Bioactive sulfur-containing sulochrin dimers and other metabolites from an alternaria sp. isolate from a Hawaiian soil sample

Article abstract of DOI:10.1021/np5005449

Polluxochrin (1) and dioschrin (2), two new dimers of sulochrin linked by thioether bonds, were purified from an Alternaria sp. isolate obtained from a Hawaiian soil sample. The structures of the two metabolites were established by NMR, mass spectrometry data, and X-ray analysis. Metabolite 1 was determined to be susceptible to intramolecular cyclization under aqueous conditions, resulting in the generation of 2 as well as another dimeric compound, castochrin (3). An additional nine new metabolites were also obtained, including four new pyrenochaetic acid derivatives (8-11), one new asterric acid analogue (13), and four new secalonic acid analogues (14-17). Bioassay analysis of these compounds revealed 1-3 displayed antimicrobial and weak cytotoxic activities.

Full text of DOI:10.1021/np5005449

Article
pubs.acs.org/jnp
Bioactive Sulfur-Containing Sulochrin Dimers and Other Metabolites
from an Alternaria sp. Isolate from a Hawaiian Soil Sample
Shengxin Cai,†,‡ Jarrod B. King, Lin Du, Douglas R. Powell, and Robert H. Cichewicz*
†,‡
†,‡
‡
,†,‡
†
‡
Natural Products Discovery Group, Institute for Natural Products Applications and Research Technologies, and Department of
Chemistry and Biochemistry, Stephenson Life Sciences Research Center, 101 Stephenson Parkway, University of Oklahoma,
Norman, Oklahoma 73019-5251, United States
*
S Supporting Information
ABSTRACT: Polluxochrin (1) and dioschrin (2), two new dimers of sulochrin linked by thioether bonds, were purified from an
Alternaria sp. isolate obtained from a Hawaiian soil sample. The structures of the two metabolites were established by NMR,
mass spectrometry data, and X-ray analysis. Metabolite 1 was determined to be susceptible to intramolecular cyclization under
aqueous conditions, resulting in the generation of 2 as well as another dimeric compound, castochrin (3). An additional nine new
metabolites were also obtained, including four new pyrenochaetic acid derivatives (8−11), one new asterric acid analogue (13),
and four new secalonic acid analogues (14−17). Bioassay analysis of these compounds revealed 1−3 displayed antimicrobial and
weak cytotoxic activities.
he Hawaiian Islands consist of a patchwork of biodiverse
habitats exhibiting a high degree of species endemism.
putative metabolites we observed could be readily attributed to
those ascribed to an Alternaria sp.
1
,2
T
Recent reports in the field of natural products have highlighted
the impact of Hawaii’s biodiversity on the likelihood for
discovering new bioactive metabolites, especially from micro-
In this report, we describe 12 new natural products from this
Alternaria sp. isolate. All of the compounds were tested in
bioassays targeting different cell types and bioactivity end
points (including antibacterial, antifungal, cancer cell cytotox-
icity, and disruption of Candida biofilm formation). Com-
pounds 1−3, which are new sulochrin dimers linked via a
thioether bridge, showed antimicrobial and weak cytotoxic
activities.
3
−7
bial sources.
For example, our research group reported the
discovery of several new bioactive metabolites from a
biosynthetically talented fungal (Aspergillus sp.) isolate obtained8
from soil collected near Waikiki Beach, Honolulu, Hawaii.
Among the compounds obtained from this fungus, the new
prenylated indole alkaloid waikialoid A was notable because of
the potent inhibition it afforded against Candida albicans
RESULTS AND DISCUSSION
8
■
biofilm formation (IC50 1.4 μM).
The Alternaria sp. isolate was prepared for scale-up chemical
analysis by culturing it for 4 weeks on a solid-state medium
composed of Cheerios breakfast cereal supplemented with a
An examination of additional samples from the Hawaiian
Islands has provided a second biosynthetically talented fungal
isolate, Wailua PDA-3, from a soil sample collected in the
vicinity of Wailua Falls. An ethyl acetate extract prepared from a
small-scale solid-phase fermentation of this fungus exhibited
antifungal activity against both C. albicans and Aspergillus
fumigatus. Subsequent LC-PDA-ESIMS profiling of the isolate’s
secondary metabolome revealed an assortment of chemically
diverse metabolites with masses ranging from approximately
9
−11
0
.3% sucrose solution.
The fungal biomass was extracted
with EtOAc, yielding a crude extract that was subsequently
processed over several types of sorbents (including silica gel,
Sephadex LH20, and C ) to provide purified metabolites 1−
18
17.
Compound 1 was obtained as a pale yellow, amorphous
powder, and its molecular formula was established as
2
00 to 700 Da. Sequencing of the isolate’s ITS region suggested
that the fungus was taxonomically similar to Alternaria
longissima. LC-PDA-ESIMS-based dereplication of natural
products reported from this genus revealed that none of the
Received: July 4, 2014
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Chart 1
+
2
C H O S based on HRESIMS data ([M + Na] ion at m/z
(δ 6.91; δ 107.6) and the gain of a new nonprotonated sp
34
30 14
H
C
7
17.1253, calcd 717.1248) indicating 20 degrees of unsatura-
carbon resonance (δC 109.1). The necessity of including a
sulfur atom in 1 as required by the molecular formula led us to
propose that the sulfur served as a thioether bridge linking the
C-2 and C-2′ carbons. Thus, the structure of the new sulochrin
dimer was proposed as illustrated (1), and the metabolite was
given the trivial name polluxochrin. Metabolite 1 shares
structural similarities to other sulochrin dimers including,
disulochrin, as well as guignasulfide, which is a demethoxy
analogue of 1.
