Anti-Cryptococcus Phenalenones and Cyclic Tetrapeptides from Auxarthron pseudauxarthron

Article abstract of DOI:10.1021/acs.jnatprod.7b00341

Auxarthrones A-E (1-5), five new phenalenones, and two new naturally occurring cyclic tetrapeptides, auxarthrides A (7) and B (8), were obtained from three different solvent extracts of cultures of the coprophilous fungus Auxarthron pseudauxarthron. Auxarthrones C (3) and E (5) possess an unusual 7a,8-dihydrocyclopenta[a]phenalene-7,9-dione ring system that has not been previously observed in natural products. Formation of 1-5 was found to be dependent on the solvent used for culture extraction. The structures of these new compounds were elucidated primarily by analysis of NMR and MS data. Auxarthrone A (1) was obtained as a mixture of chromatographically inseparable racemic diastereomers (1a and 1b) that cocrystallized, enabling confirmation of their structures by X-ray crystallography. The absolute configurations of 7 and 8 were assigned by analysis of their acid hydrolysates using Marfey's method. Compound 1 displayed moderate antifungal activity against Cryptococcus neoformans and Candida albicans, but did not affect human cancer cell lines.

Full text of DOI:10.1021/acs.jnatprod.7b00341

Article
pubs.acs.org/jnp
Anti-Cryptococcus Phenalenones and Cyclic Tetrapeptides from
Auxarthron pseudauxarthron
†
†
‡
‡
§
†
Yan Li, Qun Yue, Dinith R. Jayanetti, Dale C. Swenson, Geoffrey A. Bartholomeusz, Zhiqiang An,
‡
,†
James B. Gloer, and Gerald F. Bills*
†Texas Therapeutic Institute, The Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center at
Houston, Houston, Texas 77054, United States
‡
Department of Chemistry, University of Iowa, Iowa City, Iowa 52242, United States
§
Department of Experimental Therapeutics, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center,
Houston, Texas 77054, United States
*
S Supporting Information
ABSTRACT: Auxarthrones A−E (1−5), five new phenale-
nones, and two new naturally occurring cyclic tetrapeptides,
auxarthrides A (7) and B (8), were obtained from three
different solvent extracts of cultures of the coprophilous fungus
Auxarthron pseudauxarthron. Auxarthrones C (3) and E (5)
possess an unusual 7a,8-dihydrocyclopenta[a]phenalene-7,9-
dione ring system that has not been previously observed in
natural products. Formation of 1−5 was found to be
dependent on the solvent used for culture extraction. The structures of these new compounds were elucidated primarily by
analysis of NMR and MS data. Auxarthrone A (1) was obtained as a mixture of chromatographically inseparable racemic
diastereomers (1a and 1b) that cocrystallized, enabling confirmation of their structures by X-ray crystallography. The absolute
configurations of 7 and 8 were assigned by analysis of their acid hydrolysates using Marfey’s method. Compound 1 displayed
moderate antifungal activity against Cryptococcus neoformans and Candida albicans, but did not affect human cancer cell lines.
Cryptococcus species are the world’s most common cause of
invasive fungal infections and affect about a million people each
year, resulting in over 600 000 deaths worldwide. Approx-
amauromine, auxarthonoside, methylpenicinoline, dechloroisor-
4
−10
umbrin, rumbrin, and auxarconjugatins,
and there are no
1
prior reports of metabolites from this species.
imately a third of all HIV/AIDS-associated fatalities are due to
cryptococcal disease, exceeding the number of tuberculosis-
attributed deaths. Options for treatment of Cryptococcus
infections are limited to three classes of antifungal agents, the
polyenes, the azoles, and the pyrimidine antimetabolite
flucytosine, and are often unsatisfactory. Pharmaceutical high-
throughput screening efforts primarily have been devoted to
finding cidal agents toward Candida albicans and other Candida
species for decades, while Cryptococcus and other emerging
invasive pathogens have received only limited attention. Thus,
Bioassay-guided fractionation of this extract led to identi-
fication of three new phenalenone derivatives designated as
auxarthrones A−C (1−3), the previously described, related
11
compound FR-901235 (6), and two new cyclic tetrapeptides,
auxarthrides A (7) and B (8). After considering features of the
structures of new compounds 1−3, as well as known
compound 6, it was hypothesized that their formation might
be artifactual in nature. Further experiments supported this
hypothesis and also led to the generation of two additional new
compounds (4 and 5), which we called auxarthrones D and E.
Even though phenalenones 1−6 proved to be solvent-induced
artifacts, products 1−5 and peptides 7 and 8 are new
compounds, some of which showed antifungal activity. Herein,
we report on the identification of the producing organism, its
fermentation, solvent extraction experiments, and the separa-
tion, structure elucidation, and characterization of 1−8, as well
as the results of antifungal and cytotoxicity assays for these
compounds.
2
invasive fungal infections are a growing health concern.
