Chlorohydroaspyrones A and B, antibacterial aspyrone derivatives from the marine-derived fungus Exophiala sp.

Article abstract of DOI:10.1021/np800107c

Chlorohydroaspyrones A (1) and B (2), antibacterial aspyrone derivatives, and the previously described aspyrone (3), asperlactone (4), and penicillic acid (5) have been isolated from the broth of a marine isolate of the fungus Exophiala. The structure and absolute stereochemistry of the compounds were determined on the basis of the physicochemical data analysis and chemical reactions. Compounds 1 and 2 exhibited a mild antibacterial activity against Staphylococcus aureus, methicillin-resistant S. aureus, and multidrug-resistant S. aureus. The MIC values of each strain are as follows: compound 1 showed 62.5 μg/mL for S. aureus and 125 μg/mL for methicillin-resistant S. aureus and multidrug-resistant S. aureus, and compound 2, 62.5 μg/mL for S. aureus and methicillin-resistant S. aureus and 125 μg/mL for multidrug-resistant S. aureus.

Full text of DOI:10.1021/np800107c

1458
J. Nat. Prod. 2008, 71, 1458–1460
Chlorohydroaspyrones A and B, Antibacterial Aspyrone Derivatives from the Marine-Derived
Fungus Exophiala sp.
Dahai Zhang,† Xiudong Yang,† Jung Sook Kang,‡ Hong Dae Choi,§ and Byeng Wha Son*,†
Department of Chemistry, Pukyong National UniVersity, Busan 608-737, Korea, College of Dentistry, Pusan National UniVersity,
Busan 602-739, Korea, and Department of Chemistry, Dongeui UniVersity, Busan 614-714, Korea
ReceiVed February 17, 2008
Chlorohydroaspyrones A (1) and B (2), antibacterial aspyrone derivatives, and the previously described aspyrone (3),
asperlactone (4), and penicillic acid (5) have been isolated from the broth of a marine isolate of the fungus Exophiala.
The structure and absolute stereochemistry of the compounds were determined on the basis of the physicochemical data
analysis and chemical reactions. Compounds 1 and 2 exhibited a mild antibacterial activity against Staphylococcus
aureus, methicillin-resistant S. aureus, and multidrug-resistant S. aureus. The MIC values of each strain are as follows:
compound 1 showed 62.5 µg/mL for S. aureus and 125 µg/mL for methicillin-resistant S. aureus and multidrug-resistant
S. aureus, and compound 2, 62.5 µg/mL for S. aureus and methicillin-resistant S. aureus and 125 µg/mL for multidrug-
resistant S. aureus.
Exploitation of the marine environment has been intriguingly
successful in recent years in the search for structurally unusual and
biologically active natural products.1 We are studying fungi isolated
from marine sources for their potential to provide new natural
products.2 As part of an ongoing effort to discover biologically
active natural products from marine microorganisms,3 a marine-
derived fungus, Exophiala sp., was selected from our screening
program for further studies to examine the antimicrobial activity
of its extract against Staphylococcus aureus, methicillin-resistant
S. aureus, and multidrug-resistant S. aureus strains. This paper
describes the isolation and characterization of chlorohydroaspyrones
A (1) and B (2), as well as three known polyketides, aspyrone (3),4
asperlactone (4),4 and penicillic acid (5).5 Compounds 1 and 2
displayed moderate antibacterial effects against Staphylococcus
aureus, methicillin-resistant S. aureus, and multidrug-resistant S.
aureus strains.
