Carneic acids A and B, chemotaxonomically significant antimicrobial agents from the xylariaceous ascomycete Hypoxylon carneum

Article abstract of DOI:10.1021/np0602057

Carneic acids A and B (1, 2) are polyketide antibiotics structurally related to phomopsidin. They were isolated as major constituents of the stromata of Hypoxylon carneum, a species that had shown a highly specific secondary metabolite profile in a survey of xylariaceous ascomycetes based on HPLC profiling. Their chemical structures were elucidated by a combination of spectroscopic methods and by preparation of derivatives. An X-ray crystal structure of the dinitrobenzoate of carneic acid B methyl ester (8) was obtained, even allowing for determination of its absolute structure. The carneic acids showed weak antibacterial and moderate antifungal activities in the serial dilution assay against selected microbial organisms. They appear to be species-specific marker molecules in H. carneum from different geographic regions, but do not constitute major metabolites of more than 100 species of Xylariaceae. Their biological and chemotaxonomic significance is discussed.

Full text of DOI:10.1021/np0602057

1198
J. Nat. Prod. 2006, 69, 1198-1202
Carneic Acids A and B, Chemotaxonomically Significant Antimicrobial Agents from the
Xylariaceous Ascomycete Hypoxylon carneum
Dang Ngoc Quang,†, Marc Stadler,*,§ Jacques Fournier,‡ and Yoshinori Asakawa†
Faculty of Pharmaceutical Sciences, Tokushima Bunri UniVersity, Yamashiro-cho, Tokushima 770-8514, Japan, InterMed DiscoVery GmbH,
BioMedizinZentrum Dortmund, Otto-Hahn-Strasse 15, D-44227 Dortmund, Germany, Las Muros, F-09420 Rimont, Arie´ge, France, and
Faculty of Chemistry, Hanoi UniVersity of Education, 136 Xuan Thuy Road, Cau Giay, Hanoi, Vietnam
ReceiVed May 5, 2006
Carneic acids A and B (1, 2) are polyketide antibiotics structurally related to phomopsidin. They were isolated as major
constituents of the stromata of Hypoxylon carneum, a species that had shown a highly specific secondary metabolite
profile in a survey of xylariaceous ascomycetes based on HPLC profiling. Their chemical structures were elucidated by
a combination of spectroscopic methods and by preparation of derivatives. An X-ray crystal structure of the dinitrobenzoate
of carneic acid B methyl ester (8) was obtained, even allowing for determination of its absolute structure. The carneic
acids showed weak antibacterial and moderate antifungal activities in the serial dilution assay against selected microbial
organisms. They appear to be species-specific marker molecules in H. carneum from different geographic regions, but
do not constitute major metabolites of more than 100 species of Xylariaceae. Their biological and chemotaxonomic
significance is discussed.
Hypoxylon carneum Petch (Xylariaceae, Figure 1) is a wood-
inhabiting ascomycete, first described from Sri Lanka1 and later
also reported from the United States, Venezuela, New Zealand,2
and France.3 It is one of the few species in the large genus
Hypoxylon that is not restricted to certain geographic areas, but
was proven to have a truly cosmopolitan distribution, being present
in temperate as well as in subtropical and tropical climates. As
judged from our field studies on its occurrence in southwestern
France,3 it is apparently not rare. Because of its morphological
similarity to the common H. rubiginosum and other related species,
it has probably been frequently overlooked.3 The most striking
feature to discriminate H. carneum from its relatives can be readily
determined without any need for detailed microscopic studies: its
pigments in KOH are faint purple, while those of related species
yield dense orange, brown, or yellowish pigments in KOH.3 Also,
H. carneum has faintly yellowish granules beneath the stromata
surface, whereas those of its allies are orange, red, or brown. These
features are due to the presence of different secondary metabolites,
which was established by a HPLC profiling study of about 2500
specimens and cultures of Hypoxylon and related Xylariaceae.3-6
Recent evidence for the molecular taxonomy of Hypoxylon and
allies7 revealed that the closest affinities of H. carneum (i.e., the
clades H1 and H2 as described in ref 7), as inferred from a
molecular phylogenetic study, are with species that preferably
contain azaphilones. These clades comprise, aside from some taxa
that were not yet studied for HPLC profiles, for example, H.
cercidicola, H. petriniae,3 H. perforatum, H. pilgerianum, and H.
rubiginosum.3,4 H. carneum strongly differed from all of these
species in its HPLC profile. The common stromatal azaphilone
pigments of many Hypoxylon spp. of clades H1 and H2 as described
in ref 7 (i.e., mitorubrins, rubiginosins, and hypomiltin3,4) were not
detected by HPLC-MS, and instead 4:5:4′:5′-tetrahydroxy-1:1′-
binaphthyl (BNT, 3) and a series of apparently species-specific but
yet unknown metabolites were detected as major components.4
Interestingly, only BNT (3) and daldinins, but no mitorubrin-type
azaphilones, were also reported previously from the core species
of another side branch of clade H1, i.e., H. fuscum.
