two isoprene units via processes analogous to those proposed
for the aspoquinolones and the penigequinolones,8−10 although
these precedents would presumably incorporate tyrosine rather
than phenylalanine and also have some significant differences in
the arrangement/cyclization of the 10-carbon unit appended
to the aromatic ring of the quinolinone unit. Compounds 5−7
lack the prenyl units, but would otherwise be similarly con-
structed. In view of the results described here, it is likely that
ECD could be readily applied to assignment of the absolute
configuration of the dihydroquinolone portions of other
members of this class. In some cases, comparison with data
provided in the literature now enable such an assignment.10
However, establishment of the configurations of any centers that
Fungal Material. MYC-2048 was originally obtained by D.T.W.
from a basidioma of Rigidoporus microsporus found on a dead hardwood
branch in an alien wet forest near milepost 7 on Scenic Route 19,
Onomea Bay, Hawaii Co., Hawaii, in November 2002. This isolate was
initially identified as Aspergillus flavipes (Section Flavipedes) on the basis
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Journal of Natural Products
Article
of in vitro colony growth and micromorphology. A subculture was
deposited in the culture collection at the USDA National Center for
Agricultural Utilization Research and assigned the accession number
NRRL 58570. The culture was subjected to partial sequence analysis of
the internal transcribed spacer region (ITS) and domains D1 and D2 of
the nuclear large subunit (28S) rDNA gene using ITS5 and NL4 as
polymerase chain reaction and sequencing primers.19,20 A nucleotide-to-
nucleotide BLAST query of the GenBank database (http://www.ncbi.
nlm.nih.gov/BLAST) recovered EF669595 Aspergillus sp. NRRL 32683
(= MYC-1580; NRRL 58569)17 as the closest match to the ITS rDNA
of NRRL 58570 (100%). This undescribed species of Aspergillus
(Section Flavipedes) was also initially identified as A. flavipes and also
came from our Hawaiian collection (isolated by D.T.W. from a
basidioma of Earliella scabrosa found on a dead hardwood branch, alien
wet forest, Hilo Zoo, Hawaii Co., HI). It was shown to be a sibling
species to the clade containing Aspergillus aureofulgens NRRL 6326,
which, in turn, was originally described from a single known isolate
obtained from truffle soil in France.18 These results suggest that NRRL
58570 and NRRL 32683 represent separate Hawaiian isolates of the
same undescribed species of Aspergillus (Section Flavipedes). Because
the matching isolate has not been taxonomically described, we
characterize the Hawaiian isolate (NRRL 58570) at present only as
“Aspergillus sp.”
Aspergillus sp. SF-5044 (deposited at the College of Medical and
Life Sciences fungal strain repository, Silla University) was isolated
from an intertidal sediment sample collected from Dadaepo Beach,
Busan, Korea, in April 2006 using procedures that have been
described.15 Analysis of 28S rRNA sequences (Genbank accession
number FJ935999) and a subsequent GenBank search indicated
Aspergillus protuberus (FJ176897) and Aspergillus asperescens
(EF652495) as the closest matches, showing sequence identities of
99.64% and 98.22%, respectively. Therefore, the marine-derived fungal
strain SF-5044 was also identified as an Aspergillus sp. As was the case
for the Hawaiian isolate, it could not be assigned to species, but the
two isolates do not represent the same species.
Aspergillus sp. MYC-2048 was grown on 100 g of autoclaved rice for
30 days at 25 °C. The EtOAc extract (802 mg) of the resulting
fermentation mixture showed antifungal activity against A. flavus and
F. verticillioides. The extract also caused significant reduction in the
growth rate of fall armyworm. Aspergillus sp. SF-5044 was cultured on
110 Petri plates (90 mm), each containing 20 mL of PDA with 3%
NaCl. Plates were individually inoculated with 2 mL seed cultures of
the fungal strain and incubated at 25 °C for a period of 10 days.
Extraction of the combined agar media with EtOAc (2 L) provided an
organic phase, which was then concentrated in vacuo to yield 2.0 g of
an extract.