hydroxyl and R,ꢀ-unsaturated δ-lactonyl functionality in 1, respec-
tively. The UV spectrum of 1 revealed the presence of a conjugated
enone chromophore [272 nm (log ε 4.10)]. In the 1H NMR
spectrum, two protons were exchanged by D2O, suggesting that 1
had two hydroxyl protons [5.87 (1H, d, J ) 5.9 Hz, 5-OH); 5.16
1
(1H, d, J ) 5.5 Hz, 9-OH)]. Detailed analyses of the H and 13C
NMR spectra of 1, including DEPT, COSY, HMQC, and HMBC
experiments, revealed signals ascribable to an R,ꢀ-unsaturated
δ-lactone having 1-chloro-2-hydroxypropanyl, hydroxyl, and methyl
substituents (Table 1). The connection of functional groups in 1
was achieved on the basis of COSY and HMBC. The key COSY
correlations between H-5 and 5-OH, between H-9 and 9-OH, and
between H-6 and H3-7 were critical in establishing the positions of
5-OH, 9-OH, and 6-CH3. The diagnostic HMBC correlations, from
5-OH to C-4, C-5, and C-6, from H-4 to C-2, C-5, and C-8, from
H3-7 to C-5 and C-6, from H-8 to C-2, C-3, C-4, C-9, and C-10,
and from 9-OH to C-8, C-9, and C-10 showed the connections of
C3-C8 as well as the positions of 5,9-dihydroxy and 6-methyl
groups. These spectroscopic features revealed that compound 1 had
the general structural features of aspyrone (3).4 The NMR data of
both compounds showed similar patterns, except for the appearance
of one hydroxyl proton and the downfield shift of not only H-8
[δH 4.65 (1H, d, J ) 6.5 Hz); ∆δ ) 1.30 ppm] and C-8 [δC 62.5;
∆δ ) 8.4 ppm] but also of H-9 [δH 4.02 (1H, ddq, J ) 5.5, 6.5,
6.5 Hz); ∆δ ) 1.14 ppm] and C-9 [δC 67.9: ∆δ ) 10.3 ppm] in 1.
Thus, compound 1 was characterized as the 8-chloro-9-hydroxyl
derivative of aspyrone (3), and a direct comparison of NMR data
of 1 with those of 3 provided additional support in justifying the
planar structure shown for 1.
The relative stereochemistry of 1 was assigned by NOE and by
an analysis of vicinal proton-proton coupling constants. The NOE
correlation between H-5 and H3-7 suggested that H-5 and H-6 were
trans oriented, which was further supported by comparing the
coupling constant between H-5 and H-6 in 1 (JH5-H6 ) 8.5 Hz)
with values reported for the trans stereoisomers of aspyrone (3)
(JH5-H6 ) 8.5 Hz),4 dihydroaspyrone (JH5-H6 ) 8.0 Hz),4 and
9-chloro-8-hydroxy-8,9-deoxyaspyrone (JH5-H6 ) 9.0 Hz)6 and the
cis stereoisomer of (+)- goniotriol (JH5-H6 ) 3.2 Hz).7 From the
Chlorohydroaspyrone A (1) was obtained in the form of a
colorless oil. It showed an isotopic cluster at m/z 243 [M (35Cl) +
Na]+ (20) and 245 [M (37Cl) + Na]+ (7) with a 3:1 ratio in the
FABMS, suggesting the presence of a chlorine atom. A molecular
formula of C9H13ClO4, which gave three degrees of unsaturation,
was established by HRESIMS and 13C NMR methods. The IR
absorptions at 3344 and 1709 cm-1 indicated the presence of a
3
magnitude of the J vicinal coupling constants (6.5 Hz) between
H-8 and H-9, these two protons were assigned an erythro C8-C9
configuration.7,8 This is similar to the data for side chains of
* To whom correspondence should be addressed. Tel: +82-51-629-5592.
Fax: +82-51-629-5583. E-mail: sonbw@pknu.ac.kr.
† Pukyong National University.
3
R-pyrone and γ-butyrolactone derivatives, in which the J vicinal
‡ Pusan National University.
coupling constants for the syn and anti configurations are 2.0-4.5
§ Dongeui University.
and 7.5-9.0 Hz, respectively.7–9 To establish the stereochemistry
10.1021/np800107c CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/29/2008

Notes
Journal of Natural Products, 2008, Vol. 71, No. 8 1459
Table 1. NMR Spectroscopic Data (400 MHz, DMSO-d6) for Chlorohydroaspyrones A (1) and B (2)
chlorohydroaspyrone A (1)
δH, (J in Hz)
chlorohydroaspyrone B (2)
position
δC, mult.
δC, mult.
δH, (J in Hz)
2
3
4
5
6
7
8
9
161.9, qC
128.1, qC
148.8, CH
66.4, CH
78.2, CH
17.6, CH3
62.5, CH
67.9, CH
19.4, CH3
162.9, qC
130.4, qC
146.1, CH
66.0, CH
78.7, CH
17.9, CH3
71.2, CH
59.8, CH
18.2, CH3
6.96, d (1.7)
6.87, dd-like (2.0,1.1)
4.19, ddd (7.5, 6.0, 2.0)
4.23, dq (7.5, 6.0)
1.32, d (6.0)
4.46, ddd (5.5, 4.5, 1.1)
4.35, dq (4.5, 7.0)
1.31, d (7.0)
4.16, ddd (8.5, 6.0, 2.0)
4.20, dq (8.5, 5.9)
1.33, d (5.9)
4.65, d (6.5)
4.02, ddq (5.5, 6.5, 6.5)
1.13, d (6.5)
10
5-OH
8-OH
9-OH
5.87, d (5.9)
5.82, d (6.0)
5.73, d (5.5)
5.16, d (5.5)
of chlorohydroaspyrone A (1), a chemical synthesis of 1 was carried
out from aspyrone (3). Treatment of 3 with 37% HCl gave not
only 1 and its 8-epimer (1a) as main products but also 2 as one of
the minor products (see Experimental Section and Figure S1 in the
Supporting Information). The two products (1 and 1a) showed
coupling constants of JH8-H9 ) 6.5 Hz in 1 and JH8-H9 ) 4.5 Hz
in 1a, indicating trans and cis conformation between H-8 and H-9,
respectively.7,8 On the basis of the evidence described above, we
suppose that the two main products (1, 1a) were formed from the
nucleophilic substitution of the 8,9-epoxide by a chloride ion
through an SN1 reaction. The spectroscopic data, TLC, and HPLC
behavior of the synthetic compound (1) proved to be identical to
those observed for the natural product chlorohydroaspyrone A (see
Figure S1 in the Supporting Information). Accordingly, the absolute
stereostructure of chlorohydroaspyrone A was determined as
(8R,9S)-8-chloro-9-hydroxy-8,9-deoxyaspyrone (1).
fractions was carried out. Both 1 and 2 were detected in the original
organic crude extract and the polar fraction obtained from Si gel
column chromatography.
Compound 2 appears to be identical in its planar structure to
the chlorine-containing compound 9-chloro-8-hydroxy-8,9-deox-
yaspyrone reported by Namikoshi et al.6 The two compounds have
3
significantly different JH8-H9 vicinal coupling constants (4.5 Hz
for 2 as compared to 12.5 Hz for the compound reported by
Namikoshi et al.6), as well as optical rotations (+70 for 2 as
compared to +17.2 for the compound reported by Namikoshi et
al.6). The configurations at C-8 and C-9 in the latter compound
were suggested to be 8S* and 9R* from consideration of its
hypothetical biogenetic pathway, which is similar to that of
aspyrone; however, their absolute configurations remain unsolved.
Polyketides 3-5 were also obtained in this investigation. They
were identified by inspecting their NMR spectra and comparing
these data to literature values.4,5
Compounds 1 and 2 exhibited mild antibacterial activity against
Staphylococcus aureus, methicillin-resistant S. aureus, and multi-
drug-resistant S. aureus. The MIC values to each strain are as
follows: compound 1 showed 62.5 µg/mL for S. aureus and 125
µg/mL for methicillin-resistant S. aureus and multidrug-resistant
S. aureus, and compound 2, 62.5 µg/mL for S. aureus and
methicillin-resistant S. aureus and 125 µg/mL for multidrug-resistant
S. aureus.
Chlorohydroaspyrone B (2) was also obtained in the form of a
colorless oil. The general features of its MS, UV, IR, and NMR
spectra (Table 1) closely resembled those of chlorohydroaspyrone
A (1), except that the NMR signals including chemical shift and
coupling constants assigned to H-8 and C-8 were changed from
δH 4.65 (1H, d, J ) 6.5 Hz, H-8) and δC 62.5 (C-8) for compound
1 to δH 4.46 (1H, ddd, J ) 5.5, 4.5, 1.1 Hz, H-8) and δC 71.2
1
(C-8) for compound 2 (Table 1). Detailed analyses of the H and
13C NMR spectra of 2, including the results from DEPT, COSY,
HMQC, HMBC, and NOESY experiments, suggested that 2 is a
regioisomer of compound 1, 9-chloro-8-hydroxy-8,9-deoxyaspy-
rone. The structure of compound 2 was further supported by the
HMBC correlations between 8-OH and C-3, C-8, and C-9. Thus,
the planar structure of chlorohydroaspyrone B was confidently
assigned.