We have continued to build our collection of metabolite-
enriched fermentation extracts from unstudied coprophilous
ascomycete fungi using methods proven to promote expression
of secondary metabolism and have tested these extracts in a
growth-inhibition assay against the model pathogen Crypto-
coccus neoformans strain H99 with the objective of identifying
3
metabolites that selectively inhibit its growth in vitro. We
observed that a methyl ethyl ketone extract of cultures of an
isolate of Auxarthron pseudauxarthron (Eurotiomycetes, Ony-
genales) obtained from rabbit dung inhibited growth of
C. neoformans and C. albicans, but did not affect the growth
of Staphylococcus aureus (ATCC 4330). Only a few compounds
have been reported from members of this genus, including
Received: April 20, 2017
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.7b00341
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Article
partially resolving the components. Ultimately, the sample was
determined to be a mixture of racemic diastereomers in a 7:3
ratio, which was confirmed by integration of the best-resolved
1
H NMR resonances and the absence of optical rotation. Each
diastereomer is present in racemic form, and the ratio of the
diastereomeric forms is 7:3, so that all four different
stereoisomeric forms are present. Therefore, 2D NMR data
were collected on the mixture, and the data for the major and
minor diastereomeric forms (1a and 1b, respectively) were
considered separately for structure elucidation purposes. The
molecular formula was determined as C H O (11 degrees of
19
18
7
RESULTS AND DISCUSSION
unsaturation) on the basis of HRESIMS and NMR data (Table
■
1
13
1
). Analysis of the H, C, and HMQC NMR data for the
A methyl ethyl ketone extract from strain TTI-0363 grown in
an agitated liquid medium (MOF) inhibited growth of both
C. neoformans H99 and C. albicans ATCC 10231 in an agar
diffusion assay with moderately sized, clear inhibition zones,
while growth of S. aureus ATCC 43330 was unaffected at the
same test level. On the basis of morphological features on the
natural substratum of rabbit dung, features in culture, and
sequences of rDNA, strain TTI-0363 was identified as
Auxarthron pseudoauxarthon. Fungi of the genus Auxarthron
are typically associated with dung, soils, and keratinaceous parts
of animals, e.g., hair, skin, and hooves, and occasionally have
major diastereomeric form (1a) revealed the presence of three
exchangeable protons, including two hydrogen-bonded OH
protons (δ 12.27 and 12.79), four methyl groups (including
H
3
one O-methyl), one sp methine, 10 olefinic/aromatic carbons
(
two of which are protonated and three of which were attached
3
to oxygen), one oxygenated, nonprotonated sp carbon, and
three ketone carbons (δ 197.8, 198.8, and 208.6). These data
accounted for all of the H and C NMR resonances and
suggested that 1a possesses a tricyclic phenalene-dione
skeleton. A structure search on the phenalene-dione core
C
1
13
12−15
11
been isolated from humans.
Their sexual spores are
identified a known compound, FR-901235 (6), a sample of
formed within a spiny, cage-like hyphal structure called the
reticuloperidium, which is thought to cling to arthropods and
which was also isolated from this extract. Comparison of the
NMR spectroscopic data of 1a (Table 1) with those of 6
suggested that they possess the same 4,7,13-trihydroxy-9-
methoxy-14-methylphenalene-3,5-dione moiety, but that the
side-chain was different. In the HMBC data for 1a, correlations
from H -4′ to C-1′ and C-2′ and from H -3′ to C-1′ and C-2′
14,16
other animals to facilitate spore dispersal.
The fermentation
was subsequently scaled up to enable separation and
identification of the components responsible for the antifungal
activity.
NMR data for auxarthrone A (1) consistently showed two
sets of signals. Exhaustive efforts to separate this presumed
mixture employing column chromatography and HPLC using
various stationary and mobile phases were unsuccessful in even
3
3
indicated the presence of a 2-butanone moiety (C-1′−C-4′),
whereas a key HMBC correlation from H -4′ to C-4 indicated
3
that the 3-methyl-2-butanone moiety must be attached to C-4
of the phenalene-dione unit. Analysis of the 1D and 2D NMR
Table 1. NMR Data (500 MHz) of 1 in CDCl3
1
a (major)
1b (minor)
a
position
δC
δH mult., (J in Hz)
HMBC
δC
δH mult., (J in Hz)
1
2
3
4
5
6
7
8
9
136.8
105.6
197.8
83.4
136.8
106.2
196.9
83.3
b
b
c
c
198.8
102.6
168.0
96.6
199.8
102.1
168.0
96.3
6.41, s
6.79, s
6, 7, 10
6.39, s
6.81, s
167.1
113.6
149.9
119.1
164.9
26.3
167.1
113.6
149.7
119.4
164.8
26.3
1
1
1
1
1
1
2
3
4
9
4
7
1
0
1
2
2, 10, 13, 14
3
4
2.80, s
10, 11, 12
2.80, s
′
′
′
′
55.4
2.92, q (7.4)
4, 2′, 4′
55.7
2.92, q (7.4)
208.6
29.8
208.6
29.7
2.18, s
1.17, d (7.4)
4.01, s
1′, 2′
4, 1′, 2′
9
2.17, s
1.17, d (7.4)
4.01, s
11.2
11.2
-OCH3
-OH
-OH
3-OH
56.1
56.1
4.24, s
3, 4, 5
6, 7, 8
2, 12, 13
4.24, s
12.79, s
12.27, s
12.81, s
12.25, s
a
b,c
HMBC correlations are from proton(s) to the indicated carbon. These assignments are interchangeable.
B
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Figure 1. (A) LC-UV analysis of the different solvent extracts of cultures of the fungus Auxarthron pseudauxarthron. (B) Bioassay results for the
different solvent extracts. E: ethyl acetate extract; M: methyl ethyl ketone extract; A: acetone extract; P: 3-pentanone extract; Am: amphotericin B;
C.N.: C. neoformans; C.A.: C. albicans.
Figure 2. Thermal ellipsoid representations of 1a and 1b.
data (Table 1) for the minor diastereomeric form (1b)
established that it has the same planar structure as the major
isomer. On the basis of these data, the gross structure of
auxarthrone A was established as shown in 1. Crystals were
later obtained from the sample, enabling analysis by X-ray
crystallography. Both diastereomeric forms were observed in
the crystal, and perspective ORTEP plots of the major and
minor forms found to be present are shown in Figure 2.