Isolation. The extract from Aspergillus sp. MYC-2048 (= NRRL
58570) was partitioned between MeCN and hexanes (∼8 mL of each).
The resulting MeCN fraction (478 mg) was then chromatographed
on a silica gel column (Scientific Adsorbent, Inc.; 63−200 μm) using
a hexanes/EtOAc/MeOH step gradient (hexanes, hexanes/EtOAc,
EtOAc, EtOAc/MeOH, MeOH, ratios used: hexanes, 3:1, 1:1, 1:3; pure
EtOAc, 99:1, 49:1, 19:1 9:1; and pure MeOH) to give fifteen 100 mL
fractions. Fraction 5 (34 mg), eluted with 1:1 hexanes/EtOAc, was
further separated by reversed-phase HPLC to afford trans-dehydro-
curvularin14 (15 mg, tR 19.0 min) and aflaquinolone A (1; 4 mg, tR
20.4 min). A sample of compound 2 was also obtained from this HPLC
step (1 mg, tR 17.0 min). Fraction 6 (14 mg), eluted with 1:1 hexanes/
EtOAc, was further separated by HPLC under the same conditions
to afford aflaquinolone B (2; 4 mg, tR 17.8 min), aspochalasin J12 (3 mg,
tR 24.0 min), and an additional sample of compound 1 (2 mg). Fraction
8 (19 mg), eluted with 1:3 hexanes/EtOAc, was similarly separated by
HPLC to afford alantripinone11 (1 mg, tR 14.1 min) and methyl 3,4,5-
trimethoxy-2((2-((3-pyridinylcarbonyl)amino)benzoyl)amino)-
benzoate13 (2 mg, tR 22.2 min). Fraction 10 (17 mg), eluted with 100%
EtOAc, contained only aspochalasin I.12
resubjected to C18 flash column chromatography (4.5 × 12 cm),
eluting with a stepwise gradient of 20% to 70% MeOH in H2O
(250 mL each, 10% increment from 20%, to 50%, followed by two
additional 50% MeOH fractions, 60% MeOH, and 70% MeOH). The
fraction eluted with the first 50% MeOH in H2O elution solvent
(29.9 mg) was further purified by semipreparative reversed-phase
HPLC eluting with a gradient from 30% to 60% MeOH in H2O
(0.1% formic acid) over 40 min to yield 4 (3.5 mg, tR = 30.9 min).
The fraction eluted with the second 50% MeOH elution solvent
(35 mg) was purified by semipreparative reversed-phase HPLC
eluting with a gradient from 45% to 65% MeOH in H2O (0.1% formic
acid) over 65 min to yield compounds 3 (3.4 mg, tR = 27.5 min) and 5
(16 mg, tR = 25.1 min). The fraction from C18 flash column chro-
matography of the extract that eluted at 80% MeOH (261 mg) was
subjected to silica flash column chromatography (3.5 × 10 cm), eluting
with a stepwise gradient of 0% to 20% (v/v) MeOH in CH2Cl2 (200 mL
each, 1% increment for each fraction). The fraction eluted with 1%
MeOH (30 mg) was further purified by semipreparative reversed-phase
HPLC eluting with 75% MeOH in H2O (0.1% formic acid) to yield 6
(3.0 mg, tR = 19.5 min). Compound 7 was purified by re-HPLC of the
fraction collected between 1 and 10 min in the above HPLC procedure
using a gradient from 50% to 100% MeOH in H2O (0.1% formic acid)
over 50 min (1.5 mg, tR = 33.5 min).