We have recently checked the extracts of various Hypoxylon spp.
derived from the above studies for antimicrobial activities. The
stromatal MeOH extract of H. carneum showed significantly
stronger antifungal activities than those prepared from its aza-
philone-containing relatives (Table S1, Supporting Information).
Recently, the fungus was collected in sufficient amounts for
preparative studies. We here report the identification of 1 and 2
and preliminary studies on their biological properties.
Results and Discussion
Carneic acids A and B (1, 2) were detected in and isolated from
the stromatal methanol extract of H. carneum as described in the
Experimental Section, along with BNT (3).6 In addition, rubiginosin
A (4) and orsellinic acid (i.e., characteristic major metabolites of
many Hypoxylon spp.3,8) were detected by HPLC profiling4 in the
fractions derived from preparative chromatography after removal
of the major components (1-3). The presence of these compounds
* Corresponding author. E-mail: marc.stadler@t-online.de.
† Tokushima Bunri University.
§ InterMed Discovery GmbH.
‡ Las Muros.
Hanoi University of Education.
10.1021/np0602057 CCC: $33.50
© 2006 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/26/2006

Antimicrobial Agents from Hypoxylon carneum
Journal of Natural Products, 2006, Vol. 69, No. 8 1199
Figure 1. Stromata of Hypoxylon carneum. Left: Habit on wood. Right: Augmented section, showing perithecial mounds. Scale is indicated
by bars.
Table 1. 1H NMR Data for 1, 2, and 5-8
position
1
2
5
6
7
8
2
5.79 (d, 15.1)
7.26 (dd,10.1,
15.1)
5.79 (d, 15.1)
7.27 (dd, 9.9, 15.4)
5.79 (d, 15.3)
7.29 (dd, 10.1,
15.3)
6.09 (dd, 10.2,
15.1)
5.81 (d, 15.1)
7.28 (dd, 10.7,
15.4)
6.12 (dd, 10.3,
15.1)
5.82 (d, 15.4)
7.30 (dd, 10.4,
15.3)
6.13 (dd, 10.2,
14.8)
5.83 (d, 15.4)
7.26 (dd, 11.0,
15.4)
6.15 (dd, 11.0,
15.1)
3
4
5
6.12 (overlapped)
6.20 (dd, 10.2, 15.1)
6.16 (dd, 10.0, 15.1)
6.12 (overlapped)
6.06 (dd, 9.9, 15.1)
6.05 (dd, 10.2,
15.1)
6.07 (dd, 9.6, 15.1)
6.04 (dd, 10.4,
15.2)
6
7
8
9a
9e
10a
10e
2.46 (m)
1.29 (t, 9.9)
1.25 (m)
1.14 (m)
1.68 (m)
1.37 (m)
1.99 (dd, 3.9,
12.4)
2.51 (dd, 6.0, 10.2)
1.34 (m)
1.26 (m)
1.16 (dt, 3.6, 13.5)
1.68 (m)
1.36 (m)
2.45 (m)
1.28 (t, 9.9)
1.25 (m)
1.13 (m)
1.67 (m)
1.36 (m)
1.98 (m)
2.50 (dt, 6.0, 17.0)
1.29 (m)
1.27 (m)
1.14 (m)
1.67 (dd, 3.6, 13.7)
1.37 (m)
2.49 (dd, 6.0, 10.4)
1.44 (t, 9.9)
1.34 (m)
1.26 (m)
1.76 (m)
2.58 (dt, 6.3, 10.7)
1.44 (q, 10.7)
1.35 (m)
1.25 (m)
1.76 (m)
1.54 (m)
2.18 (m)
1.56 (m)
2.19 (m)
1.94 (quin, 4.1)
1.99 (m)
11
3.27 (dt, 4.1,
10.4)
3.71 (dt, 4.4, 10.4)
3.26 (dt, 4.4, 10.4)
3.27 (dt, 4.4, 10.4)
4.77 (dt, 4.7, 10.7)
4.79 (dt, 4.7, 10.9)
12
13
15
17
18
1.70 (m)
1.71 (m)
5.91 (brs)
2.55 (m)
5.39 (dt, 1.1, 6.6)
4.17 (dd, 6.6, 13.8)
4.12 (dd, 6.3, 13.8)
0.90 (d, 6.0)
1.57 (s)
1.70 (m)
1.72 (m)
2.18 (m)
5.46 (brs)
2.53 (d, 7.4)
5.23 (dd, 1.4, 6.8)
1.62 (d, 6.5)
2.20 (m)
5.83 (brs)
2.51 (d, 5.5)
5.22 (dd, 1.4, 6.8)
1.61 (d, 6.5)
5.83 (brd, 1.4)
2.50 (d, 5.8)
5.21 (dd, 1.1, 6.8)
1.61 (d, 6.8)
5.88 (brd, 1.4)
2.54 (d, 6.0)
5.41 (dt, 1.1, 6.6)
4.23 (dd, 6.9, 12.9)