Experimental Section
General Experimental Procedures. Optical rotation was determined
on a Perkin-Elmer model 341 polarimeter. UV/visible spectra were
measured on a Hitachi U-2001 UV/vis spectrometer. IR spectra were
recorded on a Bruker FT-IR model IFS-88 spectrometer. 1H (400 MHz)
and 13C NMR (100 MHz) spectra were obtained on a JEOL JNM-ECP
As in 1, the relative stereochemistry of metabolite 2 was deduced
by analyzing the vicinal proton-proton coupling constant and by
interpreting the NOE difference spectroscopic data. The relative
configuration of H-5 and H-6 was assigned trans on the basis of a
1
400 NMR spectrometer, using TMS or solvent peaks [DMSO-d6: H
(δ 2.50) and 13C (δ 39.5)] as reference standard. MS spectra were
obtained on a JEOL JMS-700 spectrometer. HPLC was performed on
a JASCO LC-2000 Plus system using a reversed-phase analytical
column (Alltec Appolo C18, 4.6 × 250 mm, 5 µm) with UV detection.
Fungal Isolation and Culture. The fungal strain Exophiala sp.
(family Herpotrichiellaceae) was isolated from the surface of the marine
sponge Halichondria panicea collected on Bogil Island, Jeonnam
Province, Korea, in 2006 and identified on the basis of morphological
evaluation and fatty acid methyl ester analysis (Korean Culture Center
of Microorganism, Seoul, Korea, a similarity index of 0.828). A voucher
specimen is deposited at Pukyong National University with the code
MFC353. The fungus was cultured (10 L) for 3 weeks (static) at 29 °C
in SWS medium consisting of soytone (0.1%), soluble starch (1.0%),
and seawater (100%).
4,6,7
3
large J vicinal coupling constant (7.5 Hz)
between H-5 and
H-6 and also on the basis of an NOE correlation between H-5 and
H3-7. The relative configuration of H-8 and H-9 was assigned a
syn configuration on the basis of the classical cis vicinal coupling
constant (4.5 Hz) between H-8 and H-9.8,9 Because the carbon
backbones of compounds 1 and 2 are likely to be formed by the
same biosynthetic pathway, the orientation of the hydroxyl group
of 2-chloro-1-hydroxypropanyl side chain is assumed to be the
same. This assumption was further supported by 2 derived from
the treatment of aspyrone (3) with 37% HCl (see Figure S1 in the
Supporting Information). In considering the stereochemistry of
natural product 2, we propose that the minor product 2 is also
formed through an SN1 reaction. Hence, an absolute configuration
of 5S, 6R, 8S, and 9S is proposed for 2.
Extraction and Isolation. The mycelium and broth were separated
by filtration using cheeesecloth. A crude extract from a small-scale
(100 mL) culture of an Exophiala sp. displayed antimicrobial activity
in a primary screen using S. aureus, methicillin-resistant S. aureus,
and multidrug-resistant S. aureus. This prompted the growth of a larger-
scale culture (20 L) to facilitate the purification of metabolites from
the broth extract. The filtered broth was extracted with EtOAc to afford
To rule out the possibility of the two new metabolites (1, 2)
being artifacts formed as a result of the extraction and purification
process, a careful HPLC analysis of a new extract and purified

1460 Journal of Natural Products, 2008, Vol. 71, No. 8
Notes
broth extract (1.5 g), which was subjected to Si gel flash chromatog-
raphy. Elution was performed with n-hexane-EtOAc (stepwise,
0-100% EtOAc) to yield four fractions. Fractions 3 and 4 were
separated by medium-pressure liquid chromatography (MPLC) (ODS)
using a H2O-MeOH gradient elution to afford crude compounds 3-5
and 1/2, respectively. These were further purified by HPLC (YMC,
ODS-A) utilizing a 30 min gradient program of 50% to 100% MeOH
in H2O to furnish 1 (5.2 mg), 2 (5.4 mg), 3 (60 mg), 4 (70 mg), and 5
(80 mg), respectively.
