Bioactive compounds from the seed fungus Menisporopsis theobromae BCC 3975

Article abstract of DOI:10.1021/np0601197

Eight new compounds (2-9), together with a known dithiodiketopiperazine (1), were isolated from the seed fungus Menisporopsis theobromae BCC 3975. The structures of these substances were elucidated by analyses of spectroscopic data. Compounds 1 and 4 exhibited moderate cytotoxicity against BC-1 cell lines with IC₅₀ values of 29.2 and 57.4 μM, respectively. Cytotoxicity of 1, 2, 4, and 9 against the NCI-H187 cell line showed respective IC ₅₀ values of 22.9, 20.3, 1.8, and 56.6 μM. Compounds 2 and 4 exhibited antimalarial activity with IC₅₀ values of 2.95 and 28.8 μM, respectively. Substances 1, 4, 7, 8, and 9 possessed weak antimycobacterial activity (MIC 154.8-952.3 μM), while compounds 2 and 3 showed potent antimycobacterial activity with respective MIC values of 1.24 and 7.14 μM.

Full text of DOI:10.1021/np0601197

1404
J. Nat. Prod. 2006, 69, 1404-1410
Bioactive Compounds from the Seed Fungus Menisporopsis theobromae BCC 3975
Maneekarn Chinworrungsee,*,† Prasat Kittakoop,‡ Janya Saenboonrueng,‡ Palangpon Kongsaeree,† and
Yodhathai Thebtaranonth‡
Department of Chemistry, Faculty of Science, Mahidol UniVersity, Bangkok 10400, Thailand, and National Center for Genetic Engineering and
Biotechnology (BIOTEC), NSTDA, Thailand Science Park, 113 Paholyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand
ReceiVed March 15, 2006
Eight new compounds (2-9), together with a known dithiodiketopiperazine (1), were isolated from the seed fungus
Menisporopsis theobromae BCC 3975. The structures of these substances were elucidated by analyses of spectroscopic
data. Compounds 1 and 4 exhibited moderate cytotoxicity against BC-1 cell lines with IC50 values of 29.2 and 57.4 µM,
respectively. Cytotoxicity of 1, 2, 4, and 9 against the NCI-H187 cell line showed respective IC50 values of 22.9, 20.3,
1.8, and 56.6 µM. Compounds 2 and 4 exhibited antimalarial activity with IC50 values of 2.95 and 28.8 µM, respectively.
Substances 1, 4, 7, 8, and 9 possessed weak antimycobacterial activity (MIC 154.8-952.3 µM), while compounds 2
and 3 showed potent antimycobacterial activity with respective MIC values of 1.24 and 7.14 µM.
Our previous study on the seed fungus Menisporopsis theobro-
mae BCC 41621 implied that seed fungi may be a good source of
bioactive compounds. Here we report bioactive metabolites from
another strain (BCC 3975) of M. theobromae, which produced
various classes of interesting bioactive compounds. Preliminary
screening results revealed that a crude extract of the seed fungus
M. theobromae BCC 3975 exhibited antimycobacterial activity with
an MIC value of 1.24 µM. Chemical investigation of the fungus
M. theobromae BCC 3975 led to the isolation of a known
dithiodiketopiperazine (1),2 together with eight new metabolites (2-
9). Interestingly, the fungus M. theobromae BCC 3975 produced
different classes of compounds from different culture batches. We
report herein the isolation, structure elucidation, and biological
activities of compounds 1-9.
The 1H and 13C NMR data of compound 2 were generally similar
to those of compound 1, and extensive analyses revealed that
compound 2 was a derivative of 1. The molecular formula of
compound 2, C31H36N2O9S2, was deduced from the ESITOF mass
spectrum. Signals of sp3 methines (C-12 and C-12a) in 1 were
replaced by sp2 quaternary carbons in 2. On the basis of these data
and extensive analyses of 2D NMR data, compound 2 was a
derivative of 1, the cyclohexadiene unit in 1 being dehydrogenated
to an aromatic moiety. In a similar fashion to that of 1, proton and
carbon resonances in compound 2 were successfully assigned by
analyses of spectroscopic data. The stereochemistry of 2 was
assigned by analysis of the NOESY spectrum, in which a correlation
of H-7′ and H-4′, but none between H-7′ and H-11′ methyl protons,
was observed, indicative of a trans configuration of the R,â-
unsaturated ketone moiety. An intense cross-peak was observed
between the H-1′′′ methyl protons and the R-methylene proton (δH
3.35, H-15R), but none between the H-1′′′ methyl protons and the
â-methylene proton (δH 3.28, H-15â), implying that the H-1′′′
methyl protons and the R-methylene proton (δH 3.35, H-15R) were
cofacial. A cross-peak was observed between the H-1′′ methyl
protons and H-8 in the NOESY spectrum, suggesting a cis
relationship between the H-1′′ methyl protons and H-8. The
coupling constant of 8.2 Hz for JH-5,H-5a indicated that the dihedral
angle of H-5 and H-5a was close to 0° or 180°; however, a weak
cross-peak correlation on the NOESY spectrum implied a trans
relationship between H-5 and H-5a. Furthermore, compound 2 also
showed a negative specific rotation similar to that of compound 1
and other known fungal metabolites, bis-dethiodi(methylthio)-
acetylapoaranotin3 and bis-dethiodi(methylthio)acetylaranotin.3 Com-
pound 2 is, therefore, more likely to possess the same absolute
configuration as those of 1 and its derivatives.3
Results and Discussion
Compounds 1 and 2 were isolated from the first culture batch
of broth and mycelia of the seed fungus M. theobromae BCC 3975,
respectively. The structures of compounds 1 and 2 were very
complex; however, the amounts of 1 and 2 obtained from the first
culture batch were not sufficient for further studies. Therefore, we
tried to isolate these substances in the second culture batch.
Unfortunately, the fungus did not produce compounds 1 and 2 in
the second culture batch, but instead, it produced compounds 4-7.
In the third culture batch, six metabolites, compounds 1, 3, 5, 6, 8,
and 9, were isolated from the fungus extracts. Surprisingly, the
fungus again produced compound 1 in the third culture batch, but
only a small amount of compound 1 was obtained.
The structure of compound 1 was elucidated by analyses of 1H,
13C, DEPT, 1H-1H COSY, HMQC, and HMBC spectroscopic data
and finally confirmed by literature data comparison.2 The 1H NMR
spectrum of compound 1 in CDCl3 is identical to reported data2
(see Supporting Information). However, the 1H NMR of 1 in
acetone-d6 showed better signal separation, which is more conve-
nient to assign proton signals (see Experimental Section and
Supporting Information). Although compound 1 is a known fungal
metabolite, previously isolated from the fungus Ramichloridium
The molecular formula of compound 3, C20H22N2O5S2, was
determined by ESITOFMS. In general, 1H and 13C NMR data of 3
were similar to those of compound 1, and analyses of these data
revealed that compound 3 did not possess a 3-hydroxy-2,4,6-
trimethyl-5-oxo-oct-6-enoic acid side chain, but shared the same
diketopiperazine core structure. In addition, an oxygenated H-8
methine signal in 1 (δH 4.84) was replaced by a methylene group
1
schulzeri var. schulzeri F-2440 (FERM P-10187),2 its H and 13C
1
in 3 (δH 2.98 and 3.03). Analyses of H-1H COSY and HMBC
NMR data have not yet been assigned. We have completely
assigned proton and carbon resonances in 1 (see Experimental
Section).
spectra of 3, in combination with data comparison with those of 1
and 2, led to the assignment of proton and carbon resonances in 3.
In a similar fashion to those of 1 and 2, the stereochemistry of 3
was assigned by analysis of the NOESY spectrum. Furthermore,
compound 3 showed a negative specific rotation similar to that of
compounds 1 and 2, suggesting that they shared the same absolute
configuration.
* To whom correspondence should be addressed. Phone: +662-6641000,
ext. 8221. Fax: +662-2592097. E-mail: maneekarn@swu.ac.th.
† Mahidol University.
‡ National Center for Genetic Engineering and Biotechnology.
