Antimalarial and cytotoxic depsidones from the fungus Chaetomium brasiliense

Article abstract of DOI:10.1021/np9003189

Four new depsidones, mollicellins K-N (1-4), and six known depsidones, mollicellins B (5), C (6), E (7), F (8), H (9), and J (10), along with two known sterols were isolated from the fungus Chaetomium brasiliense. Their structures were elucidated on the b

Full text of DOI:10.1021/np9003189

J. Nat. Prod. 2009, 72, 1487–1491
1487
Antimalarial and Cytotoxic Depsidones from the Fungus Chaetomium brasiliense
Primmala Khumkomkhet,† Somdej Kanokmedhakul,*,† Kwanjai Kanokmedhakul,† Chariya Hahnvajanawong,‡ and
Kasem Soytong§
Natural Products Research Unit, Department of Chemistry and Center for InnoVation in Chemistry, Faculty of Science, Khon Kaen UniVersity,
Khon Kaen 40002, Thailand, Department of Microbiology, and LiVer Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine,
Khon Kaen UniVersity, Khon Kaen 40002, Thailand, and Department of Plant Pest Management, Faculty of Agricultural Technology, King
Mongkut’s Institute of Technology Ladkrabang, Ladkrabang, Bangkok 10520, Thailand
ReceiVed May 26, 2009
Four new depsidones, mollicellins K-N (1-4), and six known depsidones, mollicellins B (5), C (6), E (7), F (8), H (9),
and J (10), along with two known sterols were isolated from the fungus Chaetomium brasiliense. Their structures were
elucidated on the basis of 1D and 2D NMR spectroscopic data and chemical transformation. Among these isolates,
1-3, 5-7, and 10 exhibited antimalarial activity against Plasmodium falciparum. Only 1 exhibited antimycobacterial
activity against Mycobacterium tuberculosis and antifungal activity against Candida albicans using in vitro assays. In
addition, 1-10 showed cytotoxicity against the KB, BC1, NCI-H187, and five cholangiocarcinoma cell lines.
The fungus Chaetomium brasiliense is one of ca. 22 Chaetomium
species that have been found in Thailand.1,2 Previous investigations
on secondary metabolites from Chaetomium species resulted in the
isolation of compounds such as benzoquinone derivatives,3 tetra-S-
methyl derivatives,4 azaphilones,5-7 bis-azaphilones7 indol-3-yl-
[13]cytochalasans, and chaetoglobosin analogues.8-14 In addition,
anthraquinone-chromanone,13 orsellinic acid, and globosumones15 have
also been reported. Chaetomium brasiliense16 has been reported to
produce chaetochalasin A17 and four depsidones, mollicellins D, H, I,
and J.18 As part of our work on bioactive constituents from Chaeto-
mium species, we noted that hexane and EtOAc extracts of C.
brasiliense, a strain isolated from Thai soil, showed in vitro antimalarial
activity against Plasmodium falciparum (IC50 3.0 and 2.9 µg/mL,
respectively), and the EtOAc extract also showed antimycobacterial
activity against Mycobacterium tuberculosis (MIC 50 µg/mL). We
report herein the isolation, structural characterization, and bioactivity
of four new depsidones (1-4), six known depsidones (5-10), and
two known sterols from C. brasiliense.
Results and Discussion
Depsidones 1-10 and two sterols were isolated as solids from
hexane, EtOAc, and MeOH extracts of dried mycelial mat of C.
brasiliense using a combination of silica gel column chromatog-
raphy (CC) and preparative TLC. Structures of the known com-
methine (including an aldehyde group), and four methyl carbons.
pounds were identified by physical and spectroscopic data mea-
The 13C NMR data together with the degrees of unsaturation
surements (IR, 1H and 13C NMR, 2D NMR, and MS) and by
revealed that 1 contained two aromatic rings in the molecule. The
comparing the data obtained with published values, as 24(R)-5R,8R-
IR absorption band at 1731 cm-1 and the 13C NMR resonance signal
epidioxyergosta-6,22-diene-3ꢀ-ol,17 ergosterol,19 and mollicellins
at δ 163.7 suggested the presence of a conjugated carbonyl ester
B, C, E, F (5-8),20 H and J (9 and 10).18 The details of physical
group.18,21 The 1H NMR data showed two singlet signals of
properties and spectroscopic data of mollicellins B, C, E, and F
aromatic protons at δ 6.72 (H-2) and 6.69 (H-6), as well as two
(5-8)20 are also presented, since they have not yet been reