An unusual oxalylated tetramic acid from the New Zealand basidiomycete Chamonixia pachydermis

Article abstract of DOI:10.1021/np050488n

An unusual oxalylated tetramic acid, pachydermin (1), has been isolated from the New Zealand basidiomycete Chamonixia pachydermis. The full structure, which was not directly accessible by NMR methods, was deduced from that of a degradation product, 5-(3-chloro-4-hydroxybenzylidene)tetramic acid (2). The degradation product 2 exhibited mild antibacterial activity against Bacillus subtilis.

Full text of DOI:10.1021/np050488n

J. Nat. Prod. 2006, 69, 151-153
151
An Unusual Oxalylated Tetramic Acid from the New Zealand Basidiomycete Chamonixia
pachydermis
Gerhard Lang,† Anthony L. J. Cole,‡ John W. Blunt,† and Murray H. G. Munro*,†
Department of Chemistry and School of Biological Sciences, UniVersity of Canterbury, PriVate Bag 4800, Christchurch, New Zealand
ReceiVed NoVember 23, 2005
An unusual oxalylated tetramic acid, pachydermin (1), has been isolated from the New Zealand basidiomycete Chamonixia
pachydermis. The full structure, which was not directly accessible by NMR methods, was deduced from that of a
degradation product, 5-(3-chloro-4-hydroxybenzylidene)tetramic acid (2). The degradation product 2 exhibited mild
antibacterial activity against Bacillus subtilis.
Within the scope of our ongoing program for the isolation of
biologically active natural products from New Zealand filamentous
fungi1 and mushrooms,2 we investigated the basidiomycete Cha-
monixia pachydermis (Boletaceae).3 This fungus is a gasteromycete
(i.e., its spores are formed inside a closed fruiting body) and has
been reported from Australia and New Zealand, where it is often
found in association with Nothofagus forest.
correlation linking the identified substructure with the unknown
part was from the vinylic proton H-5 to a carbon at δ 177.0 (C-3),
which therefore had to be located in the allylic position of the vinylic
double bond.
HPLC-DAD analysis showed that the MeOH extract of this
fungus contained gyroporin, which had been described before from
another Chamonixia species.4 The only other predominant metabo-
lite, pachydermin (1), had a UV absorption maximum at 357 nm,
suggesting an extensively conjugated carbonyl chromophore. The
molecular mass, 309 Da, was derived from the HPLC-ESIMS,
which also displayed an isotopic pattern indicative of monochlo-
rination.
The purification of 1 was complicated by the observed degrada-
tion under the acidic conditions (0.05% TFA) used for the
preparative HPLC. With a neutral eluent 1 was hardly retained in
the reversed-phase chromatography, suggesting the acidic nature
of the compound. By consecutive reversed-phase chromatography
and gel chromatography on Sephadex LH-20 it was possible to
isolate 1 as a water-soluble salt. The ESIMS (pos.) indicated a
mixture of Na+ and K+ as the counterions.
To get more information about the structure of 1, it was necessary
to identify the structure of the degradation product 2 that was
formed, even under weakly acidic conditions, e.g., the 0.05%
aqueous TFA used for HPLC. Heating of 1 in 2 M HCl led to
complete conversion into 2. From HRESIMS the molecular formula
of 2 was deduced as C11H8ClNO3, corresponding to a loss of C2O3
from the parent structure. The 1H NMR spectrum showed a mixture
of two tautomeric forms, 2 and 2′, in a 2:1 ratio. In both tautomers
the 3-chloro-4-hydroxyvinyl system observed in 1 was intact. An
additional singlet signal of one olefinic proton (5.04 ppm; H-2)
was observed in the subspectrum of 2, while that of 2′ contained a
methylene singlet (δ 3.27; H-2). In contrast to the 1H NMR
spectrum of 1, that of 2 showed exchangeable protons as sharp
singlets with HMBC and ROESY correlations useful for the
elucidation of structures 2 and 2′ (Figure 1). Chemical shifts and
HMBC correlations were consistent with 2′ being a pyrrolidine-
2,4-dione,6 and 2 the corresponding enol, substituted in the
5-position with a 3-chloro-4-hydroxybenzylidene residue. The
vinylic double bond was Z-configured, as indicated by the ROESY
interactions in 2 of the 3-OH with H-5 and H-2 and of the amide
proton to H-7 and H-11 (Figure 1).
1
The H NMR spectrum of 1 (as the free acid in DMSO-d6)
showed two doublets, H-7 at δ 7.74 and H-10 at δ 7.05, and one
doublet of doublets at δ 7.49 (H-11) with coupling constants
characteristic of a 1,3,4-trisubstituted benzene system. One ad-
ditional singlet (H-5) appeared at δ 6.41 and one broad singlet at
δ 9.88. The 13 signals in the 13C NMR spectrum, together with an
accurate mass of m/z 310.0150 for the [M + H]+ ion, supported a
molecular formula of C13H8ClNO6 for 1. Long-range H,C-couplings
of H-5 to C-7 and to C-11 revealed this to be a vinylic proton on
a double bond attached to the carbon C-6 of the aromatic ring.
The further substituents of this ring were deduced from the 13C
chemical shifts to be chlorine at C-8 and a phenolic hydroxyl group
2
3
at C-9 and confirmed by observed JCH and JCH couplings in the
HMBC spectrum. The chemical shifts of this 3-chloro-4-hydroxy-
phenyl system are in good agreement with those of 3-chloro-4-
hydroxyphenylacetamide, a known metabolite from a plant-associ-
ated fungus.5 This left about half of the molecular formula, a
C6H3NO5 unit, unaccounted for. For this part of the molecule there
were four low-field carbons (δ 179.3, 177.0, 170.3, 165.7) and two
sp2-carbons (δ 130.9, 99.4) in the 13C NMR spectrum, but there
were no associated sharp proton signals that could be used for the
assembly of a structure by 2D NMR methods. The only HMBC
With the acid degradation product 2 identified, a structure for
the natural product 1 can be proposed. The loss of C2O3 during the
conversion of 1 to 2 can be interpreted as a combined decarbony-
lation-decarboxylation reaction.7 Consequently, the presumed
structure of the natural product is that of 2 substituted in the
2-position of the pyrrole ring with an oxalyl residue (1). A possible
mechanism for the formation of 2 involves the cyclization of the
keto tautomer of 1 to the intermediate 1a, which then by consecutive
or concerted decarbonylation-decarboxylation forms 2 (Scheme
1). The tautomers 2 and 2′ are stable enough to form two peaks
with very distinct UV spectra in the HPLC. However, initially only
the peak corresponding to tautomer 2 was observed. Thus, it can
* To whom correspondence should be addressed. Tel: +64-3-3642434.
Fax: +64-3-3642429. E-mail: murray.munro@canterbury.ac.nz.
† Department of Chemistry.
‡ School of Biological Sciences.
10.1021/np050488n CCC: $33.50
© 2006 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/12/2006