4.20 (dd, 6.3, 12.9)
0.86 (d, 6.0)
1.57 (s)
5.53 (brd, 1.1)
2.64 (d, 5.5)
5.47 (dt, 1.1, 5.8)
4.92 (d, 6.9)
19
20
21
0.86 (d, 5.8)
1.54 (s)
1.57 (s)
0.86 (d, 5.8)
1.54 (d, 1.4)
1.57 (s)
0.91 (d, 6.3)
1.48 (s)
1.58 (s)
0.90 (d, 6.0)
1.51 (s)
1.78 (s)
1.66 (s)
1.65 (s)
1-OMe
benzoate
3.74 (s)
3.74 (s)
3.75 (s)
8.13 (d, 9.1)
8.25 (d, 9.1)
3.74 (s)
8.32 (d, 9.1)
8.25 (d, 9.1)
8.21 (d, 9.1)
in H. carneum is in accordance with the taxonomic relationships
between the H. rubiginosum complex and H. carneum that had
previously been established on the basis of morphological and
molecular data.3,4,7 However, 1 and 2 were not detected in any of
the fractions still available from previous work on H. rubiginosum
and allied taxa containing mitorubrin-type azaphilones,8 by the
established HPLC profiling methodology. Retrospective HPLC-MS
analysis of the data on all specimens of Xylariaceae examined
previously,3-5 using 1 and 2 as external standards, also did not
give any indication as to their presence in over 20 additional species.
However, 1 and 2 were also revealed to be major components of
the holotype material of H. carneum and other specimens.
Therefore, the taxonomic significance of carneic acids appears to
be restricted to the species level.
Extraction of the stromatal surface of H. carneum specimens
after careful detachment from the perithecial layer as described
previously4 revealed larger amounts of BNT (3) in relation to the
carneic acids (1, 2) in the resulting MeOH extracts by HPLC.
Therefore, the new compounds are presumably concentrated in the
granules embedded in waxy layers, surrounding the perithecia, while
(as in other Hypoxylon spp.4) the binaphthyl 3 is preferentially
located at the surface. The fact that 1 and 2 are not exposed to air
or light might be the reason these compounds proved stable in old
herbarium specimens for decades. It even gives some indication of
their possible biological functions as hidden “defense weapons” of
H. carneum that are only exposed if the fruiting bodies are attacked
by feeding enemies
Carneic acid A (1) was isolated as an oil. It was formulated as
C21H30O3 by HREIMS. Its IR and UV spectra exhibited absorption
bands due to a conjugated carboxylic group (3500-2300, 1687
cm-1 and 267 nm, respectively) and double bonds (1634 cm-1).
The 1H NMR spectrum of 1 (Table 1) revealed the presence of six
olefinic protons, one tertiary methyl, three vinylic methyls, and two
methylenes. The 1H-1H COSY spectra of 1 showed connectivities
from C-2 to C-13, between C-6 and C-15, and between C-17 and
C-18. Careful analysis of its HMQC and HMBC spectra suggested
that 2 contained a pentadienoic acid and 1-methylpropenyl partial
structures, which connected with the decaline skeleton at C-6 and
C-15, respectively, on the basis of HMBC correlations between
H-5 and C-6 and between H-15 and C-17. The absolute structure
of 2 was established by CD and NOESY spectra. Two double bonds
of the pentadienoic acid unit and the double bond C16-17 were all

1200 Journal of Natural Products, 2006, Vol. 69, No. 8
Quang et al.