(8S,9S)-8-chloro-9-hydroxy-8,9-deoxyaspyrone (1a, 1.0 mg). The latter
was dissolved in MeOH (1 mL), and an aliquot (10 µL) of the sample
solution was analyzed by HPLC [Alltech, Apollo C18, 10 × 250 mm,
MeOH-H2O, 2:3, UV detector (254 nm)] (see Figure S1 in the
Supporting Information).
(8S,9S)-8-Chloro-9-hydroxy-8,9-deoxyaspyrone (1a): 1H NMR
(DMSO-d6, 400 MHz) δ 7.02 (1H, d, J ) 2.7 Hz, H-4), 4.18 (1H, m,
H-5), 4.23 (1H, m, H-6), 1.32 (3H, d, J ) 6.5 Hz, H3-7), 4.66 (1H, d,
J ) 4.3 Hz, H-8), 4.00 (1H, ddq, J ) 6.5, 5.9, 4.3 Hz, H-9), 1.12 (3H,
d, J ) 5.9 Hz, H3-10), 5.88 (1H, d, J ) 5.9 Hz, 5-OH), 5.06 (1H, d,
J ) 5.9 Hz, 9-OH).
Chlorohydroaspyrone A (1): colorless oil; [R]2D0 -110 (c 0.1,
MeOH); UV (MeOH) λmax (log ε) 272 (4.10); IR (neat) νmax 3344,
1
2980, 1709, 1639, 1448, 1379, 1219, 1144, 1060 cm-1; H and 13C
Antibacterial Assay. The in Vitro antibacterial activity of the
fermentation broth and purified samples was evaluated by a conven-
tional 2-fold serial dilution method using S. aureus, methicillin-resistant
S. aureus, and multidrug-resistant S. aureus as indicator strains. A 5
mL suspension containing 105 cells per mL was used as inoculum of
the test organism. The MIC values were determined after the inoculation
for 18 h at 37 °C.2
NMR, see Table 1; EIMS 223 [M (37Cl) + 1]+ (0.18), 221 [M (35Cl)
+ 1]+ (0.5), 203 [(M + 1) - H2O]+ (0.3), 178 [M (37Cl) - CH3CHO]+
(4.4), 176 [M (35Cl) - CH3CHO]+ (15), 160 [176 (37Cl) - H2O]+ (12),
158 [176 (35Cl) - H2O]+ (37), 141 (30), 123 (100), 95 (46); FABMS
m/z 245 [M (37Cl) + Na]+ (7), 243 [M (35Cl) + Na]+ (20), 223 [M
(37Cl) + H]+ (3), 221 [M (35Cl) + H]+ (9), 178 [M (37Cl) - CH3CHO]+
(6), 176 [M (35Cl) - CH3CHO]+ (36), 156 (7), 154 (26); HRESIMS
m/z 243.0406 (calcd for C9H13ClO4Na, 243.0400).
Acknowledgment. This work was supported by a grant from Marine
Biotechnology Programs funded by Ministry of Land, Transport and
Maritime Affairs, Republic of Korea. Mass spectral data were kindly
provided by the Korea Basic Science Institute.