Aflaquinolone A (1): pale orange, amorphous solid; [α]21 +14
D
(c 0.19, MeOH); UV (MeOH) λmax (log ε) 213 (4.3), 233sh (4.2),
278 (4.1), 324 (4.1) nm; CD (43 μM, MeOH) λmax (Δε) 220 (+36),
1
252 (−41), 280 (+16), 286 (+15), and 323 (+9.5) nm; H and 13C
NMR data, see Table 1; HMBC data, NH → C-2, 3, 5, 9, 10; H-3 →
C-2, 4, 5, 11; H-8 → C-5, 6, 7, 10, 17; H-9 → C-4, 5, 6, 7, 10; H-12/
16 → C-11, 12/16, 13/15, 14; H-13/15 → C-11, 12/16, 13/15, 14;
H-14 → C-11, 12/16, 13/15; H-17 → C-6, 7, 8, 18, 19, 25; H-18 →
C-7, 19, 20, 25; Hax-20 → C-18, 19, 21, 22, 25, 26; Heq-20 → C-19, 21,
22, 24; H-21 → C-20, 22, 26; Hax-23 → C-22, 24; Heq-23 → C-19, 21,
22; Hax-24 → C-19, 23, 25; Heq-24 → C-19, 21, 22; H3-25 → C-18,
20, 23, 24; H3-26 → C-20, 21, 22; H3-27 → C-3; key NOESY data
(400 MHz; CDCl3) 4-OH ↔ H3-27; 4-OH ↔ H-3; H-3 ↔ H-12/16;
Hax-20 ↔ H3-25; Heq-20 ↔ H-18; H-21 ↔ H-17; Hax-24 ↔ Heq-23;
H3-25; Heq-24 ↔ H-17; HRESITOFMS m/z 458.1940 [M + Na]+
(calcd for C26H29NO5Na, 458.1943).
Aflaquinolone B (2): pale yellow, amorphous solid; [α]22 +20
D
(c 0.14, MeOH); UV (MeOH) λmax (log ε) 213 (4.3), 233sh (4.1),
278 (4.0), 288sh (3.9), 324 (4.0) nm; CD (54 μM, MeOH) λmax (Δε)
220 (+32), 252 (−39), 280 (+14), 286 (+12), and 323 (+7.9) nm; 1H
1
and 13C NMR data (CDCl3), see Table 1; H NMR (acetone-d6, 400
MHz) δ 9.31 (br s, NH), 9.52 (br s, OH-6), 7.43 (d, J = 8.3 Hz, H-8),
7.37−7.34 (m, 5H, H-12 through H-16), 6.62 (d, J = 17 Hz, H-17),
6.56 (d, J = 8.3 Hz, H-9), 6.30 (br s, OH-4), 6.17 (d, J = 17 Hz, H-18),
3.66 (m, H-3), 3.66 (m, H-22), 3.51 (s, H3-27), 3.23 (d, J = 4.2 Hz,
OH-22), 1.71 (m, Hax-24), 1.67 (m, H-21), 1.64 (m, Hax-23), 1.62
(m, Heq-23), 1.49 (t, J = 13 Hz, Hax-20), 1.48 (br dd, J = 13, 2.7 Hz,
Heq-24), 1.36 (dt, J = 13, 2.7 Hz, Heq-20), 1.00 (s, H3-25), 0.91 (d, J =
6.8 Hz, H3-26); key NOESY data (400 MHz; acetone-d6) H-3 ↔
H-12/16, H-17 ↔ Heq-20; H-17 ↔ H-21, H-17 ↔ Hax-23; H-17 ↔
Heq-24; additional NOESY data (400 MHz; CDCl3) 4-OH ↔ H3-27;
4-OH ↔ H-3; HRESITOFMS obsd m/z 460.2089 [M + Na]+ (calcd
for C26H31NO5Na, 460.2091).