Table 2. 13C NMR Data for 1, 2, and 5-8
position
1
2
5
6
7
8
1
2
3
4
5
6
7
8
9
10
11
12
172.4 170.8 167.9 167.8 167.8
118.0 120.4 118.4 118.9 118.8
147.3 146.8 145.3 144.9 145.0
125.8 127.5 125.8 126.3 126.2
152.4 151.9 151.1 150.1 150.2
167.6
119.4
144.5
126.8
148.8
49.8
43.3
35.2
34.4
31.7
50.3
43.2
35.5
35.0
35.8
73.5
49.9
51.6
44.5
36.8
36.5
36.6
74.0
51.0
50.2
43.2
35.5
35.0
35.9
73.5
50.2
49.9
43.0
35.4
35.0
35.9
73.3
49.9
50.3
43.2
35.2
34.5
31.7
77.9
46.6
77.6
46.4
Figure 2. ORTEP drawing of 8.
13
14
15
122.6 125.0 122.6 123.4 121.4
135.2 135.2 135.3 134.3 136.4
122.6
135.1
56.0
56.2
57.5
56.3
56.0
56.1
determined to be E-form from their coupling constants (Table 1)
and NOESY correlation between H-15 and H-17. Four protons,
H-6, H-8, H-10a, and H-12, were all axial and R-face, due to
NOESY correlations between H-6/H-8 and H-12, H-8/H10a and
H-12, and H10a/H-12; therefore, the methyl group CH3-19 was
equatorial. Furthermore, H-7, H-9a, and H-11 were also axial, but
â-face on the basis of the NOESY correlations between H-7/H-9a
and H-11, H-9a, and H-11. In addition, H-17 correlated with H-7
in the NOESY spectrum, indicating that H-15 was equatorial. This
was confirmed by the coupling constant of H-15 (d, 5.5 Hz),
suggesting that the dihedral angle of H-15 and H-6 was estimated
to be about 60°. Meanwhile, H-13 appeared as a broad singlet
because the dihedral angle between H-13 and H-12 was about 90°.9
To determine the absolute structure of 1, it was methylated to obtain
5, following by p-nitrobenzoylation to afford 7. The CD spectrum
of 7 showed positive (283 nm) and negative (243 nm) Cotton
effects, indicating the R-configuration at C-11.10 From the above
data, carneic acid A (1) was found to be 5-[5R-hydroxy-3,8R-
dimethyl-2S-(1-methylpropenyl)-1R,2S,4aR,5,6,7,8R,8aR-octahy-
dronaphthalen-1-yl]penta-2E,4E-dienoic acid.
Carneic acid B (2) was also obtained as an oil. It has the
molecular formula C21H30O4 based on HREIMS with one more
oxygen atom than 1. Its spectroscopic data were identical with those
of 1 except for the presence of a 3-hydroxy-1-methylpropenyl unit
in the place of the 1-methylpropenyl partial structure. To establish
its absolute structure, compound 2 was methylated by (CH3)3SiN2-
CH in methanol to give 6, then benzoylated by p-nitrobenzoyl
chloride to afford 8 as white crystals. Taking into account its
ORTEP drawing (Figure 2) and NOESY and CD spectra allowed
us to establish its absolute structure as 5-[5R-hydroxy-2S-(3-
hydroxy-1-methylpropenyl)-3,8-dimethyl-1R,2S,4aR,5,6,7,8R,8aR-
octahydronaphthalen-1-yl]penta-2E,4E-dienoic acid.
Crude extracts of various Hypoxylon spp. that are known to
contain azaphilones,3 as well the pure compounds 1-4, were tested
for biological activity in the serial dilution assay against several
filamentous fungi, a Gram-positive bacterium, and one yeast (Table
3 and Table S1, Supporting Information). Carneic acids (1, 2), as
well as the extract of their producer organism, showed somewhat
enhanced activity against filamentous fungi and the yeast Yarrowia
lipolytica. Bacillus subtilis was about equally affected by rubiginosin
A (4), BNT (3), and the crude extracts of the other Hypoylon spp.
studied. The stromata of all of those fungi contain azaphilones and/
or BNT (3) as major metabolites,3,4 but, according to the results of
our HPLC profiling study, they are apparently devoid of compounds
1 and 2. The results, along with the extraordinary amounts of carneic
acids present in the stromata of H. carneum (isolated yields ca.
4-5% of 1 and 2 combined, not accounting for losses during
chromatography), indicate that this species may have developed a
specific chemical defense system that is absent in its closest relatives
and may be directed toward fungi.
16
17
18
19
20
21
1-OMe
benzoates
133.6 137.4 133.7 137.2 133.4
124.4 130.9 124.3 129.3 124.7
140.9
123.8
62.5
22.7
21.7
13.6
22.8
21.7
17.6
59.6
23.2
22.1
18.7
13.6
22.8
21.7
17.7
51.5
59.5
22.8
21.7
18.4
51.5
13.7
22.7
21.7
17.8
51.1
18.4
51.6
164.4, 150.5, 164.6, 164.5,
136.0, 130.7, 150.6, 135.9,
123.6
135.1, 130.7,
123.7, 123.6
Table 3. Comparison of Biological Activities of 1-4 with
Those of Standard Antibiotics in the Serial Dilution Assay
against B. subtilis (BS, NB medium, 18 h) and Various Fungi
(YMG medium, 24 h)a
MIC [µg mL-1
]
sample
BS
YL
25
25
>100 100
100 25
MH
PG
12.