Assuming that the major form in the crystal corresponds to the
major form observed in solution, the relative configurations of
1a and 1b could be assigned as 4S*,1′R* and 4S*,1′S*,
respectively.
Auxarthrone B (2) was assigned the same molecular formula
as 1 by HRESIMS analysis. However, in this instance, only a
single set of NMR signals was observed. Interpretation of its
NMR data (Table 2) again revealed the presence of a
phenalene-dione tricyclic ring system with the same sub-
stitution pattern found in 1, except that the side-chain (C-1′−
1
1
C-4′) was different. H− H COSY correlations observed
between H -3′ and H -5′, as well as HMBC correlations from
2
3
C
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Table 2. NMR Data (500 MHz) of 2 in CDCl and 3 in DMSO-d
3
6
2
3
a
a
position
δC
δH mult. (J in Hz)
HMBC
δC
δH mult. (J in Hz)
HMBC
1
2
3
4
5
6
7
8
9
137.2
105.6
136.0
108.2
138.3
76.3
b
197.4
77.5
b
199.3
102.1
168.9
96.4
198.5
103.0
168.1
95.9
6.40, s
6.80, s
6, 7, 10
6.47, s
6.90, s
6, 7, 10
167.2
113.6
149.8
119.3
165.7
26.4
167.1
113.4
140.7
119.1
156.9
25.7
10
11
12
13
14
1′
2, 10, 13, 14
2, 10, 13, 14
2.80, s
3.26, s
10, 11, 12
2.73, s
10, 11, 12
4, 5, 2′, 3′
3, 4, 2′, 3′
50.8
3, 4, 5, 2′
42.0
2.44, d (18.7)
3
.14, d (18.7)
2
3
4
5
9
4
7
1
′
′
′
′
208.3
37.1
205.0
161.4
11.5
2.49, q (7.2)
2′, 5′
1.73, s
4.00, s
3, 2′, 3′
7.3
0.99, t (7.2)
4.01, s
2′, 3′
9
-OCH3
-OH
-OH
3-OH
56.1
56.9
9
7
3.73, s
13.28, s
12.71, s
6, 7, 8
13.48, s
2, 12, 13
11.00, brs
a
b
HMBC correlations are from proton(s) to the indicated carbon. These assignments are interchangeable.
Scheme 1. Proposed Pathways Leading to Formation of Compounds 1−6
1
7
H-3′ to C-2′ and from H -1′ to C-3, C-4, C-5, and C-2′,
obtained in optically active form. Notably, the sample of the
known related compound FR-901235 (6) obtained in this
study was also found to be optically inactive, as in the original
2
indicated the substitution pattern shown for this unit in 2. A
near-zero optical rotation value for the sample of 2 suggested
that it was also obtained as a racemic mixture. CD data acquired
for samples of both 1 and 2 supported the conclusion that both
are racemic, as the data showed only noise-level features and
did not show Cotton effects reported for similar compounds
11
report.
The molecular formula of auxarthrone C (3) was determined
to be C H O (12 unsaturations) by HRESIMS, which was 18
mass units (H O) less than that of 2. Analysis of its H and C
19
16
6
1
13
2
D
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Table 3. NMR Data (500 MHz) of 4 in CDCl3
4
a (minor)
δH mult. (J in Hz)
4b (major)
a
position
δC
δC
δH mult., (J in Hz)
HMBC
1
2
3
4
5
6
7
8
9
136.8
105.8
198.1
83.4
136.8
106.2
196.9
83.3
b
b
c
c
198.6
102.6
167.9
96.5
200.1
102.1
168.0
96.4
6.40, s
6.79, s
6.39, s
6.80, s
6, 7, 10
167.1
113.5
149.8
119.2
164.9
26.3
167.1
113.5
149.7
119.3
164.8
26.3
1
1
1
1
1
1
2
3
0
1
2
3
4
′
2, 10, 13, 14
2.79, s
2.79, s
10, 11, 12
54.3
2.97, q (7.4)
54.6
2.97, q (7.4)
3, 4, 5, 2′, 4′
′
′
211.1
36.3
211.1
36.1
2.36, m
2.36, m
2.47, m
2′, 5′
2′, 5′
4, 1′, 2′
2′, 3′
9
2
.47, m
4
5
9
7
1
′
′
11.4
7.3
1.16, d (7.4)
0.94, t (7.2)
4.01, s
11.4
7.3
1.16, d (7.4)
0.94, t (7.2)
4.01, s
-OCH3
-OH
3-OH
56.1
56.1
12.85, s
12.90, s
12.36, s
12.30, s
a
b,c
HMBC correlations are from proton(s) to the indicated carbon. These assignments are interchangeable.
NMR data again showed only a single set of signals, and
HMBC correlations (Table 2) revealed structural features
matching those of 2, except that the signals for a ketone carbon
nearly identical to those found in 1, except that the methyl
group attached to the ketone carbonyl C-2′ (δ 211.1) was
C
replaced by signals for an ethyl unit in the NMR spectra of 4,
1
1
(
δ 197.4) at C-3 and a methylene group (δ /δ 2.49/37.1) at
and this observation was further supported by relevant H− H
C
H
C
C-3′ in 2 were replaced by two olefinic carbons (δ 138.3 and
COSY correlations, as well as HMBC cross-peaks from H -5′ to
C
3
161.4) in 3, respectively, suggesting that a π-bond was formed
C-2′ and C-3′ and from H -9′ to C-2′ and C-5′. Therefore, the
2
between C-3 and C-3′ to satisfy the unsaturation requirement
of 3. This conclusion was supported by HMBC cross-peaks
structure of 4 was assigned as shown.