10.1021/np0601197 CCC: $33.50
© 2006 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/05/2006

BioactiVe Compounds from Menisporopsis theobromae
Journal of Natural Products, 2006, Vol. 69, No. 10 1405
Chart 1
The ESITOFMS of 4 established its molecular formula as
C18H14O4N2. The H NMR spectrum (CDCl3) showed signals of
derivative (4a), showing an OMe signal at δH 4.08. It should be
noted that the 7-OH group (δH 15.70) was not methylated and that
no significant changes of chemical shifts in 4a were observed when
comparing with those of 4. This information implied that the
methylation occurred at the N-OH group. Therefore, compound 4
contained two hydroxyl groups, i.e., 7-OH and N-OH. On the basis
of these data, together with spectral information from sclerominol
and flutimide,4-7 compound 4 was identified as 5-benzyl-1-hydroxy-
3-(hydroxyphenylmethylene)-3H-pyrazine-2,6-dione. Compound 4
was subsequently crystallized from MeOH, and its crystals were
subjected to X-ray crystallographic analysis, which readily con-
firmed the structure of 4 (Figure 1). Naturally occurring substances
with a 1-hydroxy-2,6-pyrazinedione skeleton are rare, and a few
examples are sclerominol and flutimide.4-7 Sclerominol showed
cytotoxicity against cancer cell lines, but demonstrated only mild
1
methylene protons at δH 4.18 (s) and a number of aromatic protons
at δH 7.36-8.19. The 13C NMR data (CDCl3) revealed 14
resonances. However, analyses of the 13C, DEPT, and HMQC
spectra of 4 indicated that it possessed 18 carbons with four sets
of overlapping signals, e.g., between C-9 and C-13, C-10 and C-12,
C-16 and C-20, and C-17 and C-19. The DEPT spectrum
demonstrated the presence of one methylene, 10 methine, and seven
quaternary carbons. Analyses of the NMR data revealed that
compound 4 consisted of a phenyl, a benzyl, and a central ring
system. The COSY spectrum demonstrated the connectivity from
H-9 to H-11 (or H-11 to H-13). The NMR signals of five protons
for each aromatic ring were assigned from the COSY spectrum,
indicating that 4 is bearing two monosubstituted aromatic rings.
Therefore, these 1-substituted aromatic rings should link to the
central core of compound 4. The HMBC spectrum of 4 exhibited
correlations from the benzylic protons (H-14) to the quaternary
carbons of the central moiety (C-5 and C-6) and to C-15 and C-16
(or C-20). The HMBC spectrum showed the correlations from H-9
(or H-13) to the deshielded oxygenated sp2 quaternary carbon (C-
7), implying that C-7 must be located next to the phenyl ring. The
downfield shift of 7-OH (δH 15.70) suggested that this OH was
hydrogen-bonded with the C-2 carbonyl, therefore supporting the
position of 7-OH in 4. A molecular formula of C18H14O4N2 and
the calculation of sites of unsaturation readily revealed that
compound 4 had 13 sites of unsaturation. The two aromatic rings
accounted for eight of these; therefore, the central part of compound
4 should contain five of these. The 13C NMR spectrum showed
four carbonyl-like signals at δC 152.7-180.3, implying the presence
of at least one ring and a double bond. Comparison of IR data of
compound 4 with those of sclerominol and flutimide4-7 revealed
that compound 4 may contain imide and CdN functionalities,
suggesting that the middle chromophore of 4 is 1-hydroxy-2,6-
pyrazinedione, as present in sclerominol and flutimide.4-7 Meth-
ylation of 4 with MeI/K2CO3 in DMF afforded a methylated
Figure 1. ORTEP plot of 5-benzyl-1-hydroxy-3-(hydroxyphenyl-
methylene)-3H-pyrazine-2,6-dione (4).

1406 Journal of Natural Products, 2006, Vol. 69, No. 10
Table 1. Biological Activities of Compounds 1-9
Chinworrungsee et al.
cytotoxicity (IC50, µM)
BC-1b
29.2
antimalaria
(IC50, µM)
antimycobacteria
(MIC, µM)
compound
KB a
>30.9
NCI-H187c
1
2
3
4
5
6
7
8
9
22.9
20.3
>46.0
1.8
>78.7
>30.3
>47.3
>49.0
56.6
1.59 ( 0.61
n ) 3
>30.9
2.95
>46.0
28.8
>78.7
>30.3
>47.3
>49.0
>95.2
154.8
1.24
7.14
310.5
>787.4
>303.0
473.9
245.1
952.3
>31.0
>46.0
>62.1
>78.7
>30.3
>47.3
>49.0
>95.2
5.41 ( 1.46
n ) 3
>31.0
>46.0
57.4
>78.7
>30.3
>47.3
>49.0
>95.2
5.93 ( 0.57
n ) 3
ellipticind
dihydroartemisinine
isoniazidef
0.004-0.014
0.29-0.66
3.43-8.58
kanamycin sulfatef
b
d
a Human epidermoid carcinoma in the mouth. Human breast cancer cells. c Human small cell lung cancer cells. Standard compound for cytotoxicity
e
f
assay. Standard antimalarial drug. Standard antimycobacterial drug.
activity against influenza virus strains, while flutimide was an
inhibitor of influenza virus endonuclease.4-7 In the present work,
compound 4 possessed antimalarial activity with an IC50 value of
28.8 µM and antimycobacterial activity with an MIC value of 310.5
µM (Table 1). Cytotoxicity of 4 against BC-1 (breast cancer) and
NCI-H187 (small cell lung cancer) cell lines was shown by IC50
values of 57.4 and 1.86 µM, respectively (Table 1).
Figure 2. ∆δ values [δ(-) - δ(+)] for the MTPA esters 6a and 6b.
The molecular formula C13H18O5 for compound 5 was determined
1
by ESITOFMS spectrum. The H NMR spectrum (CD3OD) of 5
spectrum of 6 exhibited long-range correlations of H-1 to C-3; H-2,
H-3, H-5, and H-6 to C-4; H-7 to C-5 and C-9; and H-8 to C-6
and C-1′. The J values of 15.5 and 2.2 Hz for JH-7,H-8 and JH-1′,H-2′
indicated trans geometry of a double bond and a trans-epoxide,
respectively. On the basis of these data, compound 6 was identified
as 9-hydroxy-9-(3-hydroxymethyloxiranyl)non-7-en-4-one. The ab-
solute configuration at C-9, C-1′, and C-2′ in 6 could not be
established on the basis of available spectroscopic data. However,
the absolute configuration at C-9 in compound 6 was addressed
through the use of Mosher esters.9,10 Both (R)- and (S)-MTPA esters
demonstrated an aldehydic proton resonance (δH 9.45), five olefinic
protons (δH 7.03, 6.87, 6.59, 6.44, and 6.30), four oxymethine
protons (δH 4.33, 3.43, 3.07, and 3.02), an oxygenated methylene
1
group (δH 3.78 and 3.49), and methyl protons (δH 1.85). The H
and 13C NMR spectra revealed the presence of an epoxide moiety,
showing deshielded resonances of oxygenated methine protons and
carbons at δH 3.02 and 3.07 (δC 57.4 and 57.7, respectively). The
13C NMR data revealed 13 resonances, which could be classified
by DEPT and HMQC spectra as 10 methine, one methylene, one
methyl, and one quaternary carbon. The 13C NMR spectrum showed
two sets of carbon signals, presumably indicating a mixture of
diastereoisomers. Analyses of the COSY spectrum of 5 led to the
assignments of the partial structures from H-3 along the chain to
H-10 and from H-1′ to H-3′. The HMBC spectrum showed
correlations of the aldehydic proton H-1 to C-1′, C-2, and C-3 and
from H-2′ to C-2, establishing the attachment of the aldehyde group
and the propenyl side chain to C-2 in 5. The HMBC spectrum also
displayed correlations of the methyl protons H-3′ to C-1′; H-3 to
C-1′; H-5 to C-3 and C-7; H-6 to C-4 and C-7; and H-8 to C-6.
The J values of 15.0 and 15.8 Hz for JH-4,H-5 and JH-1′,H-2′
indicated trans geometry of these double bonds, and the J value of
2.3 Hz for JH-8,H-9 suggested the presence of a trans-epoxide in
5.8 The NOESY correlation between H-1′ and H-3 revealed trans
geometry of the C-2/C-3 double bond. However, on the basis of
available data, the absolute configuration in 5 could not be
established. On the basis of these data, compound 5 was identified
as 6,7-dihydroxy-7-(3-hydroxymethyloxiranyl)-2-propenylhept-2,4-
dienal.
1
6a and 6b were separately prepared and subjected to H NMR
analysis. The ∆δ values [δ(-) - δ(+)] are shown in Figure 2,
indicating that the absolute configuration at C-9 of compound 6 is
S.
