Eremophilane-type sesquiterpenes from the fungus xylaria sp. BCC 21097

Article abstract of DOI:10.1021/np100030x

Seven new eremophilane-type sesquiterpenoids (1-5, 7, and 8) and the known mairetolide F (6) were isolated from the endophytic Xylaria sp. BCC 21097. A new furofurandione (9) was also isolated from the fungal extract. The structures of the new compounds were elucidated by analyses of NMR spectroscopic and mass spectrometry data in combination with chemical reaction studies. Eremophilanolides possessing an α-methylene-γ-lactone (1-3) exhibited moderate cytotoxic activities, while related analogues bearing an endo double bond (6 and 7) were inactive.

Full text of DOI:10.1021/np100030x

J. Nat. Prod. 2010, 73, 683–687
683
Eremophilane-Type Sesquiterpenes from the Fungus Xylaria sp. BCC 21097
Masahiko Isaka,* Panida Chinthanom, Tanapong Boonruangprapa, Nattawut Rungjindamai, and Umpava Pinruan
National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road, Klong Luang,
Pathumthani 12120, Thailand
ReceiVed January 14, 2010
Seven new eremophilane-type sesquiterpenoids (1-5, 7, and 8) and the known mairetolide F (6) were isolated from the
endophytic Xylaria sp. BCC 21097. A new furofurandione (9) was also isolated from the fungal extract. The structures
of the new compounds were elucidated by analyses of NMR spectroscopic and mass spectrometry data in combination
with chemical reaction studies. Eremophilanolides possessing an R-methylene-γ-lactone (1-3) exhibited moderate
cytotoxic activities, while related analogues bearing an endo double bond (6 and 7) were inactive.
Fungi belonging to the genus Xylaria have been the source of a
wide range of bioactive compounds. Examples include xyloketals
and xyloallenolide from Xylaria sp. (No. 2508),1,2 multiplolides
from X. multiplex BCC 1111,3 xylactam from X. euglossa,4
xylobovide and cytochalasins from X. oboVata ADA-228,5 integric
acid from a Xylaria sp.,6 and xylarenals from X. persicaria.7 As
part of our research program on the utilization of fungal sources in
Thailand, we investigated secondary metabolites of the endophytic
Xylaria sp. BCC 21097, as an extract of this fungus showed
moderate cytotoxic activity against several cancer cell lines. Scale-
up fermentation and chemical studies resulted in the isolation of
eight elemophilane-type sesquiterpenes (1-8), one of which was
assigned as the known mairetolide F (6). Furofurandione 9 was
also isolated from the extract. We report herein the detailed
isolation, structure elucidation, and biological activities of these
compounds.
Table 1. 13C NMR Data (δ) for Eremophilanolides 1-8 (125
MHz)
position
1a
2a
3b
4a
5a
6a
7b
8b
1
2
3
4
5
6
7
8
74.7
28.8
25.1
35.3
39.1
42.0
72.9
85.1
38.1
74.3
85.0
22.9
25.2
35.3
39.2
41.7
72.8
84.8
38.0
74.1
59.7
22.0
23.9
34.1
35.4
41.4
74.9
29.0
25.7
35.0
42.8
34.5
85.2
23.0
25.7
34.8
42.9
34.3
74.9
28.9
25.7
34.9
42.9
34.7
60.1
22.2
24.9
33.6
39.0
34.9
58.5
33.0
67.3
40.7
39.4
35.2
74.0 165.5 165.2 162.1 163.3 159.3
83.6
35.7
63.8
79.1
40.2
74.6
78.8
40.0
74.4
79.1
40.2
74.8
78.9
38.4
63.4
78.2
38.0
63.2
9
10
11
12
142.7 142.7 141.0 124.8 124.8 120.7 125.1 123.0
169.7 169.5 169.6 173.0 172.9 174.0 173.7 174.4
13
14
15
1-OCH3
122.1 122.1 124.4
53.5
14.2
15.0
53.5
14.0
14.9
56.8
7.3
13.9
15.0
54.7
8.2
16.1
10.2
16.5
15.1
16.2
14.9
56.8
16.2
15.3
15.2c
15.2c
a Acquired in acetone-d6. b Acquired in CDCl3. c Carbon signals were
overlapped.