25
>100 >100 >100
>100 100 >100
>100 >100 >100 >100 >100
3.12 3.12 3.12 6.25
SC
25
25
TH
50
50
carneic acid A (1) 50
carneic acid B (2) 50
BNT (3)
rubiginosin A (4)
penicillin G
actinomycin D
12.5
12.5
100
50
3.12
>100 6.25
a YL, Yarrowia lipolytica; MH, Mucor hiemalis; PG, Penicillium
griseofulVum; SC, Stachybotrys chartarum; TH, Trichoderma har-
zianum. Concentrations tested: 100, 50, 25, 12.5, 6.25, 3.1, 1, and 0.1
µg mL-1. MIC ) minimal inhibitory concentration.
structurally related compounds are the fusarielins from Fusarium11
and MK8383 from a Phoma species.9 Neither of these genera belong
to the order Xylariales or the family Xylariaceae, in which H.
carneum is included, and no structurally similar polyketides have
been reported from any member of the latter fungal taxa, according
to a recent literature survey.12 Phomopsidin was proved to be
biogenetically derived from the polyketide pathway by radiolabeling
experiments.10 A similar biogenesis of (1, 2) appears plausible, even
though they have a different stereochemistry and absolute config-
uration than phomopsidin and the other aforementioned analogues.
Since biogenetic pathways are usually stereospecific, H. carneum
may have evolved an analogous polyketide synthesis.
Despite being apparently more specific than those of the major
metabolites of other Xylariaceae, the antifungal activities of the
carneic acids (1, 2) can still be regarded as moderate at best. It
remains unclear as to whether their mode of action is the same as
that of phomopsidin and MK8383 (i.e., the cytoskeleton9). In
previous work, the stereochemistry of the decaline moiety, as well
as the presence of a free carboxyl function in the pentadienic acid
side chain, was considered to be essential for activity of this class
of fungal metabolites in the microtubule assembly assay.9,10 In the
same study, it was postulated that fusarielin A did not show such
activity since it differed in both aforementioned features, but still
that compound was shown to have antifungal effects.11 The carneic
The closest structural analogue of the carneic acids (1, 2) is
phomopsidin, an antibiotic from a marine-derived Phomopsis sp.
that was shown to inhibit microtubule assembly.9,10 Additional

Antimicrobial Agents from Hypoxylon carneum
Journal of Natural Products, 2006, Vol. 69, No. 8 1201
1
2300, 1692, 1634, 1273, 1006, 882 cm-1; H and 13C NMR spectra
acids possess a free pentadienic acid moiety, but differ from
phomopsidin in the stereochemistry of the decaline skeleton.
We reported previously4 that there are further Hypoxylon spp.,
aside from H. carneum, that are devoid of azaphilone pigments
and produce apparently specific but yet unidentified compounds
instead. The outcome of the current project suggests that it may be
rewarding to focus further on such fungal taxa in a search for further
new bioactive chemical entities. However, location and identifica-
tion of these rare species need considerable mycological expertise.
Thus, close interdisciplinary collaborations, as well as the endow-
ment of chemotaxonomy in bioprospecting, will increase the
chances to discover further untapped biological sources and novel
lead structures.13
(CD3OD), Tables 1 and 2; EIMS m/z 346 (5) [M]+, 328 (19), 297 (13),
213 (11), 205 (20), 187 (81), 157 (22), 131 (30), 121 (43), 107 (62),
81 (22), 55 (27), 44 (100); HREIMS m/z 346.2148 (calcd for C21H30O4,
346.2144).
Methylation of 1. Carneic acid A (1) (14 mg) was methylated with
(CH3)3SiN2CH (1.5 mL) in MeOH (1 mL) at 5 °C for 3 h. The reaction
mixture was purified by silica gel column chromatography, hexane-
EtOAc (4:1), to give carneic acid A methyl ester 5 (11.6 mg): 1H and
13C NMR spectra (CDCl3), Tables 1 and 2; EIMS m/z 344 (20) [M]+,
245 (22), 227 (26), 205 (27), 187 (100), 159 (34), 147 (30), 133 (35),
121 (88), 107 (52), 81 (22), 55 (22); HREIMS m/z 344.2351 (calcd for
C22H32O3, 344.2351).