Aflaquinolone C (3): pale yellow solid; [α]25 −33 (c 0.30,
D
MeOH); UV (MeOH) λmax (log ε) 203 (4.2), 212sh (4.1), 235sh
(4.0), 278 (3.8), 324 (3.8) nm; CD (70 μM, MeOH) λmax (Δε) 220
1
(+28), 252 (−30), 281 (+11), 286 (+10), and 322 (+6.8) nm; H
and 13C NMR data in CDCl3 matched those of 1 (Table 1); 13C NMR
(100 MHz, acetone-d6) δ 166.4 (C-2), 85.8 (C-3), 80.1 (C-4, 112.2
(C-5), 156.1 (C-6), 122.0 (C-7), 127.9 (C-8), 107.8 (C-9), 137.2 (C-
10), 140.3 (C-11), 129.6 (C-12/16), 127.5 (C-13/15), 129.8 (C-14),
123.6 (C-17), 135.9 (C-18), 37.9 (C-19), 48.3 (C-20), 41.7 (C-21),
212.1 (C-22), 39.1 (C-23), 39.3 (C-24), 30.8 (C-25), 14.9 (C-26),
58.9 (C-27); HMBC data, H-3 → C-2, 4, 5, 6, 11, 27; H-8 → C-5,
6, 10, 11; H-9 → C-4, 5, 6, 7, 10; H-12/16 → C-4; H-17 → C-6, 8,
19, 25; H-18 → C-7, 19, 20, 24, 25; H2-20 → C-18, 19, 21, 25, 26;
H-21 → C-20, 22, 26; H2-23 → C-22, 24; H2-24 → C-18, 19, 25;
The EtOAc extract from Aspergillus sp. SF-5044 was subjected
to C18 flash column chromatography (5 × 26 cm), eluting with a
stepwise gradient of 20%, 40%, 60%, 80%, and 100% (v/v) MeOH in
H2O (500 mL each). The fraction eluted at 60% MeOH was
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H3-25 → C-18, 19, 20, 24; H3-26 → C-20, 21, 22; H3-27 → C-3;
NOESY data H-3 ↔ H-12/16; H-17 ↔ H-23ax, H-21; H-23ax ↔ H-
24ax, H3-25, H3-26; HRESIMS m/z 434.1954 [M − H]− (calcd for
C26H28NO5, 434.1967).
Energy Minimization and ECD Calculations. All geometry
optimizations were performed in the gas phase using resolution-of-
identity (RI) approximation and a BP functional,21,22 combined with
the SV(P) basis set23 and the corresponding auxiliary basis set.24,25 No
symmetry constraints were used during the optimization. ECD spectra
were calculated for geometries obtained from the RI-BP/SV(P) calcu-
lations. Time-dependent density functional (TDDFT) calculations
were used with B3LYP functional26,27 using the RIJCOSX approxi-
mation28 and TZVP basis set23 with the corresponding auxiliary basis
set24,25 in the gas phase and in MeOH solution (COSMO solvation
model).29
A total of 80 excited states were calculated, and only singlet excited
states were considered. All of the quantum chemical calculations were
performed with ORCA version 2.7.30 ECD spectra were obtained
using SpecDis version 1.50 software.31 A broadening factor of 0.24 was
used in an effort to match the resolution level of the experimental data
as closely as possible.
Aflaquinolone D (4): pale yellow solid; [α]25 −10 (c 0.10,
D
MeOH); UV (MeOH) λmax (log ε) 204 (4.3), 212sh (4.3), 235sh
(4.2), 278 (4.0), 323 (4.0) nm; CD (47 μM, MeOH) λmax (Δε) 220
1
(+31), 252 (−32), 282 (+12), 286 (+12), and 324 (+7.6) nm; H
NMR (400 MHz, acetone-d6) δ 9.40 (br s, NH), 7.42 (d, J = 8.4 Hz,
H-8), 7.38−7.31 (m, 5H), 6.64 (d, J = 17 Hz, H-17), 6.58 (d, J = 8.4
Hz, H-9), 6.24 (d, J = 17 Hz, H-18), 3.66 (s, H-3), 3.51 (s, H3-27),
2.69 (dq, J = 13, 6.6 Hz, H-21), 2.62 (dt, J = 14, 7.3 Hz, Hax-23), 2.17
(m, Heq-23), 1.89 (m, Heq-20), 1.86 (m, Ha-24), 1.81 (m, Hb-24), 1.54
(t, J = 13 Hz, Hax-20), 1.43 (s, H3-25), 0.94 (d, J = 6.6 Hz, H3-26);
NOESY data, H-3 ↔ H-12/16; H-17 ↔ H-20ax; H-20ax ↔ H3-26;
H-21 ↔ H3-25; H-23ax ↔ H3-25; HRESIMS m/z 434.1954 [M − H]−
(calcd for C26H28NO5, 434.1967).