presence of OH and ester groups. The 1H and 13C NMR, DEPT135,
and HMQC spectroscopic data revealed that 1 contained a carbonyl
carbon (δC 169.7), an exomethylene group (δC 142.7, qC; δC 122.1,
CH2), two oxyquaternary carbons, two oxymethines, an aliphatic
quaternary carbon, a methine, four methylenes, and two methyl
groups (Table 1). The decalin unit was deduced from HMBC
correlations of the methyl protons (H3-14, s) to quaternary carbons
at δC 39.1 (C-5) and 74.3 (C-10), a methine carbon (δC 35.3, C-4),
and a methylene carbon (δC 42.0, C-6), and from an OH proton at
δH 3.57 (s) to C-10, an oxymethine (δC 74.7, C-1), and a methylene
carbon (δC 38.1, C-9). HMBC correlations from H2-6, H-8, and
the OH at δH 4.54 (s) to the quaternary olefinic carbon (δC 142.7)
indicated attachment of the exomethylene group (C-11/C-13) to
C-7 (δC 72.9). The R-methylene-γ-lactone, joined to the decalin at
C-7 and C-8, was established by HMBC correlations from the
exomethylene protons (H2-13; δH 5.94, s, and 6.24, s) and H-8 to
the carbonyl carbon (δC 169.7, C-12). The relative configuration
of 1 was based on NOESY correlations (Figure 1). The axial methyl
H3-14 exhibited cross-peaks with Hꢀ-3, Hꢀ-6, and Hꢀ-9. On the
other hand, 10-OH showed correlations to H-1 and HR-6. H-1
showed NOESY correlations with HR-2 and Hꢀ-2 with similar cross-
peak intensity, and it also exhibited an intense cross-peak with H-9R;
therefore, H-1 was equatorial. These data indicated a trans ring
junction of the decalin unit. One of the exomethylene protons (H2-
13) resonated at δH 5.94 and showed NOESY correlation to Hꢀ-6,
whereas 7-OH exhibited weak cross-peaks with HR-6 and H-8. The
NOESY correlation between H-8 and HR-9 was much stronger than
that between H-8 and Hꢀ-9. The large vicinal coupling constant
Results and Discussion
Compound 1 was the most abundant sesquiterpene constituent
of the broth extract and had the molecular formula C15H22O5, as
determined by HRESIMS. The IR spectrum exhibited absorption
bands at νmax 3526, 3456, and 1766 cm-1, which suggested the
* Corresponding author. Tel: +66-25646700, ext 3554. Fax: +66-
25646707. E-mail: isaka@biotec.or.th.
10.1021/np100030x  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/15/2010

684 Journal of Natural Products, 2010, Vol. 73, No. 4
Isaka et al.
Figure 2. ∆δ values (δS - δR) of the Mosher esters 10a and 10b.
the 1R configuration (Figure 2). The remaining question was the
configuration of the epoxide moiety of 3, 7, and 8. Since the NMR
spectroscopic data did not provide conclusive evidence of the
configuration, we examined chemical means. Treatment of 3 with
Figure 1. Key NOESY correlations for 1.
(J ) 10.7 Hz) of H-8 and Hꢀ-9 indicated the antiperiplanar
relationship. On the basis of these data, the cis junction of the
γ-lactone with the R-orientation of 7-OH and H-8 was established.
The molecular formula of compound 2 was determined by
HRESIMS as C16H24O5. The NMR spectroscopic data of 2 were
similar to those of 1, except for the presence of a methoxy group
(δC 56.8; δH 3.33, 3H, s) and the lack of the 1-OH proton resonance.
The HMBC correlations of the methoxy protons to C-1 and from
H-1 to the methoxy carbon indicated the location of the OCH3
group. H-1 resonated as a triplet with small coupling constants
(J ) 2.5 Hz) and exhibited an intense NOESY cross-peak with
HR-9; therefore, it was equatorial.
Compound 3 possessed the molecular formula C15H20O4 (HRES-
IMS). Significant differences in the 1H and 13C NMR spectroscopic
data, when compared to those of 1, were the upfield shift of H-1
(δH 3.01, br d, J ) 3.9 Hz) and the oxygenated carbons C-1 (δC
59.7) and C-10 (δC 63.8). These data implied that the 1,10-diol
moiety of 1 was replaced by the 1,10-epoxy functionality in 3.
Compound 4 possessed the same molecular formula as 1,
C15H22O5 (HRESIMS). The 1H NMR spectroscopic data lacked the
resonances of the exomethylene protons and 7-OH; instead a
hydroxymethyl group was present at δH 4.28 (2H, d, J ) 5.7 Hz,
H2-13; δC 53.5) and 3.94 (t, J ) 5.7 Hz, 13-OH). These data
indicated a rearranged allylic alcohol (C-7-C-11-C-13) moiety
with an endo olefinic bond. The proposed structure 4 was confirmed
by the HMBC correlations: from HR-9 to C-7 (δC 165.5), from H2-6
and H-8 to C-11, and from H2-13 to C-7, C-11, and C-12.
Compound 5, possessing the molecular formula C16H24O5 (HRES-
IMS), was identified as the 1-O-methyl analogue of 4. Compound
6 was assigned as the 13-deoxy analogue of 4. This compound is
identical to mairetolide F, previously isolated from the higher plant
Senecio mairetianus.8
Compound 7 possesses a 1,10-epoxy functionality, as indicated
by the similarity of the NMR spectroscopic data with those of 3,
while the γ-lactone moiety was identical to 4. Compound 8 also
possessed the 1,10-epoxy functionality, and its γ-lactone moiety
was identical to 6. The secondary alcohol functionality at C-3 (δC
67.3; δH 3.48, m) was assigned on the basis of COSY data. The
NOESY correlations of H-3 to H3-14 and H3-15 established its
ꢀ-orientation.
The absolute configuration of 1 was addressed by application of
the modified Mosher’s method.9,10 Initial attempts to prepare the
Mosher esters of 1 were unsuccessful, giving a complex mixture
of products. These results may be due to the allylic alcohol
functionality. When compound 1 was treated with LiBH4 in THF,
originally planning to achieve the reduction of the lactone,
mairetolide F (6) was obtained as the major product. This
unexpected conversion allowed us to obtain an additional quantity
of 6, which was used for the preparation of the Mosher esters. The
∆δ values of the (S)- and (R)-MTPA esters 10a and 10b indicated
1
concentrated H2SO4/MeOH gave 11 as the sole product. The H
NMR spectroscopic data of 11 in acetone-d6 resembled those of 5
except for the presence of an additional methoxy group at δH 3.29
(3H, s). Similar regioselective epoxide cleavage by acidic metha-
nolysis of mairetolides was reported previously.8 Furthermore, 2
was converted to the same compound (11) under the same reaction
conditions, which confirmed the R-epoxide configuration of 3. We
propose that 7 and 8 are also R-epoxide derivatives on the basis of
the similarity of the NMR spectroscopic data with those of 3 and
their co-occurrence with 1-6. Structural relations and the reac-
tivities mentioned above suggested that 1 and 2 were derived from
3, respectively, by hydrative epoxide cleavage and methanolysis.