152 Journal of Natural Products, 2006, Vol. 69, No. 1
Notes
chromatography on Sephadex LH-20 eluting with MeOH-H2O (1:1).
The fractions containing 1 were combined and dried in vacuo. The
residue was redissolved in H2O and further purified by vacuum liquid
chromatography on an ODS phase eluting with MeOH-H2O mixtures
(from 0% to 20% MeOH). The fractions eluting with 10% MeOH were
dried to yield pure 1 in its salt form (37.5 mg). For acquiring NMR
spectra the salt of 1 (5.7 mg) was dissolved in H2O (5 mL), acidified
with 2 M HCl, and extracted immediately with EtOAc (2 × 5 mL).
The combined EtOAc phases were dried, and the resulting free acid of
1 was dissolved in DMSO-d6.
Figure 1. ROESY (left) and HMBC correlations (right) for 2.
Scheme 1. Decarbonylation-Decarboxylation of 1 to 2
Pachydermin (1): yellow amorphous solid; UV (H2O + 0.05% TFA/
1
MeCN (54:46)) λmax (rel int) 203 (86), 254 (42), 357 (100); H NMR
(DMSO-d6, 500 MHz) δ 9.88 (1H, bs, NH), 7.74 (1H, d, J ) 2.0 Hz,
H-7), 7.49 (1H, dd, J ) 8.7, 2.0 Hz, H-11), 7.05 (1H, d, J ) 8.7 Hz,
H-10), 6.41 (1H, s, H-5); 13C NMR (DMSO-d6, 75 MHz) δ 179.3 (C,
C-12), 177.0 (C, C-3), 170.3 (C, C-13), 165.7 (C, C-1), 153.2 (C, C-9),
130.9 (C, C-4), 130.8 (CH, C-7), 129.9 (CH, C-11), 126.3 (C, C-6),
120.4 (C, C-8), 116.8 (CH, C-10), 106.4 (CH, C-5), 99.4 (C, C-2); for
HMBC data, see Supporting Information; ESIMS (pos.) m/z 310.1 [M
+ H]+, 332.1 [M + Na]+, 348.1 [M + K]+; ESIMS (neg.) m/z 307.9
[M - H]-; HRESIMS (pos.) m/z 310.0150 [M + H]+ (calcd for C13H9-
ClNO6, 310.0118).
Preparation of 2. Compound 1 (4.5 mg) was dissolved in HCl (2
M; 5 mL) and heated to 80 °C. After 15 min the reaction mixture was
cooled and extracted with EtOAc (2 × 5 mL). After evaporating the
solvent to dryness the major product was isolated by semipreparative
HPLC (Phenomenex Luna C18, 10 × 250 mm, 5 µm; solvents: A
H2O, B MeCN; linear gradient: 0 min 25% B, 15 min 65% B; 5 mL
min-1). Compound 2 was eluted at 7 min and yielded a yellow
amorphous solid (2.0 mg). NMR and HPLC analysis showed it to be
a mixture of two tautomers, 2 and 2′, in a 2:1 ratio.
Compound 2: UV (H2O + 0.05% TFA/MeCN (52:48)) λmax (rel
1
int) 204 (82), 243 (48), 341 (100); H NMR (DMSO-d6, 500 MHz) δ
11.74 (1H, s, 3-OH), 10.50 (1H, s, 9-OH), 9.53 (1H, s, NH), 7.67 (1H,
bs, H-7), 7.43 (1H, d, J ) 8.5 Hz, H-11), 7.03 (1H, d, J ) 8.5 Hz,
H-10), 6.17 (1H, s, H-5), 5.04 (1H, s, H-2); 13C NMR (DMSO-d6, 500
MHz; extracted from HSQC and HMBC data) δ 172.7 (C, C-1), 165.6
(C, C-3), 152.4 (C, C-9), 131.6 (C, C-4), 130.1 (CH, C-7), 129.0 (CH,
C-11), 126.4 (C, C-6), 119.9 (C, C-8), 116.5 (CH, C-10), 104.1 (CH,
C-5), 92.1 (CH, C-2); for HMBC and ROESY data, see Figure 1 and
Supporting Information; HRESIMS pos. (mixture of 2 and 2′) m/z
238.0220 [M + H]+ (calcd for C11H9ClNO3, 238.0271).
be assumed that the direct product of the reaction is the enol 2 and
not the ketone 2′. This supports the elimination mechanism
proposed.
While the 3-chloro-4-hydroxyphenyl residue is common to
various fungal5,8 and bacterial9 natural products, the oxalylated
tetramic acid moiety is a unique structural feature of 1. In contrast
to pachydermin (1) itself, the degradation product 2 exhibits mild
antibacterial activity against Bacillus subtilis. In an agar diffusion
assay it caused an inhibition zone of 1 mm around a disk loaded
with 40 µg of substance. It remains to be investigated if 2 is formed
also in vivo, e.g., as a response to injury of the fruiting body or
predatory attack.
Compound 2′: UV (H2O + 0.05% TFA/MeCN (58:42)) λmax (rel
1
int) 202 (100), 222 (36), 280 (6); H NMR (DMSO-d6, 500 MHz) δ
11.04 (1H, s, NH), 10.64 (1H, s, 9-OH), 7.77 (1H, bs, H-7), 7.51 (1H,
d, J ) 8.5 Hz, H-11), 7.06 (1H, d, J ) 8.5 Hz, H-10), 6.29 (1H, s,
H-5), 3.27 (2H, s, H-2); 13C NMR (DMSO-d6, 500 MHz; extracted
from HSQC and HMBC data) δ 195.4 (C, C-3), 171.7 (C, C-1), 153.1
(C, C-9), 132.9 (C, C-4), 130.4 (CH, C-7), 129.6 (CH, C-11), 125.2
(C, C-6), 120.2 (C, C-8), 116.5 (CH, C-10), 104.2 (CH, C-5),
39.1 (CH2, C-2); for HMBC and ROESY data, see Supporting
Information.
Experimental Section
Acknowledgment. This work was supported by a Fellowship within
the Postdoctoral Program of the German Academic Exchange Service
(DAAD). We thank Messrs. A. Pinkert, F. Patuel, and N. Cummings
for assistance with collecting the fungus, Mr. B. Clark for mass
spectrometric analysis, and Ms. G. Ellis for bioactivity assays.
General Experimental Procedures. UV data were extracted from
the diode array detector signal from the HPLC; only relative intensities
of absorption maxima are given. NMR spectra were recorded on a
1
Varian INOVA AS-500 spectrometer (500 and 125 MHz for H and
13C NMR, respectively), using the signals of the residual solvent protons
and the solvent carbons as internal references (δH 2.60 and δC 39.6
ppm for DMSO-d6). For HRESIMS and LC-MS detection a Micromass
LCT TOF mass spectrometer was used. Solvents for extraction and
isolation were distilled prior to use. Antimicrobial activity against
Bacillus subtilis was measured using a standard protocol.10
Supporting Information Available: Tabulated NMR data of
compounds 1, 2, and 2′ and photographs of the investigated fungus
are available free of charge via the Internet at http://pubs.acs.org.
References and Notes
Fungus. Fruiting bodies of Chamonixia pachydermis (Boletaceae)
were collected in Nothofagus forest at Bealey Spur, New Zealand, in
May 2005, and unambiguously identified by their spore shape and
fruiting body morphology.3 A voucher specimen (F-5832) has been
deposited at the School of Biological Sciences.
Extraction and Isolation. The fresh fruiting bodies (280 g wet wt)
were extracted with MeOH (2 × 1 L). The solvent was evaporated to
dryness, and the residue was taken up in H2O (250 mL), extracted first
with EtOAc (2 × 250 mL) and then with n-BuOH (8 × 150 mL). The
combined n-BuOH phases were evaporated and subjected to gel
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NP050488N