The molecular formula of C23H34O7 for compound 7 was deduced
from the ESITOFMS spectrum. The 1H NMR spectrum (CD3OD)
demonstrated resonances of an aldehydic proton (δH 10.21), two
aromatic protons (δH 7.46 and 6.81), four olefinic protons (δH 6.89,
5.78, 5.75, and 5.61), four oxymethine protons (δH 4.24, 4.10, 3.70,
and 3.45), eight methylene protons (δH 1.42-3.04), two methyl
singlets (δH 1.25 and 1.23), and a methyl doublet (δH 1.12). The
13C NMR spectrum exhibited 23 resonances, which were classified
by the DEPT spectra as 11 methine, four methylene, three methyl,
and five quaternary carbons. Analyses of the COSY spectrum
allowed the assignment of the partial structure from H-1′ to H-11′
and also showed couplings between H-4 and H-5 and between H-1′′
and H-2′′. An aldehydic proton correlated to C-1 and C-6 in the
HMBC spectrum. The HMBC correlations in 7 were also observed
from H-4 to C-2 and C-6; H-5 to C-1 and C-3; H-1′ to C-1, C-2,
and C-3′; H-2′ to C-2; H-1′′ to C-2, C-3, and C-4; and both H-4′′
and H-5′′ to C-2′′ and C-3′′. The J values of 16.1 and 15.6 Hz for
The ESITOFMS of 6 established a molecular formula of
C12H20O4. The 1H NMR spectrum (acetone-d6) of 6 showed signals
of two trans-olefinic protons (δH 5.74 and 5.53), three oxymethine
protons (δH 3.85, 2.97, and 2.83), methylene protons (δH 1.54-
3.73), and methyl protons (δH 0.86). Analyses of 13C and DEPT
spectra of 6 revealed the presence of five methine, five methylene,
one methyl, and one quaternary carbon. The deshielded carbon
J
H-1′,H-2′ and JH-5′,H-6′ indicated a trans geometry of these double
bonds. On the basis of these data, compound 7 was identified as
3-(2,3-dihydroxy-3-methylbutyl)-6-hydroxy-2-(3,4,10-trihydroxyun-
deca-1,5-dienyl)benzaldehyde. Due to the limited amount of the
material isolated, the absolute configuration of 7 could not be
established.
resonance at δC 209.3, together with the IR absorption at 1706 cm-1
,
indicated the presence of a carbonyl carbon. The HMQC spectrum
assisted in the assignment of protons attached to their corresponding
carbon, while the COSY spectrum demonstrated the connectivities
from H-1 to H-3 and from H-5 along the chain to H-3′. The HMBC
The ESITOFMS of 8 established the molecular formula as
C23H36O6. The 1H and 13C NMR spectra of 8 were similar to those
of 7, except that the aldehydic signal at δH 10.21 (δC 198.0) in
compound 7 was replaced by the oxygenated methylene resonances

BioactiVe Compounds from Menisporopsis theobromae
Journal of Natural Products, 2006, Vol. 69, No. 10 1407
cell lines with IC50 values of 29.2 and 57.4 µM, respectively.
Compounds 1, 2, 4, and 9 showed cytotoxicity against the NCI-
H187 cell line with respective IC50 values of 22.9, 20.3, 1.8, and
56.6 µM. Compounds 2 and 4 exhibited antimalarial activity with
IC50 values of 2.95 and 28.8 µM, respectively. Substances 1, 4, 7,
8, and 9 possessed weak antimycobacterial activity (MIC 154.8-
952.3 µM), while compounds 2 and 3 showed potent antimyco-
bacterial activity, with respective MIC values of 1.24 and 7.14 µM
(Table 1).
Figure 3. ∆δ values [δ(-) - δ(+)] for the MTPA esters 9a and 9b.
The seed fungus M. theobromae BCC 3975 produced different
compounds in different culture batches. Compounds 1-3 are
dithiodiketopiperazine, while compound 4 and compounds 5-9 are
pyrazinedione and polyketide, respectively. No morphological
changes of fungal culture were observed in each culture batch;
however, mutation at genetic levels of the fungus BCC 3975 could
not be evaluated at this stage. Although there is no clear explanation
why the fungus produced different classes of compound in different
cultures batches, this chemical investigation proved that seed fungi
are rich sources of bioactive compounds.
at δH 4.73 and 4.77 (δC 58.7) in compound 8. In addition, the
oxygenated methine signal at δH 3.70 (H-10′) in compound 7 was
absent in compound 8. In a similar fashion to that of 7, protons
and carbons in 8 were assigned by analyses of the COSY and
HMBC spectra. Key HMBC correlations are as follows: H-1′′ to
C-1, C-2, and C-3; H-4 to C-2, C-3, C-5, and C-6; H-5 to C-1 and
C-3; H-1′ to C-2 and C-3′; H-2′ to C-1; H-4′ and H-8′ to C-6′;
H-5′ to C-7′; H-10′ and H-11′ to C-9′; H-1′′′ to C-1, C-5, and C-6;
H-2′′′ to C-6, C-3′′′, and C-5′′′; and H-4′′′ and H-5′′′ to C-2′′′ and
C-3′′′. On the basis of these data, compound 8 was identified as
1-[6-(2,3-dihydroxy-3-methylbutyl)-3-hydroxy-2-hydroxymethylphe-
nyl]undeca-1,5-diene-3,4-diol, which was a reduced form of
compound 7. Due to the limited amount of the material isolated,
the absolute configuration of 8 could not be established.
Experimental Section
General Experimental Procedures. IR spectra and optical rotations
were measured on a Vector 22 (Bruker) spectrometer and Jasco P-1030
polarimeter, respectively. UV spectra were recorded on a Cary 1E UV-
vis spectrophotometer. 1H, 13C, 1H-1H COSY, HMQC, HMBC,
TOCSY, NOESY, and DEPT spectra were recorded on a Bruker
ADVANCE 500 D NMR spectrometer, operating at 500 MHz for
proton and 125 MHz for carbon. ESITOFMS were obtained from a
Micromass LCT mass spectrometer, and the lock mass calibration was
applied for the determination of the accurate mass.11
The ESITOFMS of 9 gave the molecular formula C10H10O5. The
1H NMR spectrum (acetone-d6) of 9 showed three aromatic protons
(δH 6.92, d, J ) 8.4 Hz; 7.07, d, J ) 7.4 Hz; and 7.59, dd, J ) 7.5
and 8.3 Hz) and three oxymethine protons (δH 4.06, dd, J ) 3.1
and 8.7 Hz; 4.59, d, J ) 8.9 Hz; and 4.98, d, J ) 3.0 Hz). The 13
C
NMR spectrum revealed 10 signals, and the DEPT spectrum
indicated the presence of six methine and four quaternary carbons.
The deshielded carbon resonance at δC 203.7, together with the IR
absorption at 1645 cm-1, suggested the presence of a carbonyl
carbon. The deshielded 8-OH resonance (δH 12.01) suggested that
this OH was hydrogen-bonded with a carbonyl, therefore supporting
the positions of 8-OH and a ketone. Analyses of the COSY spectrum
demonstrated the connectivities from H-2 to H-4 and from H-5 to
H-7. HMBC correlations were observed from H-4 to C-4a, C-5,
and C-8a; H-5 to C-7 and C-8a; H-6 to C-4a and C-8; H-7 to C-5,
C-8, and C-8a; and 8-OH to C-7 and C-8. On the basis of these
data, compound 9 was identified as 2,3,4,8-tetrahydroxy-3,4-
dihydro-2H-naphthalen-1-one. Analysis of the NOESY spectrum
provided little information concerning the relative configuration in
9 due to overlapping signals. However, the coupling constants of
8.9 and 3.0 Hz for JH-2,H-3 and JH-3,H-4 indicated that the dihedral
angle of H-2 and H-3 was close to 0° or 180° and that the dihedral
angle of H-3 and H-4 was close to 60° or 120°. The absolute
configuration of compound 9 was addressed through the use of
Mosher’s esters.9,10 Both (R)- and (S)-MTPA esters 9a and 9b were
separately prepared and subjected to 1H NMR analysis. The
deshielded methine protons of 9a at δH 6.69 (H-4) and 5.82 (H-2)
and those of 9b at δH 6.66 (H-4) and 5.79 (H-2) indicated that the
hydroxyl groups at C-4 and C-2 in 9 were esterified. The
ESITOFMS exhibited the molecular formula of 9a as C40H31O11F9-
Na [observed m/z 881.1635 (M + Na)+], similar to that of 9b
[observed m/z 881.1661 (M + Na)+]; therefore, esterification with
Mosher reagent provided tris-O-MTPA esters (compounds 9a and
9b) with ester linkages at C-2, C-4, and C-8. Moreover, the NOESY
spectrum of Mosher’s esters 9a and 9b revealed the correlation
between H-3 and H-4, but none between H-2 and H-3, implying
that H-3 and H-4 were cis oriented, while H-2 and H-3 adopted a
trans relationship. The ∆δ values [δ(-) - δ(+)] for 9a and 9b are
shown in Figure 3, indicating the S configuration at C-4. Therefore,
the absolute configuration at C-2 and C-3 in 9 were established as
R and S, respectively.