Similarly, 7 may be the precursor of 4 and 5. The 1H NMR spectrum
of the crude EtOAc extract from culture broth (pH 4.23) indicated
the presence of the major eremophilanolides 1 and 3. On the other
hand, the minor methoxy analogues 2 and 5 were possibly produced
during the chromatographic procedures. The ꢀ-epoxy isomer of 7
was previously isolated from the aerial part of Ondetia linearis
along with a number of eremophilanolides, eudesmanolides, and
guaianolides.11
Compound 9 was isolated from a fermentation batch of extended
incubation. The molecular formula of 9 was determined by
HRESIMS as C13H20O4. The IR spectrum exhibited a broad and
1
intense ester(s) band at νmax 1767 cm-1. Analysis of the H and
13C NMR, DEPT135, and HMQC data revealed that 9 contained
two carbonyls at δC 176.7 and 174.6, two oxymethines at δC 82.4
(δH 4.55, ddd, J ) 8.0, 6.3, 3.9 Hz) and 78.3 (δH 5.11, dd, J ) 6.0,
3.9 Hz), two methines at δC 49.0 (δH 3.15, dd, J ) 6.0, 1.3 Hz)
and 38.3 (δH 3.08, dq, J ) 1.3, 7.7 Hz), five methylenes, and two
methyl groups at δC 17.1 (δH 1.45, d, J ) 7.7 Hz) and 14.0 (δH
0.89, t, J ) 6.9 Hz). The planar structure was established on the
basis of COSY and HMBC correlations, and the relative configu-
ration was addressed by NOESY correlations. Intense NOESY
cross-peaks were observed from H-6a to H-6, H-3a, and H3-7. H-3a
exhibited intense and weak NOESY correlations respectively to
H3-7 and H-6. These data strongly suggested the cis ring junction
and that H3-7, H-3a, H-6, and H-6a are coplanar. Compound 9 is
a 3,7-dihydro analogue of sporothriolide, which was previously
isolated from the fungus Sporothrix sp. (strain No. 700).12
Canadensolide and dihydrocanadensolide, isolated from Peni-
cillium canadense,13 possess an n-butyl substituent instead of
the n-hexyl group in 9. These known fungal metabolites are
reported to have the (3S),3aS,6R,6aR configuration.12,14,15 The
negative optical rotation of 9 isolated in the present study ([R]25
D
-33, c 0.13, CHCl3) was similar to those of the natural and
synthetic (3S,3aS,6R,6aR)-dihydrocanadensolide ([R]D -31;
[R]20D -31, c 0.26, MeOH)13,15 and opposite those reported for
the synthetic enantiomer, (3R,3aR,6S,6aS)-dihydrocanadensolide
([R]23 +30.3, c 1.9, CHCl3; [R]D +29.8, c 0.35, CHCl3).16,17
D

Sesquiterpenes from the Fungus Xylaria
Journal of Natural Products, 2010, Vol. 73, No. 4 685
Table 2. 1H NMR Data for Eremophilanolides 1-3 (500 MHz)
position
1a
HMBC for 1
2a
3.11 t (2.5)
3b
1
2
3.62 m
C-3,5,10
3.01 br d (3.9)
R 1.94 m
R 2.14 m
R 1.94 m
ꢀ 1.50 m
C-10
ꢀ 1.73 m
ꢀ 1.89 m
3
R 1.17 m
R 1.17 m
R 1.19 m
ꢀ 1.58 dq (4.2, 12.8)
1.98 m
C-2,4
ꢀ 1.40 dq (4.1, 12.7)
1.98 m
ꢀ 1.17 m
1.74 m
4
6
R 2.17 d (13.6)
ꢀ 2.24 d (13.6)
4.77 dd (10.7, 7.5)
R 2.02 dd (14.1, 7.5)
ꢀ 1.89 dd (14.1, 10.7)
5.94 s
6.24 s
0.94 s
0.81 d (6.9)
3.84 br d (3.4)
C-4,5,7,11,14
C-5,7,8,10,11,14
C-7,9,11,12
C-1,5,7,8
R 2.16 d (13.6)
ꢀ 2.24 d (13.6)
4.76 dd (10.8, 7.4)
R 2.00 dd (14.1, 7.4)
ꢀ 1.84 dd (14.1, 10.8)
5.94 s
6.23 s
0.83 s
0.80 d (6.9)
R 1.90 d (14.4)
ꢀ 2.42 d (14.4)
4.67 dd (10.7, 7.2)
R 1.73 dd (14.2, 7.2)
ꢀ 1.88 dd (14.2, 10.7)
5.93 s
8
9
C-8,10
13
C-7,11,12
C-7,11,12
C-4,5,6,10
C-3,4,5
6.46 s
0.84 s
14
15
1-OH
1-OCH3
7-OH
10-OH
0.77 d (6.9)
3.22 s
4.53 s
3.72 s
4.54 s
3.57 s
C-6,7,8,11
C-5,9,10
3.16 s
a Acquired in acetone-d6. b Acquired in CDCl3.