Chlorohydroaspyrone B (2): colorless oil; [R]2D0 +70 (c 0.1, CHCl3);
UV (MeOH-CHCl3, 9:1) λmax (log ε) 272 (4.40); IR (neat) νmax 3379,
1
2981, 1706, 1647, 1541, 1379, 1214, 1048 cm-1; H and 13C NMR
(DMSO-d6), see Table 1; 1H NMR (C6D6, 400 MHz) δ 6.43 (1H,
deformed-d, J ≈ 2.0 Hz, H-4), 3.47 (1H, ddd, J ) 8.6, 6.5, ∼2.0 Hz,
H-5), 3.89 (1H, dq, J ) 8.6, 6.5 Hz, H-6), 1.05 (3H, d, J ) 6.5 Hz,
H3-7), 4.34 (1H, dd, J ) 6.5, 5.9 Hz, H-8), 4.41 (1H, dq, J ) 6.5, 6.5
Hz, H-9), 1.36 (3H, d, J ) 6.5 Hz, H3-10), 3.26 (1H, d, J ) 6.5 Hz,
5-OH), 1.62 (1H, d, J ) 5.9 Hz, 8-OH); 13C NMR (C6D6, 100 MHz)
δ 164.1 (qC, C-2), 130.4 (qC, C-3), 146.3 (CH, C-4), 68.0 (CH, C-5),
79.2 (CH, C-6), 18.1(CH3, C-7), 75.9 (CH, C-8), 59.7 (CH, C-9), 20.1
(CH3, C-10); EIMS m/z 223 [M (37Cl) + 1]+ (0.3), 221 [M (35Cl) +
1]+ (0.6), 203 [(M + 1) - H2O]+ (0.6), 178 [M (37Cl) - CH3CHO]+
(9), 176 [M (35Cl) - CH3CHO]+ (29), 157 (100), 139 (57), 113 (84),
95 (40), 85 (70); HRESIMS m/z 243.0404 (calcd for C9H13ClO4Na,
243.0400).
Aspyrone (3), Asperlactone (4), and Penicillic Acid (5). Spectro-
scopic data were virtually identical to those reported in the literature.4,5
Treatment of Aspyrone (3) with 37% HCl and HPLC Analysis
of the Reaction Mixture. Hydrochloric acid (37%, 0.1 mL) was added
to a solution of aspyrone (3) (5.0 mg) (0.02 mmol) in CHCl3 (1.0 mL)
at 0 °C, and then the mixture was stirred for 30 min. The reaction
mixture was then poured into H2O and extracted with EtOAc. The
EtOAc extract was washed with brine, then dried over MgSO4. After
removal of the solvent under reduced pressure, the residue was divided
into two samples for purification of reaction product and HPLC analysis
(∼1 mg less). The former was purified by silica gel column chroma-
tography (n-hexane-EtOAc, 1:5), followed by HPLC (ODS, MeOH-
H2O, 2:3) to furnish chlorohydroaspyrone A (1, 2.0 mg) and its isomer,
Supporting Information Available: 1H and 13C NMR spectra of 1
and 2 in DMSO-d6 and of 2 in C6D6, H NMR spectrum of 1a (in
DMSO-d6), and comparison of HPLC chromatograms of the reaction
mixture with those of 1 and 2. These materials are available free of
charge via the Internet at http://pubs.ac.org.
1
References and Notes
(1) Blunt, J. W.; Copp, B. R.; Hu, W.-P.; Munro, M. H. G.; Northcote,
P. T.; Prinsep, M. R. Nat. Prod. Rep. 2007, 24, 31–86.
(2) Nguyen, H. P.; Zhang, D.; Lee, U.; Kang, J. S.; Choi, H. D.; Son, B. W.
J. Nat. Prod. 2007, 70, 1188–1190.
(3) Zhang, D.; Noviendri, D.; Nursid, M.; Yang, X.; Son, B. W. Nat. Prod.
Sci. 2007, 13, 251–254.
(4) Garson, M. J.; Staunton, J.; Jones, P. G. J. Chem. Soc., Perkin Trans.
1 1984, 1021–1026.
(5) Cole, R. J.; Cox, R. H. Handbook of Toxic Fungal Metabolites;
Academic Press: New York, 1981; pp 520-526.
(6) Namikoshi, M.; Negishi, R.; Nagai, H.; Dmitrenok, A.; Kobayashi, H.
J. Antibiot. 2003, 56, 755–761.
(7) Alkofahi, A.; Ma, W.-W.; McKenzie, A. T.; Byrn, S. R.; McLaughlin,
J. L. J. Nat. Prod. 1989, 52, 1371–1373.
(8) (a) Shing, T. K. M.; Tai, V. W.-F. J. Org. Chem. 1999, 64, 2140–
2144. (b) Matsumori, N.; Kaneno, D.; Murata, M.; Nakamura, H.;
Tachibana, K. J. Org. Chem. 1999, 64, 866–876.
(9) Abrell, L. M.; Borgeson, B.; Crews, P. Tetrahedron Lett. 1996, 37,
2331–2334.
NP800107C