Aflaquinolone E (5): white solid; [α]25 −41 (c 1.1, MeOH); UV
Cell Proliferation Assay. K562 (3 × 104 cells per well), B16F10
(3 × 103 cells per well), HL-60 (1 × 105 cells per well), MDA-MB-231
(5 × 103 cells per well), and Hep3B (5 × 103 cells per well) cells were
seeded on 96-well microplates. Cell proliferation assays were carried
out using the enhanced cell viability assay kit EZ-CyTox (Daeil Lab
Service Co., Ltd.) protocol. DMSO solutions of the test compounds
were used, and cells were treated for 48 h. The absorbance (450 nm)
of each well was measured using a Power WaveX 340 (Bio-Tek
Instruments).
D
(MeOH) λmax (log ε) 208 (4.2), 252 (3.5), 295 (3.5) nm; CD (62 μM,
MeOH) λmax (Δε) 218 (+19), 245 (−23), 270 (+9.2), and 295 (+5.7)
nm; 1H and 13C NMR data, see Table 2; HMBC data, H-3 → C-2, 4,
5, 6, 11, 27; H-7 → C-5, 6; H-8 → C-5, 6, 7, 10; H-9 → C-5, 6, 7,
10; H-12/16 → C-4, 11, 14; H3-17 → C-3; HRESIMS m/z 308.0893
[M + Na]+ (calcd for C16H15NO4Na, 308.0899).
Aflaquinolone F (6): white solid; [α]25 +10 (c 0.19, MeOH); UV
D
(MeOH) λmax (log ε) 208 (4.2), 252 (3.6), 295 (3.5) nm; CD (56 μM,
MeOH) λmax (Δε) 200 (−9.5), 223 (+13), 252 (−3.2), 269 (+2.1),
and 290 (+0.8) nm; 1H and 13C NMR data, see Table 2; HMBC data,
H-3 → C-2, 4, 5, 11; H-6 → C-4, 8, 10; H-7 → C-6, 9; H-8 → C-6,
10; H-9 → C-5, 7; H-12/16 → C-4; H-13/15 → C-11, 12/16; H-14 →
C-12/16; HRESIMS m/z 256.0975 [M + H]+ (calcd for C15H14NO3,
256.0974).
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra for compounds 1−7. This material is
available free of charge via the Internet at http://pubs.acs.org.
Aflaquinolone G (7): white solid; [α]25 −6 (c 0.18, MeOH); UV
D
(MeOH) λmax (log ε) 208 (4.2), 253 (3.7), 285sh (3.3) nm; CD (57
μM, MeOH) λmax (Δε) 206 (+13), 216 (+14), 235 (−8.6), 257
AUTHOR INFORMATION
■
1
(+5.4), and 286 (−3.1) nm; H and 13C NMR data, see Table 2;
Corresponding Author
HMBC data, H-3 → C-2, 4, 11; H-6 → C-4, 8, 10; H-7 → C-5, 6, 9, 10;
H-8 → C-6, 9, 10; H-9 → C-5, 7; H-12/16 → C-4, 13/15; HRESIMS
m/z 278.0788 [M + Na]+ (calcd for C15H13NO3Na, 278.0793).
Preparation of Aflaquinolone B Mosher’s Esters. A solution of
aflaquinolone B (2; 0.2 mg) in CDCl3 (100 μL) was treated with dry
pyridine-d6 (10 μL) and R-(−)-α-methoxy-α-(trifluoromethyl)-
phenylacetyl chloride (R-MTPA-Cl, 5 μL). The mixture was allowed
to react at room temperature for 24 h in a Teflon-lined screw-cap vial,
resulting in formation of the S-MTPA ester 2a. Additional CDCl3 was
then added directly to the vial, and the resulting solution was placed
in an NMR tube for analysis. Analogous treatment of an additional
0.2 mg portion of 2 using S-MTPA-Cl afforded the R-MTPA ester 2b.
