Exochrome: Structurally Unique Chromone Dimer with Antifungal and Algicidal Activity from Exophiala SP

Article abstract of DOI:10.3987/com-07-s(w)10

A unique highly substituted chromone dimer, named exochromone (la), in addition to the known steroids ergosterol (2) and 5α,8α-epidioxyergosterol (3) were isolated from endophytic fungus Exophiala sp. The structure of the new compound was elucidated on th

Full text of DOI:10.3987/com-07-s(w)10

HETEROCYCLES, Vol. 74, 2007
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HETEROCYCLES, Vol. 74, 2007, pp. 331 - 337. © The Japan Institute of Heterocyclic Chemistry
Received, 9th June, 2007, Accepted, 26th July, 2007, Published online, 27th July, 2007. COM-07-S(W)10
EXOCHROMONE: STRUCTURALLY UNIQUE CHROMONE DIMER
WITH ANTIFUNGAL AND ALGICIDAL ACTIVITY FROM EXOPHIALA
SP.‡
Hidayat Hussain,a Karsten Krohn,a* Siegfried Draeger,b and Barbara Schulzb
‡Biologically Active Secondary Metabolites from Fungi, Part 33. Part 32 ref.[1]
aDepartment of Chemistry, Universität Paderborn, Warburger Straße 100, 33098
Paderborn, Germany
bInstitut
Spielmannstraße 7, 31806 Braunschweig, Germany
für
Mikrobiologie,
Technische
Universität
Braunschweig,
Reprint requests to Prof. Karsten Krohn. Fax: (+49)-5251-603245
E-mail: k.krohn@uni-paderborn.de
Dedicated to Prof. E. Winterfeldt on the occasion of his 75th birthday
Abstract – A unique highly substituted chromone dimer, named exochromone
(1a), in addition to the known steroids ergosterol (2) and 5α,8α-epidioxyergosterol
(3) were isolated from endophytic fungus Exophiala sp. The structure of the new
compound was elucidated on the basis of spectroscopic data. Preliminary studies
showed that exochromone is antifungal and antialgal against Microbotryum
violaceum and Chlorella fusca, respectively.
Chromones, a fused [6-6] system are a group of naturally occurring compounds that are widely
distributed in nature. They are part of a class of compounds which are now commonly called “privileged
structures” in drug discovery. These structures represent molecules capable of binding to multiple
receptors with high affinity. Whether the privileged nature of these molecules arises out of a common
structural element or is due to independent molecular characteristics is open to debate.2 Molecules
containing the chromone or benzopyranone ring have a wide range of biological activities viz., tyrosine
and protein kinase C inhibitors, anticancer, antifungal, antiviral, antitubulin, and antihypertensive agents.2
As part of our systematic search for new bioactive lead compounds from fungal sources, one new
chromone dimer, exochromone (1a), together with ergosterol (2) and 5α,8α-epidioxyergosterol (3)3,4 were
isolated from the culture of the fungus Exophiala sp.

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RESULTS AND DISCUSSION
The endophytic fungus Exophiala sp. (internal strain no. 7110), was isolated from the plant Adenocarpus
foliolosus and was cultivated at room temperature on biomalt solid agar media for 28 days.5 The 12 L of
culture media were then extracted with ethyl acetate to afford 3.5 g of a residue which was subjected to
column chromatography. Three metabolites including one new chromone dimer exochromone (1a) of
unusual connection mode were isolated from the culture extract.
OR
O
O
RO
MeO
O
OMe
9
11
10
HO
O
2
8
MeO
RO
O
OMe
1
2
7
3
4
6
5
OR
O
O
O
1a: R = H
1b: R = 4-bromobenzoate
HO
3
Figure 1. Structures of compounds isolated from Exophiala sp.
Exochromone (1a) has the molecular formula C28H28O14 as indicated by HREIMS and in agreement with
the 1H and 13C NMR spectra. The UV spectrum displayed absorptions at 214, 265, 295, 315, and 370 nm,
which are indicative of a chromone system.6 The molecular formula C28H28O14 suggested 16 double bond
equivalents. Thus, four rings must form the basic molecular skeleton of compound 1a, considering the
interconversion of the twelve double bond equivalents for four carbonyl groups and eight double bonds.
Compound 1a had a very simple 1H NMR spectrum showing only six singlets: two methine signals; one
downfield at (δ 6.65) and the other at (δ 5.21), two methoxy groups (δ 4.39 and 4.10), one methyl group (δ
2.05), and a low field signal at δ 16.16 for a strongly deshielded hydroxyl hydrogen. The presence of two
carbonyl groups was indicated by typical signals at δ 179.76,7 (C-4) and 204.7 (C-10) in agreement with the
13
IR absorption at 1664 cm-1. The C and DEPT spectra of 1a showed signals for 14 carbons indicating a
highly symmetrical molecule, comprising one methyl, two methoxy, two methine, and nine quaternary
carbon signals.
The HREIMS [M]+ molecular ion of 1a with a m/z 586.1318 indicated that the simple NMR spectra
showed only one part of a symmetric dimer. The 1D, 2D NMR and MS data presented above supported

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the conclusion that compound 1a consisted of two identical chromone units linked through a bis
acetylmethine bridge [(CH–COMe)2]. Thus, the major problem was the establishment of the linkage of
the two chromone subunits. The signal for H-9 was the key entry into the diagnostic spin system. It
occurs at a distinct chemical shift (δ 5.21) and was easily recognizable by its multiplicity compared with
those of the other protons. The signal could also be identified by its HMQC correlation to a carbon at δ
48.6 and its important HMBC correlations with the acetyl carbonyl group (δ 204.7, C-10) and C-11. The
signal for H-9 showed diagnostic long-range HMBC correlations with C-8, C-8a and C-7, confirming the
linkage to C-8 on the aromatic ring. In addition, HMBC correlations were also observed for both the
methoxy group at δ 4.39 and the benzylic hydrogen at δ 5.21 (H-9) with the carbon at δ 150.1 (C-7),
establishing the location of that methoxy group at C-7. This was further confirmed by NOE correlation
between H-9 and methoxy group at δ 4.39 (Figure 2).
OH
O
O
HO
MeO
OMe
O
10
11
9
O
MeO
HO
O
OMe
1
8
5
7
6
2
3
4
H
OH
O
1H-1H-NOE
HMBC
Figure 2. HMBC and DPFGSE-NOE correlations for exochromone (1a).
Furthermore, the assignments of the one remaining methoxy group (δ 4.10) at C-2 and hydrogen at C-3
were made based on the HMBC correlations of the methoxy group at δ 4.10 to C-2 and the hydrogen at δ
6.65 with C-2, C-3, and C-4 as well as from the NOE correlations (Figure 2).
The present data allow discussion of two remaining possibilities: either a chromone (subunit A) or a
coumarin (subunit B) (Figure 3). However, detailed analysis of the 1H and 13C NMR spectral data allowed
the unambiguous assignment of subunit B in 1a due to characteristic spectroscopic differences between
the units A and B.