Methylation of 2. Carneic acid B (2) (20 mg) was methylated with
(CH3)3SiN2CH (1.5 mL) in MeOH (1 mL) at 5 °C for 3 h. The reaction
mixture was purified by silica gel column chromatography, hexane-
EtOAc (1:1), to give carneic acid B methyl ester (6) (13.3 mg): 1H
and 13C NMR spectra (CDCl3), Tables 1 and 2; EIMS m/z 360 (3)
[M]+, 342 (24), 324 (16), 243 (19), 225 (19), 205 (39), 187 (100), 159
(50), 145 (52), 133 (43), 121 (55), 105 (57), 91 (59), 55 (25); HREIMS
m/z 360.2301 (calcd for C22H32O4, 360.2301).
Experimental Section
General Experimental Procedures. Melting points were determined
on a Yanagimoto micromelting point apparatus and are uncorrected.
Optical rotations were measured on a JASCO DIP-1000 polarimeter
with CHCl3 as solvent. UV spectra were obtained on a Shimadzu UV-
1650PC instrument in MeOH. CD spectra were measured on a JASCO
J-725 spectrometer in EtOH. IR spectra were measured on a JASCO
FT/IR-5300 spectrophotometer. The 1H and 13C NMR spectra were
recorded on a Varian Unity 600 NMR spectrometer (600 MHz for 1H
and 150 MHz for 13C), using either CDCl3 or CD3OD as solvent.
Chemical shifts are given relative to TMS (δ 0.00) as internal standard
(1H) and δ 77.0 (ppm) from CDCl3 and δ 49.0 (ppm) from CD3OD as
standards (13C). Mass spectra including high-resolution mass spectra
were recorded on a JEOL JMS AX-500 spectrometer. X-ray reflection
data were measured on a Bruker APEXII CCD area detector using Mo
KR radiation (λ ) 0.71073 Å). Column chromatography was carried
out on silica gel 60 (0.2-0.5 mm, 0.04-0.063 mm, Merck) and
Sephadex LH-20 (Amersham Pharmacia Biotech). Analytical HPLC
was carried out as reported previously,3,4 using a dual system to identify
characteristic components by HPLC with diode array detection and mass
spectrometric detection in the positive and negative electrospray modes.
Fungal Material. Stromata of H. carneum Petch were collected and
identified by J.F. in the vicinity of Rimont, Department of Arie´ge,
France, on wood and bark of Fraxinus excelsior on April 14, 2005,
and in the same locality on Salix sp. on November 3, 2005, respectively.
A voucher specimen is deposited at the Botanische Staatsammlung,
Munich, Germany (designation number JF-05048). For comparison,
the original holotype material of H. carneum (Kew, collected in 1908
from Sri Lanka),1,2 and specimens PDD 16333, PDD 59111, and PDD
62056 (collected during 1949-1991 from New Zealand)2 were also
studied by analytical HPLC. In addition, previously recorded HPLC
profiles of crude extracts and preparative fractions (spectra and
chromatograms stored in a HPLC library) were used for comparison
with that of H. carneum and compounds 1 and 2. Aliquots of crude
MeOH extracts of related azaphilone-containing Hypoxylon spp. (the
latter mostly deposited at the mycological herbarium of the Fuhlrott-
Museum, Wuppertal, Germany) were used for comparison of biological
activities. The results are compiled in Table S1 (Supporting Informa-
tion).
p-Nitrobenzoate of 5. To a solution of compound 5 (11.6 mg) in
pyridine (1 mL) were added p-nitrobenzoyl chloride (20 mg) and DMAP
(1 mg). The reactants were stirred overnight at room temperature and
concentrated to give a residue, which was separated by silica gel column
chromatography, hexane-EtOAc (4:1, 150 mL), to obtain 7 (7.3 mg):
UV (MeOH) λmax (log ꢀ) 267 (4.3); CD (EtOH) λext nm (∆ꢀ) 283
(-11.7), 243 (+ 11.2); H and 13C NMR spectra (CDCl3), Tables 1
1
and 2; EIMS m/z 493 [M]+, 327 (18), 227 (65), 187 (100), 159 (80),
148 (47), 120 (24), 105 (25), 91 (18); HREIMS m/z 493.2466 (calcd
for C29H35O6N, 493.2464).
Di-p-nitrobenzoates of 6. p-Nitrobenzoyl chloride (35 mg) and
DMAP (1.2 mg) were added to a solution of 6 (13.3 mg) in pyridine.