The 1H and 13C NMR (CDCl3) spectroscopic data of the bicyclic
bis-lactone core of 9 showed an excellent match with those
reported for synthetic dihydrocanadensolide.15–17 Therefore, the
absolute configuration of 9 was assigned as 3S,3aS,6R,6aR.
Sharmareported18 theenantioselectivesynthesisof(3S,3aS,6R,6aR)-
dihydrocanadensolide, (3S,3aS,6R,6aR)-dihydrosporothrioride
(9), and their C-3 epimers (3R,3aS,6R,6aR); however, the
reported optical rotation data were opposite and inconsistent with
those of all other reports15–17 on the synthesis of dihydroca-
nadensolides. In addition, the 13C NMR (CDCl3) spectroscopic
data of the synthetic (3S,3aS,6R,6aR)-dihydrosporothrioride18a
were significantly different from those of our natural product
(9).
Eremophilanolides 1-3, 6, and 7 were subjected to our biological
assay protocols, inclusive of cytotoxicity against cancer cell lines
(KB, MCF-7, and NCI-H187) and nonmalignant Vero cells and
antimalarial (Plasmodium falciparum K1), antituberculosis (My-
cobacterium tuberculosis H37Ra), and antifungal (Candida albi-
cans) activities. Eremophilanolides possessing an R-methylene-γ-
lactone (1-3) exhibited moderate cytotoxic activities in the range
of IC50 3.8-21 µM. Compounds 2 and 3 also displayed antimalarial
activity with respective IC50 values of 8.1 and 13 µM. Only 3 was
active against Candida albicans (IC50 7.8 µM), which suggested
that the epoxide functionality may play an important role. Related
analogues possessing an endo double bond (6 and 7) were inactive
in these assays.
L Erlenmeyer flask containing 250 mL of the same liquid medium
(PDB) and incubated at 25 °C for 4 days on a rotary shaker (200 rpm).
These secondary cultures were pooled, and each 25 mL portion was
transferred into 20 × 1 L Erlenmeyer flasks containing 250 mL of
malt extract broth (MEB; malt extract 6.0 g, yeast extract 1.2 g, maltose
1.8 g, dextrose 6.0 g, per liter). Final fermentation was carried out at
25 °C for 15 days under static conditions. The cultures were filtered to
separate broth (filtrate, pH 4.23) and mycelia (residue). Culture broth
was extracted with EtOAc (3 × 4.5 L), and the combined organic phase
was concentrated to obtain a brown gum (broth extract; 1.90 g). The
broth extract was subjected to column chromatography (CC) on
Sephadex LH-20 (3.8 × 50 cm, MeOH) to obtain seven pooled
fractions: fractions 1 (44 mg), 2 (89 mg). 3 (362 mg), 4 (1.20 g), 5
(147 mg), 6 (56 mg), and 7 (6 mg). Fraction 4 was subjected to CC on
silica gel (3.2 × 14 cm, EtOAc/CH2Cl2, step gradient elution) to obtain
20 fractions (fraction 4-1-4-20). Fraction 4-5 (8 mg) contained
2-phenylethyl alcohol. Fraction 4-7 (49 mg) was further purified by
CC on silica gel (EtOAc/hexane, 40:60) to furnish 3 (30 mg). Fraction
4-8 (8 mg) was separated using a short silica gel column (EtOAc/
hexane, 40:60) to afford 3 (2 mg) and 7 (4 mg). Fractions 4-9 (12
mg), 4-11 (140 mg), and 4-12 (29 mg) were purified by CC on silica
gel (EtOAc/hexane, 60:40) to furnish 4 (5 mg), cytochalasin E (8 mg),
and 2 (8 mg), respectively. Fraction 4-13 (37 mg) was subjected to
preparative HPLC using a reversed-phase column (Phenomenex Luna
10u C18(2) 100A, 21.2 × 250 mm, 10 µm; mobile phase MeOH/H2O,
50:50, flow rate 15 mL/min) to furnish 6 (4 mg, tR 21 min), 5 (2 mg,
tR 26 min), and 2 (2 mg, tR 37 min). Fraction 4-15 (200 mg) was purified
by CC on silica gel (EtOAc/hexane, 50:50) to afford 1 (100 mg).
Fraction 4-17 (29 mg) was purified by preparative HPLC (MeOH/H2O,
45:55) to furnish 4 (10 mg, tR 8 min). Fraction 5 was fractionated by
CC on silica gel (EtOAc/CH2Cl2, step gradient elution) to afford mellein
(26 mg) and 3,4-dihydro-3,4,8-trihydroxy-1(2H)-naphthalenone (6 mg).
Chromatographic fractionations of the MeOH extract from mycelia (400
mg) afforded cytochalasin E (7 mg) and mellein (12 mg).
Experimental Section
General Experimental Procedures. Melting points were measured
with an Electrothermal IA9100 digital melting point apparatus. Optical
rotations were measured with a JASCO P-1030 digital polarimeter.
UV spectra were recorded on a GBC Cintra 404 spectrophotometer.
FTIR spectra were taken on a Bruker VECTOR 22 spectrometer. NMR
spectra were recorded on Bruker DRX400 and AV500D spectrometers.
ESITOF mass spectra were measured with Micromass LCT and Bruker
micrOTOF mass spectrometers.