Unlike 3, the aldol product (auxarthrone E; 5) that would
arise from 4 could form as a mixture of diastereomers. LC-UV-
MS data for the 3-pentanone extract (Figure 1 and Figure S11)
did reveal a minor component peak that afforded UV and MS
data that were fully consistent with results expected for the
condensation product 5, which would be analogous to 3, but
this product was not further characterized due to sample
limitations.
In view of the demonstration that both 2 and 4 are formed
by reaction of a naturally occurring species with ketone-
containing solvents, and the relationship with 3 and 5 as
apparent aldol condensation products of 2 and 4, respectively,
efforts were undertaken to independently convert 2 to 3 by
exposure of 2 to standard basic conditions employed for such
reactions and under the acidic conditions employed for HPLC;
however, no discernible conversion of 2 to 3 was detected
under these conditions.
from H -4′ to C-3, C-2′, and C-3′. On the basis of these data,
3
structure 3 was assigned to this compound. Compound 3 was
less abundant in the extract than 1 or 2 and appears to arise
from an intramolecular aldol-type condensation of 2.
Interestingly, this ring system does not appear to have been
previously reported as a feature of a natural product.
At this stage, after considering the biosynthetic steps leading
to herqueinone in Penicillium herquei reported by the Tang
1
8
group, we hypothesized that new phenalenones 1−3 (and the
previously reported 6) are actually artifacts arising from
spontaneous addition of methyl ethyl ketone or acetone to
an unstable triketone-type product (Scheme 1). Two experi-
ments were conducted in order to test this hypothesis. When
cultures were extracted with a non-ketone solvent (EtOAc), the
phenalenone peaks were absent (Figure 1). As a further test,
and to explore a potential means of generating additional new
compounds, we employed a different, symmetrical ketone (3-
pentanone) as the initial extracting solvent, ultimately leading
to the anticipated new homologous products, which we named
auxarthrones D and E (4 and 5).
As noted above, the known compound 6 was also isolated as
a minor component in this work and was readily identified as
FR-901235 by comparison of its NMR and MS data with those
11
reported. Experiments rigorously excluding acetone from the
process showed no evidence for the presence of 6, while
preparation of an extract using acetone as the solvent clearly
afforded much more of this product (Figure 1), strongly
suggesting that the original report of 6 is also likely to have
been associated with artifactual formation, even though the
By analogy to the case of 1, auxarthrone D (4) was also
obtained as a mixture of racemic diastereomers, this time in a
ratio of 6:4. Its molecular formula was determined to be
C H O (11 unsaturations) by HRESIMS, which was 14 mass
2
0
20
7
1
13
units (CH ) more than that of 1. Analysis of its H NMR,
C
2
NMR, and HMBC data (Table 3) revealed structural features
E
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Table 4. NMR Data (500 MHz) of 7 and 8 in DMSO-d6
7
8
a
a
position
δC
δH mult. (J in Hz)
HMBC
δC
δH mult. (J in Hz)
HMBC
1
170.1
62.4
34.2
170.0
62.4
34.2
2
3
4.24, dd (11.6, 3.0)
2.95, d (15.0)
1, 3, 4, 2-NCH3
2, 4, 5/9
4.24, dd (11.6, 3.0)
2.94, d (15.0)
1, 3, 4, 2-NCH3
2, 4, 5/9
3
.45, dd (15.0, 3.0)
4, 5/9
3.46, dd (15.0, 3.0)
4, 5/9
4
5
6
7
2
1
2
3
138.4
128.6
129.0
127.0
30.7
138.4
128.6
129.0
127.0
30.7
/9
/8
7.18, d (8.2)
7.32, t (8.5)
7.22, t (8.5)
2.67, s
3, 7
7.18, d (8.2)
7.32, t (8.5)
7.22, t (8.5)
2.66, s
3, 7
4, 8/6
5/9
4, 8/6
5/9
-NCH3
1‴, 2
1‴, 2
′
′
′
171.7
59.0
171.6
59.0
3.93, dd (11.3, 3.2)
1.70, m
1′, 3′, 4′, 2′-NCH3
2′, 4′, 5′
3.94, dd (11.3, 3.2)
1.71, m
1′, 3′, 4′, 2′-NCH3
2′, 4′, 5′
37.5
37.5
1
.82, m
2′, 4′, 5′
1.83, m
2′, 4′, 5′
4
5
6
2
1
2
3
4
′
′
′
24.9
21.3
23.6
30.5
171.2
55.4
29.6
18.6
1.32, m
24.9
21.3
23.6
30.5
171.3
54.9
36.4
24.8
1.32, m
0.88, d (6.0)
0.92, d (6.0)
2.64, s
3′, 4′, 6′
3′, 4′, 5′
1, 2′
0.88, d (6.0)
0.92, d (6.0)
2.64, s
3′, 4′, 6′
3′, 4′, 5′
1, 2′
′-NCH3
″
″
″
″
4.37, dd (9.0, 6.5)
2.16, m
1″, 3″, 4″, 5″
2″, 4″, 5″
2″, 3″, 5″
4.43, dd (9.0, 6.5)
1.90, m
1″, 3″, 4″, 5″
2″, 4″, 5″
3″, 5″, 6″
2″, 3″, 5″, 6″
2″, 3″, 4″
3″, 4″
0.89, d (6.8)
0.99, m
1
.60, m
5
6
2
1
2
3
4
5
2
″
″
21.1
0.93, d (6.8)
8.11, d (9.0)
2″, 3″, 4″
1′, 2″
17.2
12.3
0.93, d (6.8)
0.88, t (6.8)
8.11, d (9.0)
″-NH
‴
‴
‴
‴
‴
1′, 2″
171.2
55.0
29.2
18.6
20.8
171.2
55.0
29.2
18.6
20.8
4.04, t (8.3)
1.92, m
1‴, 3‴, 4‴, 5‴
2‴, 4‴, 5‴
2‴, 3‴, 5‴
2‴, 3‴, 4‴
1″, 2‴
4.04, t (8.3)
1.92, m
1‴, 3‴, 4‴, 5‴
2‴, 4‴, 5‴
2‴, 3‴, 5‴
2‴, 3‴, 4‴
1″, 2‴
0.74, d (6.8)
0.64, d (6.8)
8.05, d (8.3)
0.74, d (6.8)
0.64, d (6.8)
8.02, d (8.3)
‴-NH
a
HMBC correlations are from proton(s) to the indicated carbon.
published protocol for its isolation made no mention of
1
1
acetone.