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MeO
HO
O
O
MeO
HO
O
OMe
2
3
2
3
5
4
5
4
OH OMe
OH
O
B
A
Figure 3. Structures of putative subunits A and B.
In the 13C NMR spectrum, a carbonyl resonance in compound 1a appeared at ca. δ 179.7, consistent with
the reported values for the chromone carbonyl (subunit A).7-11 By contrast, the chemical shift for the C-2
carbonyl in subunit B (coumarins) generally appears at significantly higher field (ca. δ 160)8,9,12-15. This
remarkable difference in resonance for the carbonyl carbon (ester vs. ketone type) presents a convincing
proof for the distinction between the two series of compounds. A further difference was also evident in
the 1H NMR spectrum. The appearance of a low field resonance for the strongly chelated proton at C-5 at
δ 16.16 in compound 1a is characteristic for A and excludes the possibility of a peri hydroxyl group as
present in subunit B; weekly chelated protons, as present in subunit B (C-5 hydroxyl and C-4 OMe),
usually resonate at 9 - 11.00 ppm.16
A reaction between compound 1a and 4-bromobenzoyl chloride was carried out in the expectation to
confirm the structure by X-ray single crystal analysis with a heavy atom incorporated. The
DMAP-catalyzed perbenzoylation gave a tetra-4-bromobenzoate 1b but, unfortunately, the compound did
not afford suitable crystals (CH2Cl2 : MeOH and EtOAc : n-hexane) for X-ray analysis. However, the NMR
spectra of tetra-4-bromobenzoate 1b were in total agreement with the proposed structure and thus
reconfirmed the presence of two phenolic hydroxyl groups in one chromone subunit of compound 1a.
Exochromone (1a) did not show any optical activity. This suggests that 1a exists either as a meso
compound or as a racemic mixture as suggested by Floss et al.17, Keller-Schierlein et al.,18 and Kim et
al.19 for phenazine dimers with a related mode of linkage.
In contrast to the more frequently occurring benzoquinone, naphthoquinone, and anthraquinone dimers,20
the dimeric chromone pigments are less common. A literature survey revealed that there is only one
example of C–C linked dichromone reported by Franck et al.21 Later, some dimeric or trimeric derivatives
of 2-(2-phenylethyl)chromone were reported in the literature.22,23 However, the linkage by an
acetylmethine [(CH–COMe)2 bridge between two chromone units is reported here for the first time in
naturally occurring chromones.
The two additionally isolated compounds were identified as the known compounds ergosterol (2) and
5α,8α-epidioxyergosterol (3) by comparison of their physical and spectroscopic data with those reported
in the literature.3,4

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Exochromone (1a) was tested for herbicidal, antibacterial, and antifungal activities and showed good
antifungal activity against Microbotryum violaceum and the algae Chlorella fusca, respectively (Table 1).
Table 1. Biological activity of exochromone A (1a; 50 μg applied to a filter disc) in an agar diffusion test.
Compound
algicidal antifungal antibacterial
Chla
Mb Bm
1mg/mL 1mg/mL 1mg/mL
Exochromone (1a) 5 9 gi
0
aChlorella fusca (Chl), Microbotryum violaceum (Mb) and Bacillus megaterium (Bm). The radius of zone of inhibition was
measured in mm. gi = growth inhibition, meaning that there was weak growth within the zone of inhibition.
EXPERIMENTAL
General: The endophytic fungus Exophiala sp., internal strain No. 7110, was isolated from Adenocarpus
foliolosus that had been growing on the hills of Baranco de Los Jargus, Gomera, and was cultivated on 12
L of 5% w/v biomalt solid agar media at room temperature for 28 days.5,24 For general methods and
instrumentation see ref.5,25 Melting points were determined with a Gallenkamp micro-melting point
apparatus and are uncorrected. NMR spectra were run with a Bruker Avance-500 NMR spectrometer with
TMS as internal standard. EIMS data were obtained with an MAT 8200 mass spectrometer.
Extraction and Isolation: The culture media were extracted with EtOAc to afford 3.5 g of a residue after
removal of the solvent under reduced pressure. The extract was separated into three fractions by column
chromatography (CC) on silica gel, using gradients of n-hexane/ EtOAc (90:10, 50:50, 0:100). The
fraction E (120 mg) was separated by silica gel column chromatography eluting with n-hexane-EtOAc
(3:7) to give crude compound 1a. The crude compound 1a was then purified eluting with n-hexane-
EtOAc (3:7) to give the pure natural product 1a (40 mg). Fraction B (400 mg) was separated by CC on
silica gel (98 mg) with n-hexane- EtOAc (8.5:1.5) to give compounds 2 (6.5 mg) and 3 (51 mg).
Exochromone (1a): Red pigment, mp 224 °C (uncorrected); [α]D20 + 0.00 (c 0.92, CHCl3); UV λmax, nm
(log ε): 370 (4.20), 315 (4.67), 295 (3.65), 265 (3.15), 214 (2.60) nm; IR νmax (CHCl3): 3400 (OH), 1720,
1664, 1580 (C=C); 1H-NMR (500 MHz, CDCl3): δ 16.16 (s, OH-5), 6.65 (s, 1H, H-3), 5.21 (s, 1H, H-9),
4.39 (s, 3H, OCH3-7), 4.10 (s,3H, OCH3-2), 2.05 (s, 3H, CH3CO); 13C-NMR (125 MHz, CDCl3): δ 204.7
(C-10), 179.7 (C-4), 162.0 (C-5), 167.4 (C-2), 150.1 (C-7), 149.3 (C-8a), 130.2 (C-6), 122.7 (C-8), 107.8
(C-4a), 102.4 (C-3), 61.1 (CH3-7), 56.4 (CH3-2), 48.6 (C-9), 27.9 (CH3CO); HREIMS: m/z 586.1318