Compound 2 was purified as pale yellow, block-like crystals.
1
13
tion. The H and C NMR spectra of 1 provided evidence for
approximately half of the expected proton and carbon
resonances, which were assigned as belonging to 11 non-
protonated carbons, three methines, three methyls, and three
exchangeable protons. The initial analysis of the data set led us
to suspect that 1 might be a dimeric compound. Further
examination of the H and C NMR data (Table 1) indicated
that the resonances observed for 1 were similar to those for the
known metabolite sulochrin (4), which we also purified from
the same fungal extract. The major differences between 1 and 4
as revealed by NMR were the loss of signals for a methine unit
9
13
1
13
12
HRESIMS provided a pseudomolecular ion at m/z 685.0981
+
[
M + Na] (calcd 685.0986) that supported the molecular
formula C H O S. This corresponded to 21 degrees of
33
26 13
1
13
Table 1. H (400 MHz) and 13C (100 MHz) NMR and
HMBC Data for Compound 1 (DMSO-d6)
1
unsaturation. Inspection of the H and C NMR data for 2
revealed that the spectra superficially appeared as the
superimposition of two sets of signals (Table 2) with one set
virtually identical to those generated for compound 1 and the
other set bearing substantial similarity to methyl-(1,6-
no.
/1′
δC, mult.
δH, mult.
HMBC
1
2
3
4
4
1
5
5
6
7
8
8
9
1
1
1
1
3
8
5
136.2 C
109.1 C
160.4 C
100.9 CH
157.7 C
124.3 C
107.9 CH
161.9 C
148.3 C
107.9 CH
161.9 C
109.6 C
197.8 C
56.3 CH3
22.1 CH3
167.3 C
1
4
dihydroxy-3-methylxanthone)-8-carboxylate (5), which was
also purified from the fungal extract. The additional unit of
unsaturation afforded via the incorporation of 5 in this
metabolite readily accounted for the overall difference of one
unsaturation unit between metabolites 1 and 2. The change
/2′
/3′
/4′
a/4a′
a/1a′
/5′
a/5a′
/6′
/7′
/8′
a/8a′
/9′
0/10′
1/11′
2/12′
3/13′
/3′-OH
/8′-OH
a/5a′-OH
6.52 s
6.10 s
6.10 s
2/2′, 3/3′, 1a/1a′, 4a/4a′
7/7′, 11/11′, 8a/8a′
5/5′, 11/11′, 8a/8a′
from a single C-9 diaryl ketone signal in 1 (δ 197.8) (Table 1)
C
to pyrone (δ 179.2) and diaryl (δ 197.7) ketone signals in 2
C
C
(Table 2) further supported the asymmetric dimeric nature of
the new metabolite. This assertion was subsequently confirmed
by HSQC and HMBC experiments (Figures S9 and S10), as
well as data generated from a single-crystal X-ray diffraction
experiment (Figure 1). Compound 2 has been given the trivial
name dioschrin.
Compounds 1 and 2 were determined to be susceptible to
transformation under aqueous conditions. Upon incubation for
48 h in various aqueous solvent mixtures, compound 1 was
observed by LC-PDA-ESIMS to transform into 2, which
subsequently converted to another compound (3) (Figure S1).
The transformation process was determined to be temperature
3.61 s
2.16 s
4a/4a′
5/5′, 6/6′, 7/7′
52.3 CH3
3.48 s
12/12′
10.3 br s
11.2 s
7/7′, 8/8′, 8a/8a′
5/5′, 5a/5a′, 8a/8a′
11.2 s
B
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Table 2. H (400 MHz) and 13C (100 MHz) NMR and HMBC Data for Compound 2 (DMSO-d6)
1
no.
δC, mult.
δH, mult.
HMBC
no.
δC, mult.
δH, mult.
HMBC
1
2
3
4
4
1
5
5
6
7
8
8
9
1
1
1
8
137.0 C
109.6 C
164.9 C
102.8 CH
157.2 C
118.5 C
107.8 CH
155.4 C
149.1 C
111.7 CH
161.0 C
106.1 C
179.2 C
22.4 CH3
167.4 C
53.0 CH3
1′
2′
3′
4′
4a′
1a′
5′
5a′
6′
7′
8′
8a′
9′
10′
11′
12′
13′
136.2 C
107.9 C
159.9 C
100.8 CH
157.5 C
124.4 C
107.9 CH
161.9 C
148.3 C
107.9 CH
161.9 C
109.6 C
197.7 C
56.3 CH3
22.1 CH3
166.8 C
52.2 CH3
6.80 s
6.84 s
6.63 s
2, 3, 1a, 4a
7, 10, 5a, 8a
5, 8, 10, 8a
6.50 s
6.11 s
6.11 s
2′, 3′, 1a′, 4a′
7′, 11′, 8a′
5′, 11′, 8a′
a
a
a
a
0
2.38 s
5, 6, 7
3.62 s
2.17 s
4a′
5′, 6′, 7′
1
2
3.88 s
11
-OH
12.3 br s
7, 8, 8a
3.48 s
11.2 s
11.2 s
12′
7′, 8′, 8a′
5′, 5a′, 8a′
8
′-OH
a′-OH
5
metabolite had the same planar structure as pyrenochaetic acid
15
D, but the published account of this metabolite did not
address the compound’s absolute configuration. To address this
problem, we prepared crystals of 7 for X-ray diffraction analysis
(
Figure 3). The metabolite’s absolute configuration was
Figure 1. ORTEP structure generated from the X-ray diffraction data
for a single crystal of 2.
dependent, and even mild heating for brief periods led to the
rapid loss of 1 and generation of 2 and 3. To obtain material for
structure characterization, 5.0 mg of 1 in aqueous MeOH was
held at 40 °C for 6 h, generating 3.5 mg of 3. The purified
product was examined by HRESIMS, which yielded a
1
1
Figure 2. Selected H− H COSY and HMBC correlations for
compounds 7−11.
−
pseudomolecular ion at m/z 629.0757 [M − H] (calcd
16
6
29.0754) corresponding to the molecular formula
determined to be 8S by the Hooft method. This was further
supported by comparing the ECD experimental data for 7 with
a computationally derived theoretical spectrum (Figure 4A,
1
13
C H O S. An analysis of the H and C NMR data for
32
22 12
the product (Table S1) indicated that 3 was structurally similar
13
to compounds 1 and 2. Immediately apparent was that the C
NMR diaryl ketone signal in 1 (C-9/9′, δ 197.8) had been
C
replaced by a pyrone ketone signal in 3 (C-9/9′, δ 179.1)
C
(
Tables 1 and S1). Subsequent 2D NMR experiments (Figures
S14 and S15) confirmed that these changes resulted in the
formation of a new compound that consisted of a thioether-
linked dimer of 5 connected to the sulfur atom through C-2
and C-2′. In contrast to metabolite 1, compound 3 exhibited
two nearly superimposable sets of NMR signals. The doubling
of the signals is proposed to result from the hindered rotation
of the S−C-2 and/or S−C-2′ bonds. Compound 3 has been
given the trivial name castochrin. A similar cyclization
phenomenon has been observed for the transformation of
metabolite 4 to 5, but in this case, the reaction rate was much
slower, taking upward of a week to occur. These results imply
that the sulfur atom might serve to accelerate the intra-
molecular cyclization of 1 into 2 and 3.
The molecular formula for compound 7 was determined by
HRESIMS to be C H O . An analysis of the 1D and 2D
1
3
16
5
Figure 3. ORTEP structure generated from the X-ray diffraction data
for a single crystal of 7.
NMR (in acetone-d and DMSO-d ) for 7 indicated that this
6
6
C
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Figure 4. Calculated and experimental ECD data for compounds 7 (A) and 10 (B).
refer to the Supporting Information for a discussion of how the
theoretical ECD data for 7 were prepared).
carbon skeleton as the co-occurring metabolite pyrenochaetic
acid C (6). However, the carbon and proton signals
1
7
Compound 8 was determined by HRESIMS to have the
same molecular formula as 7. Analysis of the H and C NMR
data for 8 (Tables 3 and 4) revealed that it possessed the same
attributable to an oxidized methylene (CH -10, δH 3.42; δC
2
1
13
60.4) were replaced by signals for an aliphatic methyl. Taking
into account the HRESIMS data, it was proposed that
compound 8 contained a new C-10 hydroxyl group relative
to 6. Combining the proton and carbon chemical shift patterns
1
Table 3. H NMR Data for Compounds 7−11 (400 MHz,
1
1
DMSO-d )
for 8 with the H− H COSY correlations among H-8 and H-9
and H-10, as well as the HMBC correlations between H-8 and
H-9 with C-7 (Figure 2), the position of the new hydroxyl
group was unambiguously assigned. This new metabolite has
been given the name pyrenochaetic acid E (8).
6
7
8
9
10
11
δH, mult. (J
δH, mult. (J δH, mult. (J
δH, mult. (J
δH, mult. (J
no.
in Hz)
in Hz)
7.39 s
7.44 s
in Hz)
7.40 s
7.43 s
in Hz)
in Hz)
2
6
7
8
7.36 s
7.42 s
7.31 s
7.38 s
5.31 s
7.42 s
7.49 s
Pyrenochaetic acid F (9) was purified as a colorless,
amorphous solid, and its molecular formula was established
−
to be C H O based on HRESIMS data ([M − H] ion at
15
18
6
4.25 dd
2.76 t (7.4) 2.81 t (5.7)
1.72 m 1.89 m
m/z 293.1034, calcd 293.1031). The compound exhibited
NMR resonances similar to 8 with the addition of proton and
carbon signals corresponding to an acetoxy group (Tables 3
and 4). The inclusion of the acetoxy group in the structure was
confirmed on the basis of an HMBC correlation from H-10 (δH
(
8.6, 3.9)
9
1.67 m
2.58 dq
2.82 q (7.2)
(
18.0, 7.3)
1.45 m
2.31 dq
18.0, 7.3)
(
1
1
1
1
7
0
1
2
5
0.90 t (7.4) 3.42 t (7.4) 4.03 t (5.4) 0.91 t (7.3) 1.06 t (7.2)
4
.03) to C-14 (δ 170.8) (Figure 2).
C
3.79 s
2.15 s
3.83 s
2.16 s
3.82 s
2.15 s
2.00 s
3.73 s
2.29 s
3.78 s
2.29 s
Pyrenochaetic acid G (10) was obtained as a colorless,
−
amorphous solid. An [M − H] molecular ion at m/z 251.0932
(
calcd 251.0925) in the HRESIMS spectrum was in agreement
-
5.52 br s
OH
with the molecular formula C H O . Compound 10 displayed
1
3
16
5
13
1
3-
OH
11.43 br s
13.32 br s
C NMR resonances that were similar to those for 7. The
HMBC correlation data (H-7 to C-3, C-4, C-5, and C-8, as well
Table 4. 13C NMR Data for Compounds 7−11 (100 MHz, DMSO-d6)
7
8
9
δC, mult.
10
11
no.
δC, mult.
δC, mult.
δC, mult.
δC, mult.
1
2
3
4
5
6
7
8
9
133.1 C
132.7 C
not detected
131.7 C
109.9 CH
157.4 C
132.7 C
139.1 C
124.5 CH
72.5 CH
211.9 C
31.1 CH2
8.0 CH3
56.2 CH3
19.7 CH3
167.5 C
135.3 C
110.3 CH
159.3 C
127.9 C
140.1 C
124.8 CH
196.3 C
201.7 C
29.7 CH2
7.3 CH3
56.8 CH3
19.5 CH3
166.9 C
109.5 CH
156.4 C
109.7 CH
156.0 C
109.8 CH
156.0 C
133.6 C
135.2 C
134.0 C
136.3 C
135.2 C
134.9 C
124.1 CH
209.5 C
124.2 CH
207.1 C
124.2 CH
206.6 C
78.0 CH
26.0 CH2
10.7 CH3
56.2 CH3
19.1 CH3
167.4 C
40.8 CH2
26.9 CH2
60.4 CH2
56.3 CH3
18.7 CH3
167.3 C
40.5 CH2
22.7 CH2
63.5 CH2
56.2 CH3
18.8 CH3
167.9 C
1
1
1
1
1
1
0
1
2
3
4
5
170.8 C
21.1 CH3
D
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Table 5. H (400 MHz) and 13C (100 MHz) NMR and HMBC Data for Compound 13 (DMSO-d6)
1
no.
δC, mult.
δH, mult.
HMBC
no.
1′
δC, mult.
δH, mult.
HMBC
1
2
3
4
5
6
7
8
9
4
134.2 C
126.5 C
107.6 CH
155.4 C
105.3 CH
153.7 C
56.5 CH3
165.6 C
52.5 CH3
156.1 C
2′
3′
4′
5′
6′
7′
8′
9′
10′
111.4 C
6.71 s
6.71 s
3.62 s
1, 4, 5, 8
154.7 C
109.6 CH
138.9 C
6.25 s
2′, 3′, 6′, 7′
1, 3, 6
104.3 CH
21.7 CH3
166.2 C
5.57 s
2.02 s
1′, 2′, 4′, 7′
4′, 5′, 6′
6
8
3.62 s
34.3 CH3
37.5 CH3
2.91 s
8′, 10′
8′, 9′
-OH
9.47 br s
2.85 s
3′-OH
9.81 br s
Table 6. H (400 MHz) and 13C (100 MHz) NMR Data for Compounds 14−17 (DMSO-d6)
1
1
4
15
16
17
no.
δC, mult.
δH, mult. (J in Hz)
δC, mult.
δH, mult. (J in Hz)
δC, mult.
δH, mult. (J in Hz)
δC, mult.
δH, mult. (J in Hz)
2
3
85.3 C
84.9 C
84.1 C
84.6 C
101.0 C
39.3 CH2
3.60 d (17.2)
39.3 CH2
3.58 d (17.2)
3.08 d (17.2)
40.4 CH2
3.53 d (17.3)
3.03 d (17.3)
3
.11 d (17.2)
4
4
5
6
7
8
8
9
1
1
187.4 C
106.5 C
158.9 C
118.1 C
140.3 CH
107.9 CH
157.8 C
70.4 CH
28.8 CH
33.0 CH2
196.1 C
107.5 C
158.7 C
117.5 C
141.0 CH
107.7 CH
159.1 C
87.0 CH
29.8 CH
36.1 CH2
196.2 C
107.3 C
158.6 C
117.4 C
141.1 CH
107.6 CH
158.8 C
86.1 CH
29.8 CH
36.2 CH2
196.0 C
107.6 C
160.8 C
109.9 CH
141.1 CH
115.4 C
156.2 C
85.8 CH
29.6 CH
35.5 CH2
a
6.58 d (8.6)
7.47 d (8.6)
7.39 d (8.5)
6.55 d (8.5)
7.49 d (8.5)
6.67 d (8.5)
7.47 d (8.6)
6.65 d (8.6)
a
3.95 br s
1.91 m
2.35 m
4.60 d (4.0)
2.89 m
4.55 d (5.1)
4.42 d (4.0)
0
1
2.89 m
2.42 m
2.87 dd (21.6, 9.1)
2.89 dd (17.6, 9.3)
2.31 dd (17.6, 6.0)
2.13 dd (18.0, 8.9)
2.05 dd (18.0, 5.4)
2
.28 dd (21.6, 9.1)
1
1
1
1
5
9
1
2
3
2
180.5 C
17.8 CH3
171.8 C
53.9 CH3
176.1 C
20.3 CH3
169.3 C
53.9 CH3
175.8 C
19.7 CH3
169.2 C
54.1 CH3
175.9 C
20.2 CH3
169.1 C
54.1 CH3
3
1.00 d (6.6)
1.17 d (6.5)
1.09 d (6.8)
0.97 d (7.0)
4
5
3.64 s
3.70 s
3.71 s
3.68 s
-OH
-OH
2-OH
′
11.67 s
5.80 br s
13.8 br s
11.82 s
11.79 s
11.53 s
84.8 C
84.9 C
84.9 C
85.0 C
′
39.4 CH2
3.58 d (17.2)
39.3 CH2
3.60 d (17.2)
3.11 d (17.2)
39.3 CH2
3.59 d (17.2)
3.09 d (17.2)
39.3 CH2
3.60 d (17.2)
3.12 d (17.2)
3
.09 d (17.2)
4
4
5
6
7
8
8
9
1
1
′
a′
′
′
′
′
a′
′
0′
1′
196.1 C
107.4 C
158.6 C
117.7 C
141.2 CH
107.6 CH
159.1 C
87.0 CH
29.8 CH
36.1 CH2
196.1 C
107.5 C
158.7 C
117.5 C
141.0 CH
107.7 CH
159.1 C
87.0 CH
29.8 CH
36.1 CH2
196.1 C
107.5 C
158.6 C
117.5 C
141.0 CH
107.7 CH
159.1 C
87.0 CH
29.8 CH
36.1 CH2
196.2 C
107.5 C
158.6 C
117.3 C
140.9 CH
107.7 CH
159.1 C
86.9 CH
29.8 CH
36.1 CH2
7.48 d (8.6)
6.64 d (8.6)
7.49 d (8.5)
6.67 d (8.5)
7.47 d (8.6)
6.67 d (8.6)
7.57 d (8.6)
6.67 d (8.6)
4.58 d (4.0)
2.86 m
4.60 d (4.0)
4.58 d (4.0)
4.59 d (4.2)
2.89 m
2.89 m
2.86 m
2.84 m
2.87 dd (21.6, 9.1)
2.28 dd (21.6, 9.1)