Auxarthride A (7) was obtained as a white powder with a
molecular formula of C H N O (nine degrees of unsatura-
2
7
42
4
4
1
13
tion), as determined by HRESIMS. Analysis of its H and
C
NMR (Table 4) and HSQC data revealed the presence of two
amide N-H protons (δ 8.05 and 8.11), eight methyl groups
H
including two N-methyls, two methylenes, seven methines
(four of which are heteroatom-bonded), one phenyl group, and
four carboxylic carbons (δC 170.1−171.7). These data
accounted for all of the NMR resonances of 7 and eight of
the nine unsaturations, suggesting that 7 is a monocyclic
peptide. Interpretation of H− H COSY (Figure 3) and HSQC
data of 7 established two valine (Val) residues, one N-
methylphenylalanine (N-MePhe) unit, and one N-methylleu-
cine (N-MeLeu) unit. The amino acid sequence of 7 was
proposed by using the HMBC correlations (Figure 3) from
Figure 3. Selected 2D NMR correlations for 7 and 8.
1
1
auxarthride A (7). The 1-fluoro-2,4-dinitrophenyl-5-L-alanine
amide derivatives of the amino acids present in the acid
hydrolysate of 7 were analyzed by HPLC-MS along with those
of authentic D- and L-amino acids. All of the amino acid residues
were determined to have the L-configuration (Figure S16).
The molecular formula of auxarthride B (8) was determined
to be C H N O (nine degrees of unsaturation) by
relevant N-H or N-CH protons to neighboring carboxylic and
3
methine carbons. Specifically, the signals for the N-CH of N-
3
1
MePhe, the N-CH of N-MeLeu, the N-H of Val , and the N-H
3
28 44
4
4
2
of Val were correlated to the carboxylic carbon signals of the
Val (δ 171.2), N-MePhe (δ 170.1), N-MeLeu (δ 171.7),
and Val (δC 171.2) residues, respectively, establishing the
sequence N-MePhe−N-MeLeu−Val −Val for 7. Marfey’s
method was applied to assign the absolute configurations of
HRESIMS, indicating the presence of one CH unit more
2
2
1
13
C
C
C
than that of 7. Analysis of its H and C NMR spectroscopic
1
data (Table 4) revealed structural features nearly identical to
1
2
1
those found in 7, except that the Val residue was replaced by
1
9
1
1
an Ile unit, which was confirmed by relevant H− H COSY and
the amino acid residues resulting from acid hydrolysis of
HMBC correlations (Figure 3). The absolute configurations of
F
DOI: 10.1021/acs.jnatprod.7b00341
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
all of the amino acid residues in 8 were again assigned to have
L-configurations (Figure S17) by using Marfey’s method,
although notably, as documented in the literature, this
method does not enable L-Ile to be definitively differentiated
from the L-allo-isomer.
All of these compounds were tested for antifungal activity
against C. neoformans H99 and C. albicans ATCC 10231 (Table
Cyclic peptides are among the most frequently encountered
fungal secondary metabolites. Over 40 fungal cyclic tetrapep-
tides are known, although interestingly, aside from auxarthrides
A (7) and B (8), hirsutide and tentoxins appear to be the
only previously reported fungal cyclic tetrapeptides that contain
N-methylated amino acid units.
20
28
29
5
). Phenalenone derivative 1 (i.e., the mixture of 1a and 1b)
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
acquired using a Rudolph Research AUTOPOL III automatic
polarimeter. UV data were obtained for solutions in MeOH with a
Varian Cary III UV/vis spectrophotometer. Electronic circular
dichroism data were recorded with an Olis Cary-17 spectrophotometer
1 cm cell) using MeOH as solvent. All H, C, and 2D NMR
COSY, HSQC, and HMBC) data were collected using a Bruker
Table 5. Antifungal Activities of 1−4 and 6
MIC (μg/mL)
compound
C. neoformans H99
C. albicans ATCC 10231
1
13
(
(
1
2
3
4
6
3.2
12.8
25.6
6.4
3.2
25.6
51.2
6.4
AVANCE-500 MHz spectrometer with a 5 mm triple resonance
cryoprobe at 298 K using solvent signals (CDCl : δ /δ , 7.26/77.2;
3
H
C
DMSO-d : δ /δ , 2.50/39.5) as references. The HSQC and HMBC
6
H
C
51.2
0.8
51.2
0.8
13
experiments were optimized for 145 and 8 Hz, respectively. C NMR
multiplicities were established by APT experiments and were
consistent with the position assignments in Tables 1−4. ESIMS data
were recorded on an Agilent 6120 single quadrupole LC-MS using
positive ion electrospray ionization. High-resolution mass spectra were
acquired with an Orbitrap Elite (Thermo Scientific) instrument.
HPLC separations were performed on an Agilent 1260 instrument
amphotericin B
showed a moderate inhibitory effect against both pathogens,
with MIC values of 3.2 μg/mL in each case, while the positive
control amphotericin B showed an MIC value of 0.8 μg/mL.