The reaction was performed in the same manner as described above
and led to a mixture, which was further purified by silica gel column
chromatography, hexane-EtOAc (1:1, 120 mL), to give 8 (15.6 mg)
as colorless needles (CHCl3): mp 160-165 °C; UV (MeOH) λmax (log
ꢀ) 264 (4.5); CD (EtOH) λext nm (∆ꢀ) 279 (-6.4), 246 (+ 2.9); FABMS
m/z 659 [M + H]+; HRFABMS m/z 659.2579 (cald for C36H39O10N2,
659.2605); H and 13C NMR spectra (CDCl3), Tables 1 and 2.
1
Crystal Data for 8.14 Data collection: Bruker APEX2. Cell
refinement: Bruker APEX2. Data reduction: Bruker SAINT. Comput-
ing structure refinement: SHELXL-97.15 Refinement: Full-matrix least-
squares on F2. C36H38O10N2 approximate dimensions 0.16 × 0.09 ×
0.04 mm3, MW 658.704, triclinic, P1, a ) 6.564(2) Å, b ) 15.654(6)
Å, c ) 16.701(6) Å, R ) 94.618(5)°, â ) 92.568(5)°, γ ) 100.754-
(5)°, V ) 1677.3(10) Å3, Z ) 2, µ ) 0.096 mm-1, 6697 reflections,
866 parameters; R ) 0.0869, Rw ) 0.2313, S ) 1.027.
The biological activities were determined in a conventional serial
dilution assay against various fungi and bacteria, using 48-well Nunclon
microtiter plates (Nunc, Wiesbaden, Germany). Aside from Yarrowia
lipolytica HT20 (IMD culture collection, Wuppertal/Dortmund), all test
strains were obtained from public collections (ATCC, American Type
Culture Collection, Manassas, VA; and DSM, Deutsche Sammlung von
Mikroorganismen und Zellkulturen, Braunschweig, Germany). Over-
night cultures in YMG (Y. lipolytica) or Difco nutrient broth (Bacillus
subtilis ATCC 6633) served for preparation of inoculum for nonfila-
mentous organisms. For testing of filamentous fungi (Mucor hiemalis
DSM 63298, Penicillium griseofulVum DSM 847, Stachybotrys char-
tarum DSM 2144, and Trichoderma harzianum ATCC 64870), spore
suspensions were obtained by rinsing the surface of well-grown 9-day-
old cultures propagated in Fernbach YMG agar flasks with YMG agar
containing 0.1% Tween 80. Test compounds were dissolved and diluted
in MeOH. Aliquots of these solutions were supplied to the wells in
adequate amounts to reach the desired final concentrations (100 µg
per mL and dilutions thereof; see Table 3). The solvent was evaporated
in vacuo; then cell or spore suspensions obtained from overnight
cultures of the test organisms were adjusted to 105 cells per mL and
aliquots thereof incubated with MeOH solutions of the test compounds
for 18 h (B. subtilis) or 24 h (filamentous fungi and Y. lipolytica),
respectively.
Stromata of H. carneum were detached from the substrate, air-dried,
and extracted with MeOH. The methanolic extract (781 mg) from 12
g of dried fungal material was chromatographed on SiO2 using CHCl3-
MeOH-H2O (25:2.5:0.1, 400 mL) to give four fractions. Fraction 3
(365 mg) was subjected to Sephadex LH-20 column chromatography
(CHCl3-MeOH, 1:1, 400 mL) to give carneic acid A (1) (304 mg)
and BNT (3) (25.7 mg). Fraction 4 (126 mg) was pure carneic acid B
(2). The amounts of rubiginosin A (4) and orsellinic acid3 in the crude
extract of H. carneum were estimated to be less than 0.5% each, as
judged from quantitative HPLC-UV analysis using external standards.
Those compounds were not isolated to purity.
Carneic acid A (1): colorless, amorphous solid; [R]20D -4.0 (c 0.5,
CHCl3); UV (MeOH) λmax (log ꢀ) 267 (4.4); IR (KBr) νmax 3500-
2300, 1687, 1634, 1615, 1276, 1007, 758 cm-1 1H and 13C NMR
;
spectra (CDCl3), Tables 1 and 2; EIMS m/z 330 (24) [M]+, 312 (26),
297 (11), 132 (25), 130 (28), 124 (32), 115 (100), 81 (39), 55 (28);
HREIMS m/z 330.2190 (calcd for C21H30O3, 330.2195).