2.87 dd (21.8, 8.9)
2.26 dd (21.7, 8.8)
2.86 dd (21.0, 9.3)
2.28 dd (21.5, 9.2)
2
.27 m
1
1
1
1
5
2′
3′
4′
5′
176.1 C
20.3 CH3
169.3 C
53.9 CH3
176.1 C
20.3 CH3
169.3 C
53.9 CH3
176.1 C
20.3 CH3
169.3 C
53.9 CH3
176.0 C
20.2 CH3
169.2 C
53.9 CH3
1.15 d (6.4)
1.17 d (6.5)
1.15 d (6.6)
1.15 d (6.3)
3.68 s
3.70 s
3.68 s
3.69 s
′-OH
11.81 s
11.82 s
11.81 s
11.80 s
as H-9 and H-10 to C-8) indicated that the oxidation pattern of
C-7 and C-8 in 7 was reversed in 10 (i.e., C-7 bore the hydroxyl
group and C-8 was a ketone) (Tables 3 and 4, Figure 2). The
metabolite’s absolute configuration was determined to be 7R by
comparing experimental ECD data with a theoretical spectrum
of the compound (Figure 4B, refer to the Supporting
E
dx.doi.org/10.1021/np5005449 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
1
Figure 5. Selected H− H COSY and HMBC correlations for compounds 14−17.
Information for a discussion of how the theoretical ECD data
for 10 were prepared). Thus, the structure of the new
metabolite was confirmed as illustrated for 10 and was
named pyrenochaetic acid G.
and overall planar structure of 15. It was subsequently
determined that metabolite 16 shared the same planar structure
as 15 (Figure 5). However, the presence of two distinct sets of
resonances representing the two monomeric portions of 16
denoted that this metabolite was an asymmetric diastereomer
of 15. In contrast to 15 and 16, HMBC correlations from H-7′
to C-8 and H-7 to C-6′ (Figure 5) indicated that metabolite 17
was covalently bonded via a C-8−C-6′ linkage. Since
compounds 15−17 were not active in our panel of assays
and were prone to consistent degradation during both freezer
storage and room-temperature handling, our investigation of
their relative configurations was discontinued.
The molecular formula of 11 was determined to be C H O
based on HRESIMS, which provided an [M − H] ion at m/z
49.0776 (calcd 249.0768). A comparison of the 1D NMR data
13
14
5
−
2
for 11 with 10 (Tables 3 and 4) revealed that the carbon and
proton signals attributable to the C-7 methine were missing and
had been replaced by a downfield ketone resonance (δ 196.3).
C
Compound 11 was named pyrenochaetic acid H.
Dimethylamide asterrate (13) possessed the molecular
formula C H NO as determined by HRESIMS, indicating
All the purified compounds were screened for cancer cell
19
21
7
2
0
21
22
that the metabolite possessed 10 degrees of unsaturation. The
cytotoxicity, antibacterial and antifungal activities, and
8
1
13
inhibition of fungal biofilm formation. Only compounds 1−3
exhibited anti-MRSA activity, with MIC values of 4.1, 4.9, and
compound exhibited H and C NMR resonances (Table 5)
that were similar to the known, co-occurring metabolite asterric
1
8
3
.2 μM (2.9, 3.2, and 2.0 μg/mL), respectively, whereas the
acid (12), with the major exceptions being the addition of
two new methyl groups (C-9′: δ 34.3, δ 2.91; C-10′: δ 37.5,
MIC for chloramphenicol was 5 μM (1.6 μg/mL). Metabolites
C
H
C
1
−3 also showed weak mammalian cell cytotoxicity effects
δ
2.85). Considering the chemical shift and HMBC
H
against pancreatic cancer cells (MIA PaCa-2) with IC values
correlation data for 13 (δH 2.91 and 2.85 to δ 166.2), the
50
C
of 50.8, 30.3, and 29.3 μM, respectively.
new methyl groups were rationalized to be part of a tertiary
amide that had replaced the carboxylic acid in 12.
EXPERIMENTAL SECTION
Compound 14 was obtained as a pale yellow, amorphous
■
+
solid. On the basis of the HRESIMS data ([M + H] ion at m/z
General Experimental Procedures. UV data were collected on a
Hewlett-Packard 8452A diode array spectrophotometer. IR data were
collected on a Shimadzu IR Affinity FTIR. Optical rotation data were
determined on a Rudolph Research AUTOPOL III automatic
polarimeter. NMR data were collected on a Varian 400 MHz NMR
instrument. Accurate mass data were collected on an Agilent 6538
HRESI QTOF MS coupled to an Agilent 1290 HPLC. LC-MS data
were obtained on a Shimadzu LC-MS 2020 system (ESI quadrupole)
coupled to a photodiode array detector, with a Phenomenex Kinetex
column (2.6 μm C18 column, 100 Å, 75 × 3.0 mm). The HPLC system
utilized SCL-10A VP pumps and a system controller with Luna 5 μm
C18 columns (110 Å, 250 × 21.2 mm, 10 mL/min and 110 Å, 250 × 10
mm, 4 mL/min). X-ray data were collected using a diffractometer with
a Bruker APEX CCD area detector and graphite-monochromated Mo
Kα radiation (λ = 0.710 73 Å). All solvents were of ACS grade or
better.
6
39.1708, calcd 639.1714), the molecular formula was
established as C H O requiring 18 degrees of unsaturation.
32
30 14
13
1
Analysis of H NMR, C NMR, and HSQC data (Table 6)
revealed that this metabolite possessed 17 nonprotonated
carbons, eight methines, three methylenes, and four methyl
groups. Further analysis of the 2D NMR data (Figure 5)
revealed that 14 shared the same planar structure as blennolide
1
9
G. When the NMR solvent was changed from DMSO-d to
6
CDCl (as reported in the literature), compound 14 provided
3
1
13
H and C NMR spectra that were identical to blennolide G;
however, their specific rotation values were in essence the
2
5
opposite of one another {14: [α] −68.8 (c 0.125, CHCl );
D
3
2
5
blennolide G: [α] +81.1 (c 0.29, CHCl )}. Further analysis
D
3
of the ECD data for 14 (Figure S80) revealed a Cotton effect
pattern that was the inverse of that generated for blennolide G.
Thus, metabolite 14 was determined to be the antipode of
blennolide G.
Fungal Isolate Procurement and Culture Conditions. The
fungal isolate (internal strain designation Wailua PDA-3) was obtained
from a soil sample collected in the vicinity of Wailua Falls, Hawaii. The
isolate was identified as similar to Alternaria longissima based on ITS
sequence analysis (99% sequence homology). Sequence data for the
isolate (GenBank accession no. KM088044) was compared by BLAST
analysis to sequences publicly available through the NCBI database.
The isolate was deposited in our laboratory’s fungal library. The fungal
isolate was cultured under solid-state conditions on a medium
composed of Cheerios breakfast cereal supplemented with a 0.3%
sucrose solution with 0.005% chloramphenicol. The fungus was grown
for 4 weeks at room temperature (25 °C).
Like compound 14, metabolites 15, 16, and 17 shared the
same molecular formula (C H O ), as well as many other
3
2
30 14
13
1
similarities among their H and C NMR spectral data (Table
). In spite of this, compound 15 stood out as displaying half of
6
the expected proton and carbon resonances, which indicated
that it was a symmetrical dimer. HMBC correlation data
(
Figure 5) were essential for establishing the C-6−C-6′ linkage
F
dx.doi.org/10.1021/np5005449 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
−
1
1
13
+
Extraction and Purification. The scale-up solid-phase cultures
were pooled and extracted twice overnight with EtOAc, and the
organic solvent was partitioned against water. The resultant organic
layers were concentrated under vacuum. The resulting extract (71 g)
was separated by silica gel VLC (gradient mobile phase consisting of
1047 cm ; H and C NMR see Table 6; HRESIMS [M + H] m/z
639.1708 (calcd for C H O , 639.1714).
Blennolide H (15): pale yellow, amorphous solid; [α] −75.3 (c
0.473, CHCl ); UV (MeOH) λ (log ε) 208 (4.36), 258 (4.30), 364
3.84) nm; IR (film) ν 3439, 2965, 1788, 1645, 1433, 1359, 1273,
1207, 1056 cm ; H and C NMR see Table 6; HRESIMS [M +
Na] m/z 661.1530 (calcd for C H O Na, 661.1528).
Blennolide I (16): pale yellow, amorphous solid; [α]
.035, CHCl ); UV (MeOH) λ (log ε) 206 (4.32), 256 (4.25), 360
3.79) nm; IR (film) ν 3458, 2962, 1791, 1647, 1433, 1359, 1286,
207, 1012 cm ; H and C NMR see Table 6; HRESIMS [M +
Na] m/z 661.1531 (calcd for C H O Na, 661.1528).
Blennolide J (17): pale yellow, amorphous solid; [α] −28.8 (c
.125, CHCl ); UV (MeOH) λ (log ε) 210 (4.50), 256 (4.43), 360
3.93) nm; IR (film) ν 3441, 1788, 1745, 1647, 1469, 1354, 1280,
172, 1055 cm ; H and C NMR see Table 6; HRESIMS [M +
32
31 14
2
5
D
3
max
(
max
hexane−CH Cl −MeOH), which yielded four fractions (Fr.1−Fr.4).