Fungal Material. The seed fungus M. theobromae BCC 3975 was
collected from Songkla Province, Thailand, and identified by Sayanh
Somrithipol. The culture was deposited at the BIOTEC Culture
Collection (registration no. BCC 3975), Thailand. The fungus was
grown in a potato dextrose broth and incubated for 9 days at 25 °C,
then transferred into 250 mL (1 L flask) of the same culture medium.
The culture was subsequently incubated (at 25 °C) for 24 days on a
rotary shaker (200 rpm) and harvested for further study.
Extraction and Isolation. Culture Batch 1. The culture (5 L) of
M. theobromae BCC 3975 was filtered to separate cells and broth. The
culture broth was extracted twice with an equal volume of EtOAc.
EtOAc layers were combined and evaporated to dryness, yielding 1.3
g of a crude extract. The crude EtOAc extract was purified by a
Sephadex LH-20 column (eluted with MeOH) to afford nine fractions
(A-I). Fraction D was rechromatographed on a Sephadex LH-20
column (MeOH as eluent) and further subjected to semipreparative
HPLC (C18 reversed-phase column and 50:50 MeCN-H2O as eluent)
to furnish compound 1 (35.6 mg).
Mycelia were macerated in MeOH for 2 days at room temperature.
The extract was evaporated, and H2O (100 mL) was added to the extract.
The mixture was washed with hexane (300 mL × 2). The aqueous
solution was then extracted twice with an equal volume of EtOAc.
The EtOAc layers were combined and evaporated to dryness, yielding
653.9 mg of a crude extract. The crude EtOAc extract was fractionated
using a Sephadex LH-20 column (eluted with MeOH) to provide eight
fractions (A-H), each with a volume of 100 mL. Fractions E and F
were combined and further rechromatographed on Sephadex LH-20
(eluted with MeOH) to furnish nine fractions (E1-E9). Fraction E8 was
purified by a semipreparative HPLC (C18 reversed-phase column and
50:50 MeCN-H2O as eluent) to yield compound 2 (0.7 mg).
Culture Batch 2. The culture (6 L) of M. theobromae BCC 3975
was filtered to separate cells and broth. The culture broth was extracted
twice with an equal volume of EtOAc. EtOAc layers were combined
and evaporated to dryness, yielding 2.2 g of a crude extract. The crude
EtOAc extract was subsequently chromatographed on a Sephadex LH-
20 column (MeOH as eluent) to provide nine fractions (A-I). Each
1
fraction was analyzed with H NMR technique; however, no trace of
compounds 1 and 2 was detected in any fraction.
Biological activities of compounds 1-9 are shown in Table 1.
Compounds 1 and 4 exhibited moderate cytotoxicity against BC-1
Fraction C was repeatedly purified on a Sephadex LH-20 column
(MeOH as eluent) and further purified by a semipreparative HPLC (C18

1408 Journal of Natural Products, 2006, Vol. 69, No. 10
Chinworrungsee et al.
reversed-phase column and 25:75 MeCN-H2O as eluent) to yield
3-(2,3-dihydroxy-3-methylbutyl)-6-hydroxy-2-(3,4,10-trihydroxyundeca-
1,5-dienyl)benzaldehyde (7) (1.5 mg).
H-1′′), 2.29 (3H, s, H-1′′′), 1.89 (3H, d, J ) 6.8 Hz, H-8′), 1.74 (3H,
br s, H-11′), 1.17 (3H, d, J ) 6.9 Hz, H-10′), 1.13 (3H, d, J ) 7.0 Hz,
H-9′); 13C NMR (acetone-d6, 125 MHz) δC 204.5 (1C, s, C-5′), 174.2
(1C, s, C-1′), 168.7 (1C, s, C-14), 163.0 (1C, s, C-7), 140.2 (1C, d,
C-3), 138.2 (1C, d, C-1), 138.3 (1C, d, C-7′), 137.7 (1C, s, C-6′), 135.6
(1C, s, C-8a), 132.3 (1C, d, C-11), 123.4 (1C, d, C-10), 121.5 (1C, d,
C-9), 111.1 (1C, s, C-15a), 106.4 (1C, d, C-4), 77.5 (1C, s, C-7a),
74.7 (1C, d, C-12), 74.6 (1C, d, C-8), 73.4 (1C, d, C-3′), 72.2 (1C, d,
C-5), 70.7 (1C, s, C-14a), 67.6 (1C, d, C-12a), 60.9 (1C, d, C-5a),
43.2 (1C, d, C-2′), 42.8 (1C, d, C-4′), 40.8 (1C, t, C-15), 14.6 (1C, q,
C-8′), 14.5 (1C, q, C-1′′′), 14.3 (1C, q, C-1′′), 14.2 (1C, q, C-10′),
11.2 (1C, q, C-9′), 11.0 (1C, q, C-11′); ESITOFMS m/z 669.1923 (M
+ Na)+, calcd for C31H38N2O9S2Na 669.1916.
Fraction E was rechromatographed on a Sephadex LH-20 column
and eluted with MeOH to provide 13 fractions (E1-E13), which were
further purified by a Sephadex LH-20 column and a semipreparative
HPLC (C18 reversed-phase column and 25:75 MeCN-H2O as eluent).
Separation of fraction E6 by a Sephadex LH-20 column (MeOH as
eluent) and a semipreparative HPLC (C18 reversed-phase column and
25:75 MeCN-H2O as eluent) yielded 12.5 mg of 9-hydroxy-9-(3-
hydroxymethyloxiranyl)non-7-en-4-one (6). Fraction E8 was subse-
quently subjected to a Sephadex LH-20 column (MeOH as eluent),
followed by a semipreparative HPLC (C18 reversed-phase column and
25:75 MeCN-H2O as eluent), to afford 6,7-dihydroxy-7-(3-hydroxym-
ethyloxiranyl)-2-propenylhepta-2,4-dienal (5) (3.8 mg). Crystallization
of fraction F from MeOH gave 5-benzyl-1-hydroxy-3-(hydroxyphe-
nylmethylene)-3H-pyrazine-2,6-dione (4) (22.4 mg).
Culture Batch 3. The culture (6 L) of M. theobromae BCC 3975
was filtered to separate cells and broth. The culture broth was extracted
twice with an equal volume of EtOAc. The EtOAc layers were
combined and evaporated to dryness, yielding 2.4 g of a crude extract.
The crude EtOAc extract was subjected to a Sephadex LH-20 column
and eluted with MeOH, yielding nine fractions (A-I). Fraction C was
rechromatographed on a Sephadex LH-20 column and eluted with
MeOH to give fractions C1-C10. Fractions C6 and C7 were combined
and further purified by a Sephadex LH-20 column (MeOH as eluent)
to give fractions C6,1-C6,9. Separation of fraction C6,5 by a semiprepara-
tive HPLC (C18 reversed-phase column and 25:75 MeCN-H2O as
eluent) yielded 4.35 mg of 1-[6-(2,3-dihydroxy-3-methylbutyl)-3-
hydroxy-2-hydroxymethylphenyl]undeca-1,5-diene-3,4-diol (8). Fraction
Compound 2: colorless needles (MeOH-acetone); [R]24 -237.9
D
(c 0.03, CHCl3); UV (MeOH) λmax (log ꢀ) 206 (4.3), 261 (3.6), and
293 (3.5) nm; 1H (acetone-d6, 500 MHz) δH 7.27 (1H, dd, J ) 7.4, 8.1
Hz, H-10), 7.11 (1H, d, J ) 7.5 Hz, H-9), 6.96 (1H, d, J ) 8.3 Hz,
H-11), 6.91 (1H, br q, J ) 6.3 Hz, H-7′), 6.84 (1H, br s, H-1), 6.44
(1H, dd, J ) 8.3, 2.3 Hz, H-3), 5.86 (1H, ddd, J ) 8.2, 2.0, 2.0 Hz,
H-5), 5.21 (1H, br d, J ) 8.6 Hz, H-5a), 5.15 (1H, br s, H-8), 4.78
(1H, dd, J ) 8.3, 1.9 Hz, H-4), 4.22 (1H, dd, J ) 6.3, 5.7 Hz, H-3′),
3.46 (1H, m, H-4′), 3.35 (1H, br d, J ) 15.5 Hz, H-15R), 3.28 (1H, br
d, J ) 15.5 Hz, H-15â), 2.56 (1H, m, H-2′), 2.38 (3H, s, H-1′′′), 2.29
(3H, s, H-1′′), 1.89 (3H, d, J ) 6.8 Hz, H-8′), 1.75 (3H, br s, H-11′),
1.18 (3H, d, J ) 6.9 Hz, H-10′), 1.14 (3H, d, J ) 7.0 Hz, H-9′); 13C
NMR (acetone-d6, 125 MHz) δC 204.5 (1C, s, C-5′), 174.3 (1C, s, C-1′),
168.7 (1C, s, C-14), 163.0 (1C, s, C-7), 148.5 (1C, s, C-12), 147.2
(1C, s, C-12a), 140.3 (1C, d, C-3), 138.6 (1C, d, C-1), 138.2 (1C, s,
C-7′), 137.7 (1C, s, C-6′), 135.5 (1C, s, C-8a), 129.8 (1C, d, C-10),
119.8 (1C, d, C-11), 118.1 (1C, d, C-9), 110.8 (1C, s, C-15a), 106.6
(1C, d, C-4), 77.5 (1C, s, C-7a), 76.0 (1C, d, C-8), 73.4 (1C, d, C-3′),
72.4 (1C, d, C-5), 70.5 (1C, s, C-14a), 61.2 (1C, d, C-5a), 43.3 (1C, d,
C-2′), 42.8 (1C, d, C-4′), 40.5 (1C, t, C-15), 14.6 (2C, q, C-8′, C-1′′),
14.4 (1C, q, C-1′′′), 14.3 (1C, q, C-10′), 11.2 (1C, q, C-9′), 11.0 (1C,
q, C-11′); ESITOFMS m/z 667.1752 (M + Na)+, calcd for C31H36N2O9S2-
Na 667.1760.