Fungal Material. The fungus used in this study was isolated from
the palm Licuala spinosa in Trang Province, Thailand, and it was
deposited in the BIOTEC Culture Collection as BCC 21097 on April
10, 2006. This fungus was identified as a Xylaria sp. on the basis of
the gene sequence data of the 18S rDNA and ITS genes by one of the
authors (N.R.).
Fermentation and Isolation (Batch 2). The fermentation and
isolation was repeated to obtain additional quantities of the minor
metabolites. The fungus BCC 21097 was fermented using the same
procedure as described above, but the final fermentation was extended
to 30 days using 14 L (64 × 250 mL) of MEB medium. The EtOAc
extract from culture broth (6.0 g) was fractionated by chromatographic
methods as described above to furnish eremophilanes 7 (3 mg) and 8
(3 mg), furofurandione 9 (50 mg), cytochalasins E (223 mg) and K (9
mg), mellein (29 mg), 3,4-dihydro-3,4,8-trihydroxy-1(2H)-naphthale-
none (6 mg), 2-phenylethyl alcohol (43 mg), and 2-(4-hydroxyphenyl)-
ethyl alcohol (9 mg).
1ꢀ,7r,10r-Trihydroxyeremophil-11(13)-en-12,8ꢀ-olide (1): color-
Fermentation and Isolation (Batch 1). The fungus BCC 21097
was maintained on potato dextrose agar at 25 °C. The agar was cut
into small plugs and inoculated into 2 × 250 mL Erlenmeyer flasks
containing 25 mL of potato dextrose broth (PDB; potato starch 4.0 g,
dextrose 20.0 g, per liter). After incubation at 25 °C for 4 days on a
rotary shaker (200 rpm), each primary culture was transferred into a 1
less solid; mp 194-196 °C; [R]24D -65 (c 0.105, MeOH); UV (MeOH)
λ
max (log ε) 205 (3.94) nm; IR (KBr) νmax 3526, 3456, 1766 cm-1; 1H
NMR (500 MHz, acetone-d6) and 13C NMR (125 MHz, acetone-d6),
Tables 1 and 2; HRESIMS m/z 305.1366 [M + Na]+ (calcd for
C15H22O5Na 305.1359).

686 Journal of Natural Products, 2010, Vol. 73, No. 4
Isaka et al.
Table 3. 1H NMR Data for Eremophilanolides 4-8 (500 MHz)
position
4a
5a
6a
7b
8b
1
2
3.71 m
3.19 t (2.6)
R 1.97 m
ꢀ 1.79 ddt (14.2, 4.1, 2.5)
R 1.24 m
ꢀ 1.43 dq (4.1, 13.6)
2.13 m
R 2.50 br d (12.9)
ꢀ 2.83 d (12.9)
5.08 dd (11.0, 7.3)
R 2.18 dd (12.9, 7.3)
ꢀ 2.00 dd (12.9, 11.0)
4.28 br s
3.70 br s
R 2.17 m
ꢀ 1.55 m
R 1.25 m
ꢀ 1.62 dq (4.1, 13.2)
2.15 m
R 2.49 br d (12.8)
ꢀ 2.52 d (12.8)
5.05 m
R 2.16 dd (12.7, 7.5)
ꢀ 2.01 dd (12.7, 10.8)
1.75 t (1.5)
3.08 d (4.0)
R 2.00 m
3.06 d (4.4)
R 2.17 m
ꢀ 1.56 m
R 1.25 m
ꢀ 1.62 dq (4.1, 12.8)
2.15 m
R 2.52 d (12.9)
ꢀ 2.83 d (12.9)
5.09 dd (10.9, 7.2)
R 2.19 dd (13.0, 7.2)
ꢀ 2.06 dd (13.0, 10.9)
4.28 d (5.7)
R 1.91 dd (15.6, 8.7)
ꢀ 2.47 ddd (15.6, 7.9, 4.5)
ꢀ 3.48 m
ꢀ 1.96 m
3
R 1.25 m
ꢀ 1.27 m
4
6
1.90 m
1.82 m
R 2.26 d (13.6)
ꢀ 2.93 d (13.6)
4.98 dd (11.1, 7.0)
R 1.91 dd (12.8, 7.0)
ꢀ 2.07 dd (12.8, 11.1)
4.41 s
R 2.25 br d (13.6)
ꢀ 2.78 d (13.6)
4.91 dd (11.0, 7.0)
R 1.86 dd (12.8, 7.0)
ꢀ 1.97 dd (12.8, 11.0)
1.85 t (1.6)
8
9
13
14
0.94 s
0.83 s
0.91 s
0.86 s
0.84 s
15
0.85 d (6.9)
0.84 d (6.9)
0.87 d (7.0)
0.82 d (6.9)
0.98 d (6.9)
1-OH
1-OCH3
3-OH
10-OH
13-OH
3.97 d (4.2)
3.96 br s
3.26 s
1.37 br d (6.1)
3.76 s
3.94 t (5.7)
3.92 s
3.94 br s
3.73 br s
not observed
a Acquired in acetone-d6. b Acquired in CDCl3.
7r,10r-Dihydroxy-1ꢀ-methoxyeremophil-11(13)-en-12,8ꢀ-olide
(2): colorless solid; mp 181-184 °C; [R]25D -61 (c 0.11, MeOH); UV
C-8), 25.3 (CH2, C-9), 22.5 (CH2, C-12), 17.1 (CH3, C-7), 14.0 (CH3,
C-13); HRESIMS m/z 263.1256 [M + Na]+ (calcd for C13H20O4Na
263.1254).
(MeOH) end absorption; IR (KBr) νmax 3484, 3357, 1740, 1019 cm-1
;
1H NMR (500 MHz, acetone-d6) and 13C NMR (125 MHz, acetone-
d6), Tables 1 and 2; HRESIMS m/z 319.1527 [M + Na]+ (calcd for
C16H24O5Na 319.1521).
Reaction of 1 with LiBH4. To a solution of 1 (5.2 mg) was added
LiBH4 (10 mg), and the mixture was stirred for 2.5 h. The reaction
was terminated by addition of H2O and extracted three times with
EtOAc. The organic layer was concentrated under reduced pressure to
leave a colorless solid (5.6 mg), which was purified using a short silica
gel column (EtOAc/CH2Cl2, 30:70) to furnish the major reaction product
1r,10r-Epoxy-7r-hydroxyeremophil-11(13)-en-12,8ꢀ-olide (3):
colorless solid; mp 150-152 °C; [R]25 -112 (c 0.11, MeOH); UV
D
(MeOH) end absorption; IR (KBr) νmax 3436, 1765, 1745, 1629, 1001
cm-1; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3),
Tables 1 and 2; HRESIMS m/z 287.1251 [M + Na]+ (calcd for
C15H20O4Na 287.1254).
1
(4.0 mg), whose MS and H NMR data were identical to those of 6.
Preparation of the MTPA Esters 10a and 10b. Compound 6 (1.0
mg; obtained by LiBH4 reduction of 1) was treated with (-)-(R)-
MTPACl (5 µL) in pyridine (0.2 mL) at room temperature for 16 h.
The mixture was diluted with EtOAc and washed with H2O and 1 M
NaHCO3, and the organic layer was concentrated in vacuo. The residue
was purified by HPLC (MeCN/H2O, 40:60) to obtain the (S)-MTPA
ester 10a (1.0 mg). Similarly, (R)-MTPA ester 10b was prepared from
6 (0.9 mg) and (+)-(S)-MTPACl. Although the conversion of 6 into
10b was poor, separation of the crude reaction products by HPLC
furnished a pure sample of 10b (0.2 mg). The assignments of protons
for 10a and 10b were achieved by analysis of COSY and NOESY data.
(S)-MTPA Ester 10a: colorless gum; 1H NMR (400 MHz, CDCl3)
δ 7.47-7.37 (5H, m, phenyl of MTPA), 4.94 (1H, m, H-8), 4.91 (1H,
m, H-1), 3.55 (3H, br s, OCH3 of MTPA), 2.39 (1H, d, J ) 12.8 Hz,
Hꢀ-6), 2.38 (1H, d, J ) 12.8 Hz, HR-6), 2.16 (1H, m, HR-2), 1.98 (1H,
m, H-4), 1.94 (1H, dd, J ) 13.1, 7.0 Hz, HR-9), 1.78 (1H, m, Hꢀ-2),
1.75 (3H, br s, H-13), 1.39 (2H, m, HR-3 and Hꢀ-3), 1.28 (1H, dd, J )
13.1, 10.8 Hz, Hꢀ-9), 0.85 (3H, d, J ) 6.9 Hz, H-15), 0.45 (3H, s,
H-14); HRESIMS m/z 505.1805 [M + Na]+ (calcd for C25H29O6F3Na,
505.1808).
1ꢀ,10r,13-Trihydroxyeremophil-7(11)-en-12,8ꢀ-olide (4): color-
less solid; mp 147-150 °C; [R]24D -65 (c 0.105, MeOH); UV (MeOH)
λmax (log ε) 223 (4.19) nm; IR (KBr) νmax 3516, 3429, 1708, 1662,
1012 cm-1; 1H NMR (500 MHz, acetone-d6) and 13C NMR (125 MHz,
acetone-d6), Tables 1 and 3; HRESIMS m/z 305.1349 [M + Na]+ (calcd
for C15H22O5Na 305.1359).
10r,13-Dihydroxy-1ꢀ-methoxyeremophil-7(11)-en-12,8ꢀ-olide (5):
colorless amorphous; [R]27D -52 (c 0.145, MeOH); UV (MeOH) λmax
(log ε) 222 (3.99) nm; IR (KBr) νmax 3424, 1731, 1674 cm-1; 1H NMR
(500 MHz, acetone-d6) and 13C NMR (125 MHz, acetone-d6), Tables
1 and 3; HRESIMS m/z 297.1698 [M + H]+ (calcd for C16H25O5
297.1697).
1ꢀ,10r-Dihydroxyeremophil-7(11)-en-12,8ꢀ-olide (mairetolide F,
6): colorless solid; mp 164-165 °C; [R]27D -84 (c 0.08, MeOH); UV
(MeOH) λmax (log ε) 223 (4.09) nm; IR (KBr) νmax 3425, 1725, 1678,
1034 cm-1; 1H NMR (500 MHz, acetone-d6) and 13C NMR (125 MHz,
acetone-d6), Tables 1 and 3; HRESIMS m/z 289.1414 [M + Na]+ (calcd
for C15H22O4Na 289.1410).