The other phenalenones (2−4 and 6) showed weaker anti-
Cryptococcus and anti-Candida activity, with MIC values ranging
from 6.4 to 51.2 μg/mL. These compounds were also evaluated
for cytotoxicity against two human cancer cell lines, but none
showed significant cytotoxic effects against MDA-MB-231 or
MDA-MB-451 breast cancer cell lines (IC50 > 20 μM in each
case).
(
Agilent, USA) equipped with a variable-wavelength UV detector.
Fungal Strain and Identification. Strain TTI-0363 was isolated
from ascomata formed on rabbit dung (Figure S1), likely from desert
cottontail (Sylvilagus audubonii), collected in October 2015 along U.S.
Route 385, between Marathon and the north entrance of Big Bend
National Park, Brewster County, Texas. Dung samples were
rehydrated with deionized water and incubated at room temperature
in deep-dish Petri plates lined with moist filter paper until ascomata
formed on the dung surface. Ascospores and asci were dissected from
mature ascomata and transferred to cornmeal-dextrose agar
supplemented with 50 μg/mL chlortetracycline and streptomycin
sulfate. Agar plates with dissected ascospores and asci were sealed and
incubated overnight at 45 °C to promote ascospore germination.
Germinated ascospores were then transferred to YMA medium (1%
maltose extract, 0.2% yeast extract, and 2% agar) to establish axenic
cultures. The isolate was assigned the accession number TTI-0363 in
Texas Therapeutics Institute’s culture collection at the Brown
Foundation Institute of Molecular Medicine, University of Texas
Health Science Center at Houston, Houston, TX, USA.
21
Phenalenones comprise a unique class of polyketides.
Members of this group of natural products have been reported
from both higher plants and microbial sources and have shown
significant biological activities, including antimicrobial and
cytotoxic effects. To date, more than 80 fungal-derived
phenalenones have been characterized, some of which are
homodimers and heterodimers, reflecting substantial chemical
2
1
1
8,21−25
diversity.
Auxarthrones A−E (1−5) are new variants in
this class. Structurally, they are most closely related to the co-
11
isolated compound FR-901235 (6), a compound previously
identified from extracts of Paecilomyces carneus cultures. As
discussed above, 1−6 are not natural products, but are derived
from a natural precursor upon exposure to ketone-containing
solvents. However, auxarthrones C (3) and E (5) possess a
novel 7a,8-dihydrocyclopenta[a]phenalene-7,9-dione ring sys-
tem not previously observed in natural products, and synthetic
compounds with this ring system have only very rarely been
The strain was identified as a species of Auxarthron based on the
following combination of characteristics (Figure S1). The ascomata
were buff- to cinnamon-colored, spherical, and consisting of a loosely
interwoven reticuloperidium. The peridial hyphae were yellowish
brown to rusty brown, finely to coarsely asperate, with short spines
(10−100 μm long) forming on the angled joints of the peridial
hyphae. The asci formed in the center of the reticuloperidium were
globose to subgloblose and dissolved quickly. The ascospores were
globose and pale yellow, with the wall ornamented by a punctate
reticulum, and had an average diameter of 3.3 ± 0.3 μm (n = 20). The
short peridial spines, small ascospores, and lack of a conidial state
distinguished the strain as A. pseudauxarthron. A. pseudauxthron was
first described from rabbit dung in Utah and has been reported from
2
6,27
reported.
Compounds 1−6 share the same putative
biosynthetic precursor heptaketide and intermediate triketone,
subsequently undergoing acetone, 2-butanone, or 3-pentanone
addition at C-6 to generate 1, 2, 4, and 6, while 3 and 5 are
further aldol condensation products of 2 and 4, respec-
3
0,31
Texas and Argentina.
1
8,21,22
tively.
The production of phenalenone oxidative cleavage
To corroborate the morphological identification, we estimated its
approximate phylogenetic relationships based on the internal tran-
scribed spacer (ITS) and large subunit (LSU) rDNA sequences. DNA
extraction from cultured mycelium and DNA cloning and sequencing
artifacts leading to the formation of naphthalic anhydrides has
been discussed previously, as has the possibility that certain
metabolites of this type might be formed as artifacts.
However, potential for reactions of fermentation products
from phenalenone biosynthesis with ketone-containing solvents
21
3
2
protocols were the same as previously described. The DNA sequence
was accessioned in GenBank (KY014424, TTI-0363, ITS + LSU).
Several recent phylogenetic studies of the Onygenales and the genus
Auxarthron have focused on defining the generic boundaries and
placement of new species within the existing phylogenetic frame-
(or other reagents) seems to have been neither fully verified
nor explored in detail. Ultimately, even though the new
phenalenones isolated in this work proved to be artifactual in
nature, some of them showed antifungal activity.
1
2,13,15
work.
The sequence of the ITS region of TTI-0363 was
intercalated within previous alignments of ITS sequences from other
G
DOI: 10.1021/acs.jnatprod.7b00341
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Auxarthron species. A maximum likelihood analysis of this alignment
a narrow-frame algorithm. All non-hydrogen atoms were refined with
anisotropic displacement parameters, and all hydrogen atoms were
placed in idealized positions and refined as riding atoms with the
relative isotropic parameters. Data were corrected for absorption
effects using the multiscan method (SADABS). The ratio of minimum
to maximum apparent transmission was 0.968. The calculated
minimum and maximum transmission coefficients (based on crystal
size) are 0.9740 and 0.9980. The 24 943 measurements yielded 3382
independent reflections after equivalent data were averaged, and
Lorentz and polarization corrections were applied. The final
refinement gave R = 0.0529 and wR = 0.1504 [I > 2σ(I)]. The
33
was executed in Mega 6.0 and resulted in a phylogenetic tree with
topology similar to those previously reported (Figure S2). On the basis
of alignment of its ITS sequence, TTI-0363 fell among a group of
strains that have been loosely referred to as the A. kuehnii
12,13,15
complex
with the nearest match being a strain from soil in
Utah identified as A. kuehnii (RV 22810, Figure S2). TTI-0363 fell
outside of a monophyletic branch of the tree with the type strain of
A. kuehnii and among a loosely organized branch that included the ex-
type strain of A. pseudauxarthron and other strains that have been
previously referred to as A. kuehnii. The phylogenetic heterogeneity of
strains identified as A. kuehnii has prompted investigators to consider
this species as a complex that includes A. pseudauxthron, A. pseudor-
eticulum, and other possibly unrecognized species. Therefore, we refer
to TTI-0363 as A. pseudauxarthron based on its morphological features
and its phylogenetic proximity to the ex-type strain.
1
2
crystal structure was found to contain a mixture of the (4R′,1′S′)
diastereomer (Occ. = 0.772(3)) and the (4R′,1′R′) diastereomer (Occ.
=
0.228(3)).
Auxarthrone B (2): yellow, amorphous solid; UV (MeOH) λ
max
1
13
(
log ε) 248 (4.58), 330 (4.51) nm; H and C NMR and HMBC data
Fermentation. Agar plugs from YMA cultures were inoculated
into 250 mL Erlenmeyer flasks containing 50 mL of SMYA seed
medium (1% Bacto neopeptone, 4% maltose, 1% yeast extract, and
+
see Table 2; HRESIMS m/z 359.1126 [M + H] (calcd for C H O ,
1
9
19
7
3
59.1125).
Auxarthrone C (3): yellow, amorphous solid; UV (MeOH) λ
max
0
.4% agar). Seed media were incubated for 5 days with agitation at 220
1
13
(log ε) 219 (4.20), 258 (4.40), 334 (4.01) nm; H and C NMR and
rpm at 23 °C. A scaled-up fermentation was then carried out in 10
+
HMBC data see Table 2; HRESIMS m/z 341.1012 [M + H] (calcd
Erlenmeyer flasks (500 mL) each containing 100 mL of MOF
for C H O , 341.1020).
34
19 17
6
medium consisting of mannitol 75 g; oat flour 15 g; yeast extract 5 g;
L-glutamic acid 4 g; and 2-(N-morpholino)ethanesulfonic acid 16.2 g
Auxarthrone D (4): green, amorphous solid; UV (MeOH) λ (log
max
1
13
ε) 217 (4.23), 255 (4.14), 331 (3.76) nm; H and C NMR and
in 1 L of deionized H O. A 5 mL amount of seed medium was
+
2
HMBC data see Table 3; HRESIMS m/z 373.1290 [M + H] (calcd
inoculated into each flask and cultivated at 25 °C with agitation at 220
rpm for 14 days.
Extraction and Isolation. The fermented cultures of TTI-0363
were combined and extracted repeatedly with methyl ethyl ketone (3
for C H O , 373.1287).
2
0
21
7
FR-901235 (6): H and 13C NMR and MS data were consistent with
1
11
literature values.
Auxarthride A (7): white powder; [α]20 −149 (c 0.07, MeOH);
D
×
1.0 L), and the organic solvent was evaporated to dryness under
vacuum to afford a crude extract (3.0 g), which was fractionated by
1
13
UV (MeOH) λmax (log ε) 206 (4.14) nm; H and C NMR and
+
HMBC data see Table 4; HRESIMS m/z 487.3273 [M + H] (calcd
silica gel VLC using n-hexane−EtOAc gradient elution. The fraction
for C H N O , 487.3279).
Auxarthride B (8): white powder; [α]
27
43
4
4
(
300 mg) eluted with 40% to 60% EtOAc were combined and further
20
−165 (c 0.07, MeOH);
1 13
D
separated by Sephadex LH-20 column chromatography using 1:1
UV (MeOH) λmax (log ε) 206 (4.19) nm; H and C NMR and
CH Cl −MeOH as the eluent, and resulting subfractions were
2
2
+
HMBC data see Table 4; HRESIMS m/z 501.3429 [M + H] (calcd
combined and further purified by semipreparative RP HPLC (Agilent
Zorbax SB-C18 column; 5 μm; 9.4 × 250 mm; gradient elution 55−
for C H N O , 501.3435).
28
45
4
4
Marfey’s Analysis of Auxarthride A (7) and Auxarthride B
7
0% MeCN in H O with 0.1% formic acid over 35 min; 2 mL/min) to
12
2
(
8). A sample (ca. 1.0 mg) of the target compound (7 or 8) was
afford 3 (2.5 mg, t 10.1 min), 6 (1.5 mg, t 13.4 min), 1 (12.0 mg, t
R
R
R
hydrolyzed by heating in 6 N HCl (1 mL) at 110 °C for 24 h. Upon
removal of excess HCl under vacuum, the hydrolysate was placed in a
1
5.5 min), 2 (14.5 mg, t 17.4 min), 7 (8.5 mg, t 23.6 min), and 8
R
R
(
10.5 mg, t 30.2 min).