C6,6 was purified by a silica gel column (95:5 CH2Cl2-MeOH as eluent)
to provide 9-hydroxy-9-(3-hydroxymethyloxiranyl)non-7-en-4-one (6)
(12.6 mg). Fraction C8 was rechromatographed on a Sephadex LH-20
column (MeOH as eluent) and finally with a semipreparative HPLC
(C18 reversed-phase column and 25:75 MeCN-H2O as eluent), furnish-
ing 6,7-dihydroxy-7-(3-hydroxymethyloxiranyl)-2-propenylhepta-2,4-
dienal (5) (3.8 mg).
Compound 3: yellow solid; [R]24 -103.3 (c 0.19, CHCl3); UV
D
Fraction D was rechromatographed on a Sephadex LH-20 column
(MeOH as eluent) followed by a semipreparative HPLC (C18 reversed-
phase column and 25:75 MeCN-H2O as eluent) to give compound 1
(3.4 mg). Fraction G was rechromatographed on a Sephadex LH-20
column (MeOH as eluent) followed by a silica gel column (95:5 CH2-
Cl2-MeOH as eluent) to yield compound 3 (3.2 mg) and 2,3,4,8-
tetrahydroxy-3,4-dihydro-2H-naphthalen-1-one (9) (9.3 mg).
Bioassays. Antimalarial activity was evaluated against the parasite
Plasmodium falciparum (K1, multidrug-resistant strain), which was
cultured continuously according to the method of Trager and Jensen.12
Quantitative assessment of antimalarial activity in vitro was determined
by means of the microculture radioisotope technique based upon the
method described by Desjardins et al.13 The inhibitory concentration
(IC50) represents the concentration that causes 50% reduction in parasite
growth, as indicated by the in vitro uptake of [3H]hypoxanthine by P.
falciparum. An antimalarial drug, dihydroartemisinin, was used as a
positive control (see Table 1). The cytotoxicity of compound 1 was
determined employing the colorimetric method as described by Skehan
and co-workers.14 A standard compound, ellipticine, was used as a
positive control in the cytotoxicity assay (see Table 1). The antimy-
cobacterial activity was assessed against Mycobacterium tuberculosis
H37Ra using the microplate Alamar Blue assay (MABA).15 The
reference compounds for the antimycobacterial assay were isoniazid
and kanamycin sulfate (see Table 1).
(MeOH) λmax (log ꢀ) 204 (4.0), 260 (3.5), and 295 (3.4) nm; 1H NMR
(CDCl3, 500 MHz) δH 6.58 (1H, dd, J ) 2.1, 2.1 Hz, H-1), 6.25 (1H,
dd, J ) 7.7, 2.4 Hz, H-3), 5.99 (1H, m, H-9), 5.93 (1H, m, H-10), 5.79
(1H, br d, J ) 9.7 Hz, H-11), 4.95 (1H, dd, J ) 8.3, 2.0 Hz, H-4),
4.94 (1H, m, H-12), 4.89 (1H, m, H-12a), 4.87 (1H, m, H-5a), 4.71
(1H, ddd, J ) 7.7, 2.1, 2.1 Hz, H-5), 3.10 (1H, br d, J ) 15.2 Hz,
H-15R), 3.04 (1H, br d, J ) 15.2 Hz, H-15â), 3.03 (1H, ddd, J )
15.5, 1.9, 1.9 Hz, H-8R), 2.98 (1H, br d, J ) 15.3 Hz, H-8â), 2.31
(3H, s, H-1′′); 2.27 (3H, s, H-1′); 13C NMR (CDCl3, 125 MHz) δC
167.3 (1C, s, C-14), 166.7 (1C, s, C-7), 138.1 (1C, d, C-1), 138.0 (1C,
d, C-3), 131.2 (1C, s, C-8a), 130.6 (1C, d, C-11), 123.0 (1C, d, C-10),
120.7 (1C, d, C-9), 110.9 (1C, d, C-4), 107.8 (1C, s, C-15a), 74.5 (1C,
d, C-12), 73.5 (1C, s, C-7a), 72.7 (1C, d, C-5), 68.9 (2C, d, C-12a and
C-14a), 64.0 (1C, d, C-5a), 39.3 (1C, t, C-15), 38.7 (1C, t, C-8), 15.0
(1C, q, C-1′), 14.8 (1C, q, C-1′′); ESITOFMS m/z 457.0869 (M + Na)+,
calcd for C20H22N2O5S2Na 457.0868.
Compound 4: yellow needles (MeOH); mp 168-171 °C; UV
(MeOH) λmax (log ꢀ) 204 (4.6), 251 (4.2), and 365 (4.3) nm; IR (CHCl3
solution) νmax 2954, 2924, 2854, 1674, 1584, 1535, 1456, 1376, 1355,
1153, 761, 719, and 690 cm-1; 1H NMR (CDCl3, 500 MHz) δH 15.70
(1H, br s, 7-OH), 8.19 (2H, d, J ) 8.3 Hz, H-9 and H-13), 7.62 (1H,
t, J ) 7.4 Hz, H-11), 7.43 (2H, t, J ) 7.7 Hz, H-10 and H-12), 7.40
(1H, t, J ) 6.35 Hz, H-18), 7.39 (2H, t, J ) 6.7 Hz, H-17 and H-19),
7.36 (2H, d, J ) 7.6 Hz, H-16 and H-20), 4.18 (2H, s, H-14); 13C
NMR (CDCl3, 125 MHz) δC 180.3 (1C, s, C-7), 161.6 (1C, s, C-2),
152.7 (1C, s, C-6), 147.3 (1C, s, C-5), 136.4 (1C, s, C-15), 133.5 (1C,
d, C-11), 132.0 (2C, d, C-9 and C-13), 131.5 (1C, s, C-8), 129.8 (2C,
d, C-16 and C-20), 128.7 (2C, d, C-17 and C-19), 128.2 (2C, d, C-10
and C-12), 126.9 (1C, d, C-18), 115.5 (1C, s, C-3), 39.1 (1C, t, C-14);
ESITOFMS m/z 321.0865 (M - H)-, calcd for (C18H14O4N2-H)-
321.0876.