(R)-MTPA Ester 10b: colorless gum; 1H NMR (400 MHz, CDCl3)
δ 7.44-7.38 (5H, m, phenyl of MTPA), 5.01 (1H, m, H-8), 4.84 (1H,
m, H-1), 3.47 (3H, br s, OCH3 of MTPA), 2.41 (1H, d, J ) 13.0 Hz,
Hꢀ-6), 2.39 (1H, d, J ) 13.0 Hz, HR-6), 2.07 (1H, dd, J ) 12.9, 7.0
Hz, HR-9), 2.06 (1H, m, HR-2), 1.92 (1H, m, H-4), 1.78 (3H, br s,
H-13), 1.75 (1H, m, Hꢀ-2), 1.55 (1H, m, Hꢀ-9), 1.26 (1H, m, HR-3),
1.09 (1H, dq, J ) 4.1, 13.7 Hz, Hꢀ-3), 0.79 (3H, d, J ) 6.8 Hz, H-15),
0.43 (3H, s, H-14); HRESIMS m/z 505.1318 [M + Na]+ (calcd for
C25H29O6F3Na, 505.1308).
1r,10r-Epoxy-13-hydroxyeremophil-7(11)-en-12,8ꢀ-olide (7): col-
orless solid; mp 121-124 °C; [R]25 -126 (c 0.11, MeOH); UV
D
(MeOH) λmax (log ε) 220 (4.19) nm; IR (KBr) νmax 3424, 1758, 1011
cm-1; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3),
Tables 1 and 3; HRESIMS m/z 287.1251 [M + Na]+ (calcd for
C15H20O4Na 287.1254).
1r,10r-Epoxy-3r-hydroxyeremophil-7(11)-en-12,8ꢀ-olide (8): col-
orless solid; mp 202-203 °C; [R]26 -126 (c 0.145, MeOH); UV
D
(MeOH) λmax (log ε) 219 (4.06) nm; IR (KBr) νmax 3489, 1727, 1684,
1
1038 cm-1; H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz,
Synthesis of 11. Compound 3 (1.4 mg) was dissolved in concentrated
H2SO4/MeOH, 1:9 (0.4 mL), and the solution was stirred at room
temperature for 16 h. The mixture was evaporated without heating,
and the residue was diluted with EtOAc (5 mL) and washed with H2O
(3 × 3 mL). The organic layer was concentrated under reduced pressure,
and the residue was purified by CC on silica gel to obtain 11 (0.5 mg).
1ꢀ,13-Dimethoxy-10r-hydroxyeremophil-7(11)-en-12,8ꢀ-olide (11):
CDCl3), Tables 1 and 3; HRESIMS m/z 287.1251 [M + Na]+ (calcd
for C15H20O4Na 287.1254).
Compound 9: colorless solid; mp 92-93 °C; [R]26D -50 (c 0.0975,
1
MeOH), [R]25 -33 (c 0.13, CHCl3); IR (KBr) νmax 1767 cm-1; H
D
NMR (500 MHz, CDCl3) δ 5.11 (1H, dd, J ) 6.0, 3.9 Hz, H-6a), 4.55
(1H, ddd, J ) 8.0, 6.3, 3.9 Hz, H-6), 3.15 (1H, dd, J ) 6.0, 1.3 Hz,
H-3a), 3.08 (1H, dq, J ) 1.3, 7.7 Hz, H-3), 1.91 (1H, m, Ha-8), 1.82
(1H, m, Hb-8), 1.50-1.45 (2H, m, H-9), 1.45 (3H, d, J ) 7.7 Hz,
H-7), 1.38-1.35 (2H, m, H-10), 1.32-1.28 (4H, m, H-11 and H-12),
0.89 (3H, t, J ) 6.9 Hz, H-13); 13C NMR (125 MHz, CDCl3) δ 176.7
(C, C-4), 174.6 (C, C-2), 82.4 (CH, C-6), 78.3 (CH, C-6a), 49.0 (CH,
C-3a), 38.3 (CH, C-3), 31.5 (CH2, C-11), 28.9 (CH2, C-10), 28.8 (CH2,
1
colorless, amorphous; H NMR (400 MHz, acetone-d6) δ 5.12 (1H,
dd, J ) 10.8, 7.3 Hz, H-8), 4.10-4.08 (2H, m, H-13), 3.92 (1H, s,
10-OH), 3.29 (3H, s, 13-OCH3), 3.26 (3H, s, 1-OCH3), 3.20 (1H, m,
H-1), 2.75 (1H, d, J ) 13.0 Hz, Hꢀ-6), 2.53 (1H, br d, J ) 13.0 Hz,
HR-6), 2.19 (1H, dd, J ) 12.8, 7.3 Hz, HR-8), 2.13 (1H, m, H-4), 2.01
(1H, dd, J ) 12.8, 10.8 Hz, Hꢀ-8), 1.97 (1H, m, HR-2), 1.79 (1H, m,

Sesquiterpenes from the Fungus Xylaria
Journal of Natural Products, 2010, Vol. 73, No. 4 687
(4) Wang, X.-N.; Tan, R.-X.; Liu, J.-K. J. Antibiot. 2005, 58, 268–270.
(5) Abate, D.; Abraham, W.-R.; Meyer, H. Phytochemistry 1997, 44,
1443–1448.
(6) Singh, S. B.; Zink, D.; Polishook, J.; Valentino, D.; Shafiee, A.;
Silverman, K.; Felock, P.; Teran, A.; Vilella, D.; Hazuda, D. J.;
Lingham, R. B. Tetrahedron Lett. 1999, 40, 8775–8779.