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(Calcd. 586.1328 for C28H28O14); EIMS (rel. int.): m/z 586 [M]+ (100), 544.3 (88), 501.3 (77), 401.2 (66),
372.2 (15), 298.1 (14), 83.1 (17), 43.0 (47), 31.0 (25).
4-Bromobenzoylation of 1a to 1b: A solution of phenol 1a (8 mg, 0.5 mmol) in pyridine (2 mL) was
treated with 4-bromobenzoyl chloride (0.5 mg, 1 mmol) and 4-(dimethylamino)pyridine (DMAP) (2 mg).
The mixture was stirred for 10 h at 21 °C and then poured into cold 2 N HCl (5 mL) and stirred for 1 h to
hydrolyze the excess 4-bromobenzoyl chloride. The aqueous phase was extracted three times with CH2Cl2
(5 mL); the organic phase was washed with 2 N NaHCO3 (5 mL) to remove 4-bromobenzoic acid, and dried
with Na2SO4. The solvent was removed at reduced pressure followed by separated with chromatographed
on silica gel (solvent EtOAc/petrol ether, 4:6 to 6:4) to afford 1b. Red solid, mp 207 °C (uncorrected); UV
λ
max, nm (log ε): 365 (4.12), 317 (4.13), 265 (3.18), 220 (2.90) nm; IR νmax (CHCl3): 1720, 1585 (C=C);
1H-NMR (500 MHz, CDCl3): δ 7.90 (d, J = 8.1 Hz, 2H, H-2', H-6'), 7.88 (d, J = 8.1 Hz, 2H, H-2'', H-6''),
7.68 (d, J = 8.1 Hz, 2H, H-3', H-5'), 7.65 (d, J = 8.1 Hz, 2H, H-3'', H-5''), 6.64 (s, 1H, H-3), 5.18 (s, 1H,
H-9), 4.35 (s, 3H, OCH3-7), 4.07 (s, 3H, OCH3-2), 2.02 (s, 3H, CH3CO).
Bioactivity Tests: Agar diffusion test:
The tested compound 1a was dissolved in acetone at a concentration of 1 mg/mL. 50 μL of the solution
were pipetted onto a sterile filter disc, which was placed onto an appropriate agar growth medium for the
respective test organism and subsequently sprayed with a suspension of the test organism.26 The test
organisms were Escherichia coli (NB), Bacillus megaterium (NB), Microbotryum violaceum (MPY) and
Chlorella fusca (MPY). The radius of zone of inhibition was measured in mm.
ACKNOWLEDGEMENTS
We thank BASF AG and the Bundesministerium für Bildung und Forschung (BMBF), project no.
03F0360A for sponsoring our research work.
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