Compound 1: colorless needles (MeOH-acetone); mp 87-89 °C;
[R]24 -135.5 (c 0.13, CHCl3); UV (MeOH) λmax (log ꢀ) 205 (4.5),
D
224 (4.4), and 265 (3.8) nm; IR (CHCl3 solution) νmax 3425, 2927,
1
1728, 1642, 1392, 1191, and 1124 cm-1; H NMR (acetone-d6, 500
MHz) δH 6.90 (1H, br q, J ) 6.9 Hz, H-7′), 6.79 (1H, dd, J ) 2.1, 2.1
Hz, H-1), 6.42 (1H, dd, J ) 8.3, 2.3 Hz, H-3), 6.19 (1H, dd, J ) 3.2,
3.8 Hz, H-9), 5.94 (1H, ddd, J ) 9.8, 4.7, 2.7 Hz, H-10), 5.82 (1H,
ddd, J ) 8.1, 2.1, 2.1 Hz, H-5), 5.71 (1H, br d, J ) 9.8 Hz, H-11),
5.13 (1H, br d, J ) 8.0 Hz, H-5a), 5.08 (1H, dd, J ) 13.6, 2.1 Hz,
H-12a), 4.92 (1H, s, OH-12), 4.87 (1H, br d, J ) 13.7 Hz, H-12), 4.84
(1H, s, H-8), 4.74 (1H, dd, J ) 8.3, 1.9 Hz, H-4), 4.20 (1H, dd, J )
6.0, 11.6 Hz, H-3′), 4.08 (1H, d, 5.6 Hz, OH-3′), 3.45 (1H, dq, J )
6.9, 6.9 Hz, H-4′), 3.21 (1H, br d, J ) 15.6 Hz, H-15R), 3.04 (1H,
ddd, J ) 15.6, 2.0, 2.0 Hz, H-15â), 2.53 (1H, m, H-2′), 2.32 (3H, s,
Methylation of 5-Benzyl-1-hydroxy-3-(hydroxylphenylmethyl-
ene)-3H-pyrazine-2,6-dione (4). To a solution of 5-benzyl-1-hydroxy-
3-(hydroxylphenylmethylene)-3H-pyrazine-2,6-dione (4) (2.5 mg) in
DMF (1 mL) were added K2CO3 (10 mg) and MeI (0.3 mL), and the
mixture was left stirring at room temperature for 20 h. The mixture
was dried under vacuum, then dissolved in EtOAc (8 mL) and
subsequently washed with H2O (5 × 8 mL). A crude reaction mixture

BioactiVe Compounds from Menisporopsis theobromae
Journal of Natural Products, 2006, Vol. 69, No. 10 1409
was subjected to a Sephadex LH-20 column (eluted with MeOH) to
obtain the methylated derivative (4a) (1.9 mg): 1H NMR (CDCl3, 500
MHz) δH 8.09 (2H, d, J ) 8.5 Hz, H-9 and H-13), 7.55 (1H, t, J ) 7.4
Hz, H-11), 7.37 (4H, t, J ) 8.0 Hz, H-10, H-12, H-17, and H-19),
7.35 (1H, t, J ) 8.0 Hz, H-18), 7.33 (2H, d, J ) 7.5 Hz, H-16, and
H-20), 4.11 (2H, s, H-14), 4.09 (3H, s, OMe); 13C NMR (CDCl3, 125
MHz) δC 178.9 (1C, s, C-7), 164.9 (1C, s, C-2), 153.7 (1C, s, C-6),
149.7 (1C, s, C-5), 136.6 (1C, s, C-15), 135.1 (1C, s, C-8), 133.1 (1C,
d, C-11), 131.7 (2C, d, C-9, and C-13), 129.9 (2C, d, C-16 and C-20),
128.6 (2C, d, C-17 and C-19), 128.1 (2C, d, C-10 and C-12), 126.7
(1C, d, C-18), 111.0 (1C, s, C-3), 64.6 (1C, q, OMe), 39.1 (1C, t, C-14);
ESITOFMS m/z 337.1188 (M + H)+, calcd for (C19H16O4N2+H)+
337.1188.
EtOAc layer was dried, yielding the (S)-(-)-MTPA ester of 6 (ca. 2.5
mg). Preparation of the (R)-(+)-MTPA ester of 6 from (S)-(+)-R-
methoxy-R-trifluoromethylphenylacetyl chloride was conducted in the
same manner as that of the (S)-(-)-MTPA ester derivative. The S-(-
)-MTPA ester (6a) was obtained as a colorless oil: 1H NMR (CDCl3,
500 MHz) δH 7.52 (4H, m, aromatic protons of MTPA), 7.41 (6H, m,
aromatic protons of MTPA), 5.82 (1H, ddd, J ) 15.2, 6.7, and 6.7 Hz,
H-7), 5.40 (1H, br dd, J ) 15.4 and 7.3 Hz, H-8), 5.20 (1H, dd, J )
7.0 and 7.0 Hz, H-9), 4.61 (1H, dd, J ) 12.3 and 3.3 Hz, H-3′), 4.26
(1H, dd, J ) 12.3 and 6.8 Hz, H-3′), 3.59 (3H, s, OMe), 3.56 (3H, s,
OMe), 3.20 (1H, m, H-2′), 3.08 (1H, dd, J ) 6.9 and 2.0 Hz, H-1′),
2.45 (2H, t, J ) 7.3 Hz, H-5), 2.37 (2H, t, J ) 7.4 Hz, H-3), 2.30 (2H,
dt, J ) 7.0 and 7.0 Hz, H-6), 1.61 (2H, sext, J ) 7.4 Hz, H-2), and
0.92 (3H, t, J ) 7.4 Hz, H-1); ESITOFMS m/z 683.2076 (M + Na)+,
calcd for (C32H34O8F6Na)+ 683.2056. The R-(+)-MTPA ester (6b) was
obtained as a colorless oil: 1H NMR (CDCl3, 500 MHz) δH 7.52 (4H,
m, aromatic protons of MTPA), 7.41 (6H, m, aromatic protons of
MTPA), 5.93 (1H, ddd, J ) 15.2, 6.6, and 6.6 Hz, H-7), 5.53 (1H, br
dd, J ) 15.6 and 7.7 Hz, H-8), 5.32 (1H, dd, J ) 6.1 and 6.7 Hz,
H-9), 4.61 (1H, dd, J ) 12.4 and 3.2 Hz, H-3′), 4.20 (1H, dd, J ) 12.4
and 5.0 Hz, H-3′), 3.54 (6H, br s, OMe), 3.12 (1H, m, H-2′), 3.08 (1H,
dd, J ) 5.9 and 1.9 Hz, H-1′), 2.49 (2H, t, J ) 7.3 Hz, H-5), 2.37 (2H,
t, J ) 7.3 Hz, H-3), 2.35 (2H, dt, J ) 6.7 and 6.8 Hz, H-6), 1.60 (2H,
m, H-2), and 0.92 (3H, t, J ) 7.4 Hz, H-1); ESITOFMS m/z 683.2050
(M + Na)+, calcd for (C32H34O8F6Na)+ 683.2056.
X-ray Crystal Structure Analysis of Compound 4. Crystal data
for 4: C28H14N2O4‚CH3OH, MW 354.36, monoclinic, P21/c, a )
4.7417(11) Å, b ) 17.886(9) Å, c ) 20.692 (5) Å, â ) 94.847(10)°,
V ) 1748.6(11) Å,3 Dx ) 1.346 g/cm3, Z ) 4, F(000) ) 744. A total
of 11 101 reflections, of which 2050 were unique reflections (1614
observed, |Fo| > 4σ|Fo|), were measured at room temperature from a
0.25 × 0.15 × 0.10 mm3 yellow-orange crystal using graphite-
monochromated Mo KR radiation (λ ) 0.71073 Å) on a Bruker-Nonius
kappaCCD diffractometer. The crystal structure was solved by direct
methods using SIR-97, and then all atoms except hydrogen atoms were
refined anisotropically by full-matrix least-squares methods on F2 using
SHELXL-97 to give a final R-factor of 0.0530 (Rw ) 0.1156 for all
data). Crystallographic data of compound 4 have been deposited at the
Cambridge Crystallographic Data Centre under the reference number
CCDC 296815.
Compound 7: amorphous solid; [R]24D -53.6 (c 0.05, MeOH); UV
(MeOH) λmax (log ꢀ) 206 (4.3), 225 (4.2), 264 (3.8), and 349 (3.5) nm;
1H NMR (CD3OD, 500 MHz) δH 10.21 (1H, s, CHO), 7.46 (1H, d, J
) 8.6 Hz, H-4), 6.89 (1H, d, J ) 16.1 Hz, H-1′), 6.81 (1H, d, J ) 8.6
Hz, H-5), 5.78 (1H, dd, J ) 6.6, 15.3 Hz, H-6′), 5.75 (1H, dd, J ) 6.3,
16.0 Hz, H-2′), 5.61 (1H, dd, J ) 6.8, 15.6 Hz, H-5′), 4.24 (1H, dd, J
) 5.7, 5.1 Hz, H-3′), 4.10 (1H, dd, J ) 5.8, 5.7 Hz, H-4′), 3.70 (1H,
m, H-10′), 3.45 (1H, d, J ) 10.1 Hz, H-2′′), 3.04 (1H, br d, J ) 13.9
Hz, H-1′′), 2.50 (1H, dd, J ) 10.8, 14.0 Hz, H-1′′), 2.11 (2H, m, H-7′),
1.42 (4H, m, H-8′ and H-9′), 1.25 (3H, s, H-4′′), 1.23 (3H, s, H-5′′),
1.12 (3H, d, J ) 6.2 Hz, H-11′); 13C NMR (CD3OD, 125 MHz) δC
198.0 (1C, d, CHO), 161.0 (1C, s, C-6), 142.6 (1C, s, C-2), 140.0 (1C,
d, C-2′), 139.0 (1C, d, C-4), 133.1 (1C, d, C-6′), 129.8 (1C, s, C-3),
129.3 (1C, d, C-5′), 125.8 (1C, d, C-1′), 118.5 (1C, s, C-1), 115.2 (1C,
d, C-5), 78.5 (1C, d, C-2′′), 75.4 (1C, d, C-3′), 75.3 (1C, d, C-4′), 72.6
(1C, s, C-3′′), 67.0 (1C, d, C-10′), 38.2 (1C, t, C-9′), 33.7 (1C, t, C-1′′),
32.0 (1C, t, C-7′), 25.0 (1C, t, C-8′), 25.0 (1C, q, C-4′′), 23.1 (1C, q,
C-5′′), 22.1 (1C, q, C-11′); ESITOFMS m/z 421.2222 (M - H)-, calcd
for (C23H34O7-H)- 421.2227.