(7) Smith, C. J.; Morin, N. R.; Bills, G. F.; Dombrowski, A. W.; Salituro,
G. M.; Smith, S. K.; Zhao, A.; MacNeil, D. J. J. Org. Chem. 2002,
67, 5001–5004.
(8) Pe´rez-Castorena, A. L.; Arciniegas, A.; Guzma´n, S. L.; Villasen˜or,
J. L.; Romo de Vivar, A. J. Nat. Prod. 2006, 69, 1471–1475.
(9) Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1973, 95, 512–519.
(10) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem.
Soc. 1991, 113, 4092–4096.
(11) Zdero, C.; Bohlmann, F. Phytochemistry 1989, 28, 1653–1660.
(12) Krohn, K.; Ludewig, K.; Aust, H.-J.; Draeger, S.; Schluz, B. J. Antibiot.
1994, 47, 113–118.
(13) McCorkindale, N. J.; Wright, J. L. C.; Brian, P. W.; Clarke, S. M.;
Hutchinson, S. A. Tetrahedron Lett. 1968, 727, 730.
(14) (a) Anderson, R. C.; Fraser-Reid, B. Tetrahedron Lett. 1978, 3233,
3236. (b) Anderson, R. C.; Fraser-Reid, B. J. Org. Chem. 1985, 50,
4786–4790.
Hꢀ-2), 1.43 (1H, dq, J ) 4.1, 13.0 Hz, Hꢀ-3), 1.25 (1H, m, HR-3), 0.84
1
(3H, d, J ) 6.9 Hz, H-15), 0.81 (3H, s, H-14); H NMR (500 MHz,
CDCl3) δ 5.13 (1H, t, J ) 9.1 Hz, H-8), 4.17 (1H, d, J ) 12.2 Hz,
Ha-13), 4.15 (1H, d, J ) 12.2 Hz, Hb-13), 3.38 (3H, s, 13-OCH3),
3.27 (3H, s, 1-OCH3), 3.07 (1H, m, H-1), 2.72 (1H, d, J ) 13.0 Hz,
Hꢀ-6), 2.47 (1H, br d, J ) 13.0 Hz, HR-6), 2.13 (2H, d, J ) 9.1 Hz,
HR-8 and Hꢀ-8), 1.91 (1H, m, H-4), 1.86 (1H, m, Hꢀ-2), 1.79 (1H, m,
HR-2), 1.47 (1H, dq, J ) 4.5, 13.1 Hz, Hꢀ-3), 1.29 (1H, m, HR-3), 0.88
(3H, d, J ) 6.8 Hz, H-15), 0.82 (3H, s, H-14); HRESIMS m/z 333.1674
[M + Na]+ (calcd for C17H26O5Na 333.1672).
Biological Assays. Assay for activity against Plasmodium falciparum
(K1, multidrug-resistant strain) was performed using the microculture
radioisotope technique.19 Growth inhibitory activity against Mycobac-
terium tuberculosis H37Ra and cytotoxicity to Vero cells (African green
monkey kidney fibroblasts) were performed using the green fluorescent
protein microplate assay (GFPMA).20 Antifungal activity against
Candida albicans and anticancer activities against KB cells (oral human
epidermoid carcinoma), MCF-7 cells (human breast cancer), and NCI-
H187 cells (human small-cell lung cancer) were evaluated using the
resazurin microplate assay.21
(15) Mulzer, J.; Kattner, L. Angew. Chem., Int. Ed. Engl. 1990, 29, 679–
680.
(16) Nubbemeyer, U. J. Org. Chem. 1996, 61, 3677–3686.
(17) Chen, M.-J.; Narkunan, K.; Liu, R.-S. J. Org. Chem. 1999, 64, 8311–
8318.
(18) (a) Sharma, G. V. M.; Gopinath, T. Tetrahedron 2003, 59, 6521–
6530. (b) Sharma, G. V. M.; Gopinath, T. Tetrahedron Lett. 2001,
42, 6183–6186.
Acknowledgment. Financial support from the Bioresources Research
Network, National Center for Genetic Engineering and Biotechnology
(BIOTEC), is gratefully acknowledged.
Supporting Information Available: NMR spectra of 1-9 and
biological activities of 1-3, 6, and 7. This material is available free of
charge via the Internet at http://pubs.acs.org.
(19) Desjardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chulay, J. D.
Antimicrob. Agents Chemother. 1979, 16, 710–718.
(20) Changsen, C.; Franzblau, S. G.; Palittapongarnpim, P. Antimicrob.
Agents Chemother. 2003, 47, 3682–3687.
(21) O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. Eur. J. Biochem. 2000,
267, 5421–5426.
References and Notes
(1) Lin, Y.; Wu, X.; Feng, S.; Jiang, G.; Luo, J.; Zhou, S.; Vrijmoed,
L. L. P.; Jones, E. B. G.; Krohn, K.; Steingro¨ver, K.; Zsila, F. J. Org.
Chem. 2001, 66, 6252–6256.
(2) Lin, Y.; Wu, X.; Feng, S.; Jiang, G.; Zhou, S.; Vrijmoed, L. L. P.;
Jones, E. B. G. Tetrahedron Lett. 2001, 42, 449–451.
(3) Boonphong, S.; Kittakoop, P.; Isaka, M.; Pittayakhajonwut, D.;
Tanticharoen, M.; Thebtaranonth, Y. J. Nat. Prod. 2001, 66, 6252–
6256.
NP100030X