−1
1
13
2
2
Fr.2 (eluted with 9:1 MeOH−CH Cl ) was subjected to C VLC
+
2
2
18
32 30 14
(
4
H O−MeOH) to give four subfractions. Subfraction 1 (eluted with
25
2
−35.0 (c
D
:6 H O−MeOH) was further purified by semipreparative HPLC
2
0
3 max
(
mobile phase 1:1 H O−MeOH with 0.1% formic acid in water) to
2
(
1
max
afford compounds 6 (2.0 mg), 7 (15.0 mg), 8 (2.5 mg), 9 (2.8 mg), 10
3.0 mg), 11 (2.3 mg), and 12 (6.0 mg). Subfraction 3 (eluted with
:2 H O−MeOH) was applied to a Sephadex LH20 column and
−1
1
13
(
+
32
30 14
8
2
25
D
eluted with 1:1 CH Cl −MeOH. Further purification by semi-
2
2
0
3
max
preparative HPLC (mobile phase 6:4 to 9:1 MeOH−H O with
2
(
1
0.1% formic acid in water) yielded 1 (8.0 mg), 2 (7.5 mg), 4 (25 mg),
5 (14 mg), 13 (6.5 mg), 14 (4.0 mg), 15 (5.1 mg), 16 (4.7 mg), and
17 (5.0 mg).
max
−1
1
13
+
Na] m/z 661.1527 (calcd for C H O Na, 661.1528).
32 30 14
X-ray Crystal Structure Analysis of 2 and 7. A yellow plate-
shaped crystal of dimensions 0.46 × 0.26 × 0.08 mm of 2 and a
colorless prism-shaped crystal of dimensions 0.44 × 0.32 × 0.18 mm of
7 were selected for structural analysis. Intensity data for these two
compounds were collected using a diffractometer with a Bruker APEX
ccd area detector and graphite-monochromated Mo K radiation
(0.710 73 Å). Details of the X-ray crystal structure analysis for 2 and 7
are available in the Supporting Information. The X-ray crystallographic
data for 2 and 7 have been deposited with the Cambridge
Crystallographic Data Center under accession numbers CCDC
Polluxochrin (1): pale yellow, amorphous solid; UV (MeOH) λmax
(
2
log ε) 210 (4.27), 286 (3.81), 346 (3.54) nm; IR (film) νmax 3600,
−1 1
953, 1712, 1641, 1514, 1454, 1344, 1211, 1091, 1022, 831 cm ; H
13
+
and C NMR see Table 1; HRESIMS [M + Na] m/z 717.1253
(
calcd for C H O SNa, 717.1248).
34 30 14
Dioschrin (2): pale yellow, block-like crystals; UV (MeOH) λmax
(
2
log ε) 208 (4.37), 238 (4.34), 364 (4.05) nm; IR (film) νmax 3600,
−1 1
930, 1712, 1645, 1585, 1514, 1365, 1209, 1089, 1020, 831 cm ; H
13
+
and C NMR see Table 2; HRESIMS [M + Na] m/z 685.0981
(
calcd for C H O SNa, 685.0986).
33 26 13
Castochrin (3): pale yellow powder; UV (MeOH) λmax (log ε) 210
4.39), 246 (4.12), 298 (3.56), 364 (3.90) nm; IR (film) νmax−3610,
9
93668 and 993669, respectively. These data can be accessed free of
(
2
charge at http://www.ccdc.cam.ac.uk/.
1
1
935, 1739, 1699, 1651, 1514, 1365, 1269, 1211, 1008, 827 cm ; H
ECD Calculation Details of 7 and 10. Conformational analyses
1
3
−
and C NMR see Table S1; HRESIMS [M − H] m/z 629.0757
23
were carried out using Spartan’10. Geometry, frequency, and ECD
(
calcd for C H O S, 629.0754).
Pyrenochaetic acid D (7): colorless cubic crystals; [α] −5.0 (c
.18, EtOH); UV (MeOH) λmax (log ε) 212 (4.14), 252 (3.68), 302
32 21 12
24
25
calculations were applied at the DFT level with Gaussian 09. SpecDis
D
1
.60 was used to average single ECD or UV−vis spectra after
0
(
25
Boltzmann statistical weighting.
3.32) nm; CD (MeOH) λmax (Δε) 254 (6.82) 317 (−2.50); IR (film)
−1 1
Cytotoxicity Assay. Mammalian cell cytotoxicity assays were
performed on pancreatic cancer (MIA PaCa-2) cells by adding 5000
cells per well into 96-well plates. The cells were allowed to adhere
νmax 2972, 1699, 1541, 1456, 1413, 1315, 1226, 1097, 974 cm ; H
and 13C NMR see Tables 3 and 4; HRESIMS [M − H]− m/z
2
51.0931 (calcd for C H O , 251.0925).
13 15 5
Pyrenochaetic acid E (8): colorless, amorphous solid; UV (MeOH)
λmax (log ε) 212 (4.12), 246 (3.61), 298 (3.23) nm; IR (film) ν
overnight at 37 °C in a humidified incubator (5% CO atmosphere).
2
Test compounds were diluted in DMSO and added to the wells so that
the final concentration of DMSO per well did not exceed 1% by
volume. The plates containing treated and control cells were incubated
max
13
−1 1
2
941, 1697, 1541, 1456, 1413, 1315, 1234, 1095 cm ; H and
C
−
NMR see Tables 3 and 4; HRESIMS [M − H] m/z 251.0931 (calcd
26
for C H O , 251.0925).
13
15
5
for 48 h, and cell viability was determined by MTT assay.