Acknowledgment. Financial support of JSPS (Japan Society for
the Promotion of Science) for granting a postdoctoral fellowship to
D.N.Q. (No. P04162) is greatly acknowledged. Thanks are also due to
Carneic acid B (2): colorless, amorphous solid; [R]20D -1.3 (c 1.0,
CHCl3); UV (MeOH) λmax (log ꢀ) 266 (4.5); IR (KBr) νmax 3500-

1202 Journal of Natural Products, 2006, Vol. 69, No. 8
Quang et al.
can also be found at http://pyrenomycetes.free.fr/hypoxylon/in-
dex.htm.
(4) Hellwig, V.; Ju, Y.-M.; Rogers, J. D.; Fournier, J.; Stadler, M. Mycol.
Prog. 2005, 4, 39-54.
(5) See: Stadler, M.; Ju, Y.-M.; Rogers, J. D. Mycol. Res. 2004, 108,
239-256, and a complete list of Xylariaceae taxa so far studied by
HPLC in the Supporting Information.
(6) Stadler, M.; Wollweber, H.; Mu¨hlbauer, A.; Asakawa, Y.; Hashimoto,
T.; Rogers, J. D.; Ju, Y.-M.; Wetzstein, H.-G.; Tichy, H.-V. Mycol.
Res. 2001, 105, 1191-1205.
(7) Hsieh, H.-M.; Ju. Y.-M.; Rogers,. J. D. Mycologia 2005, 97, 844-
865.
Dr. M. Tanaka, S. Takaoka, and Y. Okamoto (TBU, Japan) for
recording NMR, X-ray, and mass spectra, respectively. We thank Ms.
A. Tomita (TBU) for her excellent experimental assistance. Further-
more, we greatly appreciate the kind cooperation of the curators of the
Royal Botanic Gardens (Kew, UK, especially Dr. B. Aguirre-Hudson)
and the mycological herbarium of Landcare Research (PDD, Auckland,
New Zealand, especially Dr. E. H. C. McKenzie), the New York
Botanical Gardens (especially Ms. E. Bloch), and Dr. L. Vasilyeva,
ISSB, Vladivostok, Russia, and all other colleagues and public
institutions who had kindly provided specimens on loan in the preceding
studies3,4,6 that had helped to establish the specificity of the metabolite
profile of H. carneum.
(8) Quang, D. N.; Hashimoto, T.; Stadler, M.; Asakawa, Y. J. Nat. Prod.
2004, 67, 1152-1155.
(9) Namikoshi, M.; Kobayashi, H.; Yoshimoto, T.; Meguro, S.; Akano,
K. Chem. Pharm. Bull. 2000, 48, 1452-1457.
Supporting Information Available: Table S1, summarizing the
antimicrobial activities of crude extracts of various Hypoxylon species.
Table S2, containing a list of species so far examined in the course of
our polythetic taxonomic study, using HPLC profiling, morphological,
ultrastructural, and molecular data. Additional references are given
where those data were previously published. In addition, a CIF file
containing the X-ray data of compound 8 (corresponding to Figure 2)
is provided. This material is available free of charge via the Internet at
http://pubs.acs.org.
(10) Kobayashi, H.; Meguro, S.; Yoshimito, T.; Namikoshi, M. Tetrahe-
dron 2003, 59, 455-459.
(11) Kobayashi, H.; Sunaga, R.; Furihata, K.; Morisaki, N.; Iwasaki, S. J
Antibiot. 1995, 48, 42-52.
(12) Stadler, M.; Hellwig, V. Recent Res. DeV. Phytochem. 2005, 9, 41-
93.
(13) Larsen, T. O.; Smedsgaard, J.; Nielsen, K. F.; Hansen, M. E.; Frisvad,
J. C. Nat. Prod. Rep. 2005, 22, 672-695.
(14) Crystallographic data for the structure reported in this paper have
been deposited at the Cambridge Crystallographic Data Centre
(deposition number CCDC 604567). Copies of the data can be
obtained, free of charge, on application to the Director, CCDC, 12
Union Road, Cambridge CB2 1EZ, UK (fax: +44-(0) 1223-336033
or e-mail: deposit@ccdc.cam.ac.uk).
References and Notes
(1) Petch, T. Ann. R. Bot. Gard. (Peradeniya) 1924, 8, 119-166.
(2) Ju, Y.-M.; Rogers, J. D. A reVision of the genus Hypoxylon;
Mycologia Memoir no. 20; APS Press: St. Paul, MN, 1996.
(3) (a) Stadler, M.; Wollweber, H.; Fournier, J. Mycotaxon 2004, 90,
187-211. (b) Images and morphological descriptions of all European
species treated herein, as well as an outline of their chemotaxonomy,
(15) Sheldrick, G. M. SHELXL97, Program for the Refinement of Crystal
Structures; University of Go¨ttingen: Germany. 1997.
NP0602057