Compound 5: yellow solid; [R]24 +5.9 (c 0.16, MeOH); UV
D
(MeOH) λmax (log ꢀ) 206 (3.6), 225 (3.6), and 271 (3.3) nm; IR (KBr)
1
νmax 3406, 2927, 1716, 1635, 1454, 1384, 1261, and 1087 cm-1; H
and 13C NMR data of 5 exhibited 2 sets of signals, designated as
1
superscript a and b, respectively. H NMR (CD3OD, 500 MHz) δH
9.45a,b (1H, d, J ) 1.5 Hz, H-1), 7.04a (1H, ddd, J ) 15.0, 11.6, 1.5
Hz, H-4), 7.03b (1H, ddd, J ) 15.0, 11.6, 1.5 Hz, H-4), 6.88a (1H, d,
J ) 11.5 Hz, H-3), 6.87b (1H, d, J ) 11.4 Hz, H-3), 6.59b (1H, dq, J
) 16.0, 6.7 Hz, H-2′), 6.58a (1H, dq, J ) 16.7, 6.7 Hz, H-2′), 6.50a
(1H, dd, J ) 15.1, 5.4 Hz, H-5), 6.44b (1H, dd, J ) 15.0, 5.9 Hz, H-5),
6.30a,b (1H, dd, J ) 15.8, 1.4 Hz, H-1′), 4.33b (1H, dd, J ) 6.0, 6.0
Hz, H-6), 4.30a (1H, dd, J ) 7.1, 7.1 Hz, H-6), 3.80a (1H, dd, J )
12.9, 2.7 Hz, H-10), 3.78b (1H, dd, J ) 12.8, 2.7 Hz, H-10), 3.50a
(1H, dd, J ) 12.0, 6.0 Hz, H-10), 3.49b (1H, dd, J ) 12.6, 5.3 Hz,
H-10), 3.43b (1H, dd, J ) 5.6, 5.7 Hz, H-7), 3.40a (1H, dd, J ) 5.2,
6.5 Hz, H-7), 3.11a (1H, dd, J ) 5.0, 2.3 Hz, H-8), 3.09a (1H, dddd, J
) 5.1, 2.5, 2.5, 2.5 Hz, H-9), 3.07b (1H, dddd, J ) 5.2, 2.5, 2.5, 2.5
Hz, H-9), 3.02b (1H, dd, J ) 5.6, 2.3 Hz, H-8), 1.85a,b (1H, dd, J )
6.7, 1.6 Hz, H-3′); 13C NMR (CD3OD, 125 MHz) δC 196.3a,b (1C, d,
C-1), 148.8a (1C, d, C-3), 148.6b (1C, d, C-3), 145.7a (1C, d, C-5),
144.8b (1C, d, C-5), 137.4b (1C, s, C-2), 137.3a (1C, s, C-2), 134.6b
(1C, d, C-2′), 134.5a (1C, d, C-2′), 127.4b (1C, d, C-4), 127.0a (1C, d,
C-4), 122.2b (1C, d, C-1′), 122.2a (1C, d, C-1′), 75.5b (1C, d, C-7),
75.1a (1C, d, C-7), 74.8b (1C, d, C-6), 74.4a (1C, d, C-6), 62.7a (1C, t,
C-10), 62.5b (1C, t, C-10), 57.7b (1C, d, C-9), 57.6a (1C, d, C-9), 57.4b
(1C, d, C-8), 57.4a (1C, d, C-8), 19.6a,b (1C, q, C-3′); ESITOFMS m/z
277.1051 (M + Na)+, calcd for (C13H18O5+Na)+ 277.1052.
Compound 8: white needles (MeOH-CHCl3); mp 79-81 °C; [R]24
D
-14.7 (c 0.16, MeOH); UV (MeOH) λmax (log ꢀ) 206 (4.4) and 290
(3.5) nm; IR (KBr) νmax 3375, 2955, 2927, 2855, 1646, 1585, 1457,
1
1384, 1282, 1061, 1000, and 976 cm-1; H NMR (acetone-d6-D2O,
9:1, v/v, 500 MHz) δH 7.01 (1H, d, J ) 8.3 Hz, H-5), 6.67 (1H, d, J
) 16.3 Hz, H-1′), 6.66 (1H, d, J ) 8.2 Hz, H-4), 5.74 (1H, dd, J )
6.5, 16.3 Hz, H-2′), 5.72 (1H, ddd, J ) 1.0, 6.8, 14.3 Hz, H-6′), 5.61
(1H, ddd, J ) 1.3, 6.2, 16.8 Hz, H-5′), 4.77 (1H, d, J ) 12.3 Hz,
H-1′′), 4.73 (1H, d, J ) 12.3 Hz, H-1′′), 4.14 (1H, ddd, J ) 5.8, 5.2,
1.1 Hz, H-3′), 4.08 (1H, dd, J ) 5.9, 5.7 Hz, H-4′), 3.45 (1H, dd, J )
1.8, 10.2 Hz, H-2′′′), 2.97 (1H, dd, J ) 1.8, 13.9 Hz, H-1′′′), 2.41 (1H,
dd, J ) 10.2, 13.9 Hz, H-1′′′), 2.00 (2H, m, H-7′), 1.36 (2H, m, H-8′),
1.27 (4H, m, H-9′ and H-10′), 1.18 (3H, s, H-4′′′), 1.17 (3H, s, H-5′′′),
0.84 (2H, t, J ) 7.0 Hz, H-11′); 13C NMR (acetone-d6-D2O, 9:1, v/v,
125 MHz) δC 154.8 (1C, s, C-3), 138.3 (1C, s, C-1), 135.6 (1C, d,
C-2′), 132.1 (1C, d, C-6′), 130.3 (1C, d, C-5), 130.0 (1C, d, C-5′),
129.4 (1C, s, C-6), 128.6 (1C, d, C-1′), 124.0 (1C, s, C-2), 113.9 (1C,
d, C-4), 78.7 (1C, d, C-2′′′), 75.8 (1C, d, C-3′), 75.2 (1C, d, C-4′),
72.4 (1C, s, C-3′′′), 58.7 (1C, t, C-1′′), 34.8 (1C, t, C-1′′′), 32.2 (1C,
t, C-7′), 31.3 (1C, t, C-10′), 28.8 (1C, t, C-8′), 25.2 (1C, q, C-4′′′),
24.0 (1C, q, C-5′′′), 22.3 (1C, t, C-9′), 13.4 (1C, t, C-11′); ESITOFMS
m/z 407.2425 (M - H)-, calcd for (C23H36O6-H)- 407.2434.
Compound 6: colorless oil; [R]24D -1.8 (c 0.08, MeOH); IR (KBr)
νmax 3399, 3018, 2964, 2933, 2877, 1706, 1408, 1377, 1216, 1125,
1
1079, 972, 751, and 667 cm-1; H NMR (acetone-d6, 500 MHz) δH
5.74 (1H, dtd, J ) 15.5, 6.7, 1.3 Hz, H-7), 5.53 (1H, dddd, J ) 15.5,
5.8, 1.4, 1.4 Hz, H-8), 3.85 (1H, br s, H-9), 3.73 (1H, dd, J ) 2.1,
12.4 Hz, H-3′), 3.48 (1H, dd, 4.9, 12.4 Hz, H-3′), 2.97 (1H, ddd, J )
5.1, 2.2, 2.2 Hz, H-2′), 2.83 (1H, dd, J ) 2.2, 5.8 Hz, H-1′), 2.50 (2H,
t, J ) 7.4 Hz, H-5), 2.41 (2H, t, J ) 7.3 Hz, H-3), 2.26 (2H, dt, J )
6.8, 6.7 Hz, H-6), 1.54 (2H, sext, J ) 7.3 Hz, H-2), 0.86 (3H, t, J )
7.4 Hz, H-1); 13C NMR (acetone-d6, 125 MHz) δC 209.3 (1C, s, C-4),
131.1 (1C, d, C-7), 130.4 (1C, d, C-8), 72.6 (1C, d, C-9), 62.2 (1C, t,
C-3′), 59.1 (1C, d, C-1′), 56.5 (1C, d, C-2′), 44.6 (1C, t, C-3), 42.1
(1C, t, C-5), 26.8 (1C, t, C-6), 17.4 (1C, t, C-2), 13.6 (1C, q, C-1);
ESITOFMS m/z 251.1252 (M + Na)+, calcd for (C12H20O4+Na)+
251.1259.