*Tel: 319-335-1361. Fax: 319-335-1270. E-mail: james-gloer@
uiowa.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Research in the University of Iowa lab was supported by grants
from the National Science Foundation (CHE-0718315 and
CHE-11011847 to J.B.G. and AGS 0927944 to J.B.). Assistance
from the staff of the University of Iowa NMR and Mass
Spectrometry Facilities is gratefully acknowledged. Valuable
assistance from an anonymous reviewer in recognizing a significant
error in the original manuscript is appreciated. Acquisition of the
HRESITOFMS and 600 MHz NMR instruments was made
possible by grants from the National Institutes of Health (S10
RR023384 and S10 RR025500, respectively). The USDA is an
equal opportunity provider and employer. Research carried out at
Silla University and KRIBB was supported by the grants from the
Global R&D Center (GRDC) Program funded by the Korean
Ministry of Education, Science & Technology (MEST) and from
the Marine Biotechnology Program funded by the Ministry of
Land, Transport, and Maritime Affairs of the Korean Government.
1
S-MTPA ester 2a: key H NMR data (400 MHz, CDCl3) δ 0.736 (d,
J = 6.9 Hz, H3-26), 0.947 (s, H3-25), 1.29 (m, Hax-23), 1.71 (m,
1
Heq-23). R-MTPA ester 2b: key H NMR data (400 MHz, CDCl3)
δ 0.822 (d, J = 6.9 Hz H3-26), 0.891 (s, H3-25), 1.23 (m, Hax-23), 1.64
(m, Heq-23). Precise δ values for other potentially relevant signals
(e.g., H-21) could not be established due to signal overlap.
Sodium Borohydride Reduction of Aflaquinolone A. A 0.7 mg
sample of aflaquinolone A (1) was transferred to a 0.5 mL Reacti-vial.
To this vial were added 0.75 mg of NaBH4 and 75 μL of MeOH, and a
small triangular stir bar was placed into the vial. The vial was then
sealed under Ar, and the course of the reaction was monitored by TLC
(9:1 Et2O/MeOH). After 1 h, all of the starting material had reacted.
The solvent was then evaporated with air flow and worked up using
aqueous NH4Cl and ether. The ether layer was collected and
1
evaporated to dryness. H NMR analysis of the ether-soluble material
indicated the presence of a mixture of products, which were separated
by reversed-phase HPLC (C18, column: 4.6 × 250 mm, 1.0 mL/min,
50−100% MeCN in H2O over 25 min), resulting in the collection of
DEDICATION
■
1
Dedicated to Dr. Gordon M. Cragg, formerly Chief, Natural
Products Branch, National Cancer Institute, Frederick, Maryland,
for his pioneering work on the development of natural product
anticancer agents.
two fractions (tR = 11.2 and 12.8 min). H NMR analysis of the first
fraction to elute exhibited signals matching those of aflaquinolone B (2),
indicating that reduction of 1 to 2 had occurred. HPLC co-injection
with a sample of 2 supported this conclusion.
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REFERENCES
■
(1) Gloer, J. B. Applications of Fungal Ecology in the Search for New
Bioactive Natural Products. In The Mycota, Vol. IV, 2nd ed.; Kubicek,
C. P.; Druzhinina, I. S., Eds.; Springer-Verlag: New York, 2007; pp
257−283.
(2) Schmidt, L. E.; Deyrup, S. T.; Baltrusaitis, J.; Swenson, D. C.;
Wicklow, D. T.; Gloer, J. B. J. Nat. Prod. 2010, 73, 404−408.
(3) Wicklow, D. T.; Jordan, A. M.; Gloer, J. B. Mycol. Res. 2009, 113,
1433−1442.
(4) Greshock, T. J.; Grubbs, A. W.; Jiao, P.; Wicklow, D. T.; Gloer, J.
B.; Williams, R. M. Angew. Chem., Int. Ed. 2008, 47, 3573−3577.
(5) Sy, A. A.; Swenson, D. C.; Gloer, J. B.; Wicklow, D. T. J. Nat.
Prod. 2008, 71, 415−419.
(6) Shim, S. H.; Swenson, D. C.; Gloer, J. B.; Dowd, P. F.; Wicklow,
D. T. Org. Lett. 2006, 8, 1255−1228.
(7) Lee, S. U.; Asami, Y.; Lee, D.; Jang, J.-H.; Ahn, J.-S.; Oh, H.