Pyrenochaetic acid F (9): colorless, amorphous solid; UV (MeOH)
λmax (log ε) 210 (4.19), 298 (3.34) nm; IR (film) ν 2966, 1737,
Antibacterial Assay. Compounds were tested against methicillin-
resistant Staphylococcus aureus (MRSA) strain ATCC 700787, which
also exhibits reduced susceptibility to vancomycin. A stock culture was
diluted in brain heart infusion broth and delivered into presterilized
max
13
−1 1
1
687, 1546, 1454, 1413, 1238, 1097, 1039 cm ; H and C NMR see
−
Tables 3 and 4; HRESIMS [M − H] m/z 293.1034 (calcd for
C H O , 293.1031).
1
5
17
6
9
6-well plates. Stocks prepared in DMSO were added to each well of
25
Pyrenochaetic acid G (10): colorless, amorphous solid; [α]
D
the 96-well plate (1% final DMSO concentration). Optical density
measurements (OD600) of the cultures were determined on a Tecan
Infinite M200 plate reader. The plates were incubated for 17 h in a
humidified incubator at 37 °C. Plates were removed and orbitally
shaken, and a final OD600 value was recorded. The final OD600
reading was subtracted from the initial OD600 reading to obtain the
change in OD600 (antimicrobial activity was determined based on the
change in optical density).
−
11.4 (c 0.18, MeOH); UV (MeOH) λmax (log ε) 216 (4.20), 248
(
(
1
3.79), 296 (3.37) nm; CD (MeOH) λmax (Δε) 244 (6.48), 275
−1.85); IR (film) ν 2965, 1699, 1577, 1456, 1413, 1313, 1226,
max
−1
1
13
091 cm ; H and C NMR see Tables 3 and 4; HRESIMS [M −
H] m/z 251.0932 (calcd for C H O , 251.0925).
−
13
15
5
Pyrenochaetic acid H (11): colorless, amorphous solid; UV
MeOH) λmax (log ε) 212 (4.19), 270 (3.88), 326 (3.46) nm; IR
(
(
−1 1
film) νmax 2981, 1685, 1548, 1460, 1311, 1255, 1096 cm ; H and
1
3
−
C NMR see Tables 3 and 4; HRESIMS [M − H] m/z 249.0776
(
calcd for C H O , 249.0768).
13
13
5
ASSOCIATED CONTENT
■
Dimethylamide asterrate (13): pale yellow, amorphous solid; UV
MeOH) λmax (log ε) 212 (4.33), 284 (3.32), 310 (3.34) nm; IR
S
Supporting Information
(
(
*
−
1
1
1
13
1
1
film) ν 3439, 1625, 1469, 1357, 1249, 1205, 1149, 1060 cm ; H
max
NMR ( H, C, H− H COSY, HSQC, and HMBC) and
HRESIMS data of 1−3, 7−11, and 13−17, X-ray crystal
structure analysis for 2 and 7, ECD data for compound 14, and
detailed analyses of calculated and experimental ECD data for
compounds 7 and 10. This material is available free of charge
via the Internet at http://pubs.acs.org.
13
+
and C NMR see Table 5; HRESIMS [M + H] m/z 376.1392 (calcd
for C H NO , 376.1391).
19
22
7
25
(
−)-Blennolide G (14): pale yellow, amorphous solid; [α] −68.8
D
(
3
(
c 0.125, CHCl ); UV (MeOH) λ (log ε): 206 (4.26), 260 (3.97),
3 max
40 (3.85) nm; CD (MeOH) λmax (Δε) 211 (8.37), 223 (14.2), 328
−3.35); IR (film) νmax 2967, 1788, 1739, 1612, 1435, 1361, 1213,
G
dx.doi.org/10.1021/np5005449 | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
O’Neill, D. P.; DiStasio, R. A., Jr.; Lochan, R. C.; Wang, T.; Beran, G.
J.; Besley, N. A.; Herbert, J. M.; Lin, C. Y.; Van Voorhis, T.; Chien, S.
H.; Sodt, A.; Steele, R. P.; Rassolov, V. A.; Maslen, P. E.; Korambath,
P. P.; Adamson, R. D.; Austin, B.; Baker, J.; Byrd, E. F.; Dachsel, H.;
Doerksen, R. J.; Dreuw, A.; Dunietz, B. D.; Dutoi, A. D.; Furlani, T. R.;
Gwaltney, S. R.; Heyden, A.; Hirata, S.; Hsu, C. P.; Kedziora, G.;
Khalliulin, R. Z.; Klunzinger, P.; Lee, A. M.; Lee, M. S.; Liang, W.;
Lotan, I.; Nair, N.; Peters, B.; Proynov, E. I.; Pieniazek, P. A.; Rhee, Y.
M.; Ritchie, J.; Rosta, E.; Sherrill, C. D.; Simmonett, A. C.; Subotnik, J.
E.; Woodcock, H. L., 3rd; Zhang, W.; Bell, A. T.; Chakraborty, A. K.;
Chipman, D. M.; Keil, F. J.; Warshel, A.; Hehre, W. J.; Schaefer, H. F.,
3rd; Kong, J.; Krylov, A. I.; Gill, P. M.; Head-Gordon, M. Phys. Chem.
Chem. Phys. 2006, 8, 3172−3191.
AUTHOR INFORMATION
Corresponding Author
E-mail: rhcichewicz@ou.edu. Tel: 405-325-6969. Fax: 405-
25-6111.
■
*
3
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Research reported in this publication was supported by the
National Institutes of Health (1R01AI085161), NSF (CHE-
130835), a Challenge Grant from the Office of the Vice
President for Research at the University of Oklahoma, and an
award through the Shimadzu Equipment Grant Program.
■
0
(24) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci,
B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H.
P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.;
Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima,
T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.;
Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin,
K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.;
Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega,
N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.;
Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.;
Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.;
Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.;
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