R
2
mL reaction vial and treated with a 1% solution of 1-fluoro-2,4-
Similarly, 100 mL of fermented cultures of TTI-0363 were extracted
dinitrophenyl-5-L-alanine amide (FDAA; 150 μL) in acetone, followed
repeatedly with 3-pentanone (3 × 100 mL), and the solvent was
evaporated to dryness under vacuum to afford a crude extract (300
mg), which was fractionated on a Grace RP C18 column (10−100%
MeOH in water over 35 min; flow rate was 20 mL/min) coupled to a
Grace Reveleris X2 flash chromatography system. Fractions with
desired compounds were combined and further purified by semi-
preparative RP HPLC (Agilent Zorbax SB-C18 column; 5 μm; 9.4 ×
by 1.0 M NaHCO (40 μL). The reaction mixture was heated at 45 °C
3
for 1.5 h, cooled to room temperature, and acidified with 2.0 N HCl
(20 μL). Similarly, the standard L- and D-amino acids were also
derivatized with FDAA following the same procedure. The derivatives
of the hydrolysates and the standard amino acids were subjected to
HPLC-ESIMS analysis (Agilent Eclipse Plus C18 column; 5 μm, 4.6 ×
1
50 mm; 1.0 mL/min) at 35 °C using the following gradient program:
2
50 mm; gradient elution 55−64% MeCN in H O with 0.1% formic
2
solvent A, H O (0.1% formic acid); solvent B, MeCN (0.1% formic
acid over 21 min; 2 mL/min) to afford compound 4 (5.3 mg, t 19.75
2
R
acid); linear gradient, 15−45% of B in A over 60 min with UV
detection at 340 nm. The retention times and ESIMS data for FDAA
derivatives of the hydrolysates and the standard amino acids are
summarized in Figures S16 and S17.
min). A metabolite with LCMS and LCUV spectra consistent with
structure 5 was detected, but was not present in sufficient quantities to
enable its isolation.
Auxarthrone A (1): yellow needles; mp 166−168 °C; UV (MeOH)
1
13
Antifungal Assays. In vitro antifungal activity was measured
according to National Committee for Clinical Laboratory Standards
λmax (log ε) 248 (4.58), 330 (4.51) nm; H and C NMR and HMBC
+
data see Table 1; HRESIMS m/z 381.0944 [M + Na] (calcd for
3
6
(
NCCLS) recommendations. The minimum inhibitory concen-
C H O Na, 381.0945).
X-ray Crystallographic Analysis of 1. Upon crystallization
1
9
18
7
3
5
tration (MIC) was determined by means of the serial dilution method
in 96-well plates with YM (1% maltose extract, 0.2% yeast extract, and
from CHCl −H O (30:1) using the vapor diffusion method, colorless
3
2
0
.4% agar) as the test medium. Amphotericin B was used as the
crystals were obtained for 1. A crystal (0.020 × 0.110 × 0.230 mm)
was separated from the sample and mounted on a glass fiber, and data
were collected using a Bruker-Nonius Kappa ApexII diffractometer
with graphite-monochromated Mo Kα radiation, λ = 0.710 73 Å at
positive control. Test compounds were dissolved in DMSO and
serially diluted in growth medium. Visual end point and optical density
readings of microplate wells were measured relative to positive and
negative controls. The strains were incubated at 25 °C, and the MICs
were determined at 24 h for C. albicans ATCC 10231 and at 48 h for
C. neoformans H99. Viability was determined with the aid of a plate
reader using PrestoBlue resazurin dye (Life Technologies) as the
viability indicator. The spectrophotometric MIC value was defined as
the lowest concentration of a test compound that resulted in a culture
1
50(2) K. Crystal data: C H O ·H O, M = 376.35, space group
19 18 7 2
orthorhombic, P2(1)2(1)2(1); unit cell dimensions a = 10.1479(10)
Å, b = 10.1826(10) Å, c = 10.2083(10) Å, V = 857.44(15) Å , Z = 2,
Dcalcd = 1.458 mg/m , μ = 0.115 mm , F(000) = 396. A total of 1461
frames were collected. The total exposure time was 20.29 h. The
frames were integrated with the Bruker SAINT software package using
3
3
−1
H
DOI: 10.1021/acs.jnatprod.7b00341
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.7b00341.
■
(
S
6
(
Liu, F.; Zhang, W.-Y.; Chen, G.; Bai, J.; Hua, H.-M.; Wang, H.-F.; Pei,
Y.-H. Tetrahedron Lett. 2016, 57, 645−649.
Crystallographic data (CIF)
H and C NMR spectra of 1−4, 7, and 8, and
LCESIMS data for FDAA derivatives of the acid
1
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AUTHOR INFORMATION
Corresponding Author
E-mail: Gerald.F.Bills@uth.tms.edu.
Phongpaichit, S.; Borwornwiriyapan, K.; Sakayaroj, J. J. Nat. Prod.
014, 77, 2375−2382.
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M. H. G. J. Nat. Prod. 2005, 68, 1303−1305.
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L.; Tam, J.; Rich, D. U.; Durbin, R. D. Biochem. Biophys. Res. Commun.
■
2
(
1
(
*
ORCID
James B. Gloer: 0000-0002-9261-7571
Gerald F. Bills: 0000-0003-2352-8417
1
Notes
(
The authors declare no competing financial interest.
1
973, 53, 653−658.
ACKNOWLEDGMENTS
■
(30) Anonymous. CBS-KNAW Fungal Biodiversity Center Culture
Collection. In 2016.
(31) Godeas, A. M. Mycopathologia 1972, 46, 189−204.
(32) Jayanetti, D. R.; Yue, Q.; Bills, G. F.; Gloer, J. B. J. Nat. Prod.
This work was supported by University of Texas Health Science
Center at Houston new faculty start-up funds, the Kay and Ben
Fortson Endowment (to G.B.), and a grant from the NIH (R01
GM121458).
2
(
015, 78, 396−401.
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Mol. Biol. Evol. 2013, 30, 2725−2729.
34) Mandala, S. M.; Harris, G. H. Isolation and characterization of
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DOI: 10.1021/acs.jnatprod.7b00341
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