J. Nat. Prod. 2011, 74, 1284−1287.
(8) Scherlach, K.; Hertweck, C. Org. Biomol. Chem. 2006, 4, 3517−
3520.
(9) Kimura, Y.; Kusano, M.; Koshino, H.; Uzawa, J.; Fujioka, S.; Tani,
K. Tetrahedron Lett. 1996, 37, 4961−4964.
(10) Kusano, M.; Koshino, H.; Uzawa, J.; Fujioka, S.; Kawano, T.;
Kimura, Y. Biosci. Biotechnol. Biochem. 2000, 64, 2559−2568.
(11) Larsen, T. O.; Frydenvang, K.; Frisvad, J. C.; Christophersen, C.
J. Nat. Prod. 1998, 61, 1154−1157.
(12) Zhou, G. X.; Wijeratne, E. M. K.; Bigelow, D.; Pierson, L. S. III;
VanEtten, H. D.; Gunatilaka, A. A. L. J. Nat. Prod. 2004, 67, 328−332.
(13) Arai, K.; Shimizu, S.; Yamamoto, Y. Chem. Pharm. Bull. 1981,
29, 1005−1012.
(14) Munro, H. D.; Musgrave, O. C.; Templeton, R. J. Chem. Soc. C
1967, 10, 947−948.
(15) Hoye, T. R.; Jeffrey, C. S.; Shao, F. Nat. Protoc. 2007, 2, 2451−
2457.
(16) Bajpai, R.; Curran, D. P. J. Am. Chem. Soc. 2011, 133, 20435−
20443.
(17) He, J.; Lion, U.; Sattler, I.; Gollmick, F. A.; Grabley, S.; Cai, J.;
Meiners, M.; Schunke, H.; Schamann, K.; Dechert, U.; Krohn, M.
̈
J. Nat. Prod. 2005, 68, 1397−1399.
(18) Peterson, S. W. Mycologia 2008, 100, 205−226.
(19) O’Donnell, K. Sydowia 1996, 48, 57−70.
(20) White, T. J.; Bruns, T.; Lee, S.; Taylor, J. W. In PCR Protocols: a
Guide to Methods and Applications; Innis, M. A.; Gelfand, D. H.;
Sninsky, J. J.; White, T. J., Eds.; Academic Press: New York, 1990; pp
315−322.
(21) Perdew, J. P. Phys. Rev. B 1986, 33, 8822−8824.
(22) Becke, A. D. Phys. Rev. A 1988, 38, 3098−3100.
(23) Schaefer, A.; Horn, H.; Ahlrichs, R. J. Chem. Phys. 1992, 97,
2571−2577.
(24) Eichkorn, K.; Weigend, F.; Treutler, O.; Ahlrichs, R. Theor.
Chim. Acta 1997, 97, 119−124.
̈
(25) Eichkorn, K.; Treutler, O.; Ohm, H.; Haser, M.; Ahlrichs, R.
̈
Chem. Phys. Lett. 1995, 240, 283−289.
(26) Becke, A. D. J. Chem. Phys. 1993, 98, 5648−5652.
(27) Lee, C.; Yang, W.; Parr, R. G. Phys. Rev. B 1988, 37, 785−789.
(28) Neese, F.; Wennmohs, F.; Hansen, A.; Becker, U. Chem. Phys.
2009, 356, 98−109.
(29) Sinnecker, S.; Rajendran, A.; Klamt, A.; Diedenhofen, M.;
Neese, F. J. Phys. Chem. A 2006, 110, 2235−2245.
(30) Neese, F. ORCA, Version 2.7.0; Universitat Bonn: Bonn,
̈
Germany, 2010.
(31) Bruhn, T.; Hemberger, Y.; Schaumloffel, A.; Bringmann, G.
̈
SpecDis, Version 1.50; Universitat Wurzburg: Wurzburg, Germany,
2010.
̈
̈
̈
472
dx.doi.org/10.1021/np200958r | J. Nat. Prod. 2012, 75, 464−472