Compound 9: brown solid; mp 79-81 °C; [R]24 +31.4 (c 0.24,
D
MeOH); UV (MeOH) λmax (log ꢀ) 214 (4.1), 258 (3.8), and 335 (3.4)
nm; IR (KBr) νmax 3417, 2924, 2854, 1645, 1458, 1291, 1229, and
1
1087 cm-1; H NMR (acetone-d6, 500 MHz) δH 12.01 (1H, s, 8-OH),
Preparation of MTPA Ester Derivatives of 6. A reaction mixture
consisting of 6 (ca. 2.3 mg), pyridine (300 µL), and (R)-(-)-R-methoxy-
R-trifluoromethylphenylacetyl chloride (40 µL) was left standing at
room temperature for 6 h. The mixture was dried under vacuum, then
dissolved in 5 mL of EtOAc and subsequently washed with H2O. The
7.59 (1H, dd, J ) 7.5, 8.3 Hz, H-6), 7.07 (1H, d, J ) 7.4 Hz, H-5),
6.92 (1H, d, J ) 8.4 Hz, H-7), 4.98 (1H, d, J ) 3.0 Hz, H-4), 4.59
(1H, d, J ) 8.9 Hz, H-2), 4.06 (1H, dd, J ) 3.1, 8.9 Hz, H-3); 13C
NMR (acetone-d6, 125 MHz) δC 203.7 (1C, s, C-1), 162.0 (1C, s, C-8),
143.5 (1C, s, C-4a), 137.1 (1C, d, C-6), 120.3 (1C, d, C-5), 116.9 (1C,

1410 Journal of Natural Products, 2006, Vol. 69, No. 10
Chinworrungsee et al.
d, C-7), 114.3 (1C, s, C-8a), 73.4 (1C, d, C-2), 73.1 (1C, d, C-3), 69.5
(1C, d, C-4); ESITOFMS m/z 233.0422 (M + Na)+, calcd for
(C10H10O5+Na)+ 233.0426.
References and Notes
(1) Chinworrungsee, M.; Kittakoop, P.; Isaka, M.; Maithip, P.; Supothina,
S.; Thebtaranonth, Y. J. Nat. Prod. 2004, 67, 689-692.
(2) Kawashima, A.; Yoshimura, Y.; Mizutani, T.; Hanada, K.; Omura,
S. Jpn. Kokai Tokkyo Koho 1990, Patent No. JP 2062880 (19900302).
(3) (a) Seya, H.; Nozawa, K.; Nakajima, S.; Kawai, K.; Udagawa, S. J.
Chem. Soc., Perkin Trans. 1 1986, 109-116. (b) Kawahara, N.;
Nozawa, K.; Nakajima, S.; Kawai, K.; Yamazaki, M. J. Chem. Soc.,
Chem. Commun. 1989, 951-952. (c) Kawahara, N.; Nozawa, K.;
Yamazaki, M.; Nakajima, S.; Kawai, K. Heterocycles 1990, 30, 507-
515. (d) Hegde, V. R.; Dai, P.; Patel, M.; Das, P. R.; Puar, M. S.
Tetrahedron Lett. 1997, 38, 911-914. (e) Neuss, N.; Nagarajan, R.;
Molloy, B. B.; Huckstep, L. L. Tetrahedron Lett. 1968, 9, 4467-
4471.
(4) Savard, M. E.; Melzer, M. S.; Boland, G. J.; Bensimon, C.; Blackwell,
B. A. J. Nat. Prod. 2003, 66, 306-309.
(5) Hensens, O. D.; Goetz, M. A.; Liesch, J. M.; Zink, D. L.; Raghoobar,
S. L.; Helms, G. L.; Singh, S. B. Tetrahedron Lett. 1995, 36, 2005-
2008.
(6) Singh, S. B. Tetrahedron Lett. 1995, 36, 2009-2012.
(7) Singh, S. B.; Tomassini, J. E. J. Org. Chem. 2001, 66, 5504-5516.
(8) Pretsch, E.; Bu¨hlmann, P.; Affolter, C. Structure Determination of
Organic Compounds: Tables of Spectral Data; Springer-Verlag:
Berlin, 2000; p 204.
Preparation of MTPA Ester Derivatives of 9. A mixture consisting
of 9 (ca. 1.2 mg), pyridine (300 µL), and (R)-(-)-R-methoxy-R-
trifluoromethylphenylacetyl chloride (40 µL) was left standing at room
temperature for 6 h. The mixture was dried under vacuum, then
dissolved in 5 mL of EtOAc and subsequently washed with H2O. The
EtOAc layer was dried, yielding the (S)-(-)-MTPA ester of 9 (ca. 1.3
mg). Preparation of the (R)-(+)-MTPA ester of 9 from (S)-(+)-R-
methoxy-R-trifluoromethylphenylacetyl chloride was conducted in the
same manner as that of the (S)-(-)-MTPA ester derivative. The S-(-
)-MTPA ester (9a) was obtained as a colorless oil: 1H NMR (CDCl3,
500 MHz) δH 7.71 (1H, dd, J ) 7.9 and 8.0 Hz, H-6), 7.58 (1H, br d,
J ) 7.7 Hz, H-5), 7.44 (6H, m, aromatic protons of MTPA), 7.37 (9H,
m, aromatic protons of MTPA), 7.18 (1H, dd, J ) 7.9 and 1.0 Hz,
H-7), 6.69 (1H, d, J ) 3.5 Hz, H-4), 5.82 (1H, d, J ) 11.1 Hz, H-2),
4.68 (1H, m, H-3), 3.65 (3H, s, OMe), 3.57 (3H, s, OMe), and 3.56
(3H, s, OMe); ESITOFMS m/z 881.1635 (M + Na)+, calcd for
(C40H31O11F9Na)+ 881.1620. The R-(+)-MTPA ester (9b) was obtained
as a colorless oil: 1H NMR (CDCl3, 500 MHz) δH 7.75 (1H, dd, J )
7.9 and 7.9 Hz, H-6), 7.63 (1H, br d, J ) 7.9 Hz, H-5), 7.46 (6H, m,
aromatic protons of MTPA), 7.39 (9H, m, aromatic protons of MTPA),
7.18 (1H, dd, J ) 7.8 and 1.1 Hz, H-7), 6.66 (1H, d, J ) 3.4 Hz, H-4),
5.79 (1H, d, J ) 11.1 Hz, H-2), 4.46 (1H, m, H-3), 3.77 (3H, s, OMe),
3.68 (3H, s, OMe), and 3.44 (3H, s, OMe); ESITOFMS m/z 881.1661
(M + Na)+, calcd for (C40H31O11F9Na)+ 881.1620.
(9) Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1973, 95, 512-519.
(10) Sullivan, G. R.; Dale, J. A.; Mosher, H. S. J. Org. Chem. 1973, 38,
2143-2147.
(11) Eckers, C.; Wolff, J. C.; Haskins, N. J.; Sage, A. B.; Giles, K.;
Bateman, R. Anal. Chem. 2000, 72, 3683-3688.
Acknowledgment. M.C. acknowledges the Thailand Graduate
Institute of Science and Technology (TGIST) for the student’s grant,
and P.K. thanks The Thailand Research Fund (TRF) for financial
support. Bioresources Research Network, National Center for Genetic
Engineering and Biotechnology, is gratefully acknowledged.
(12) Trager, W.; Jensen, J. B. Science 1976, 193, 673-675.
(13) Desjardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chulay, J. D.
Antimicrob. Agents Chemother. 1979, 16, 710-718.
(14) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.;
Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J.
Natl. Cancer Inst. 1990, 82, 1107-1112.
(15) Collins, L.; Franzblau, S. G. Antimicrob. Agents Chemother. 1997,
41, 1004-1009.
Supporting Information Available: 1H and 13C NMR spectra of
compounds 1-9. This material is available free of charge via the
Internet at http://pubs.acs.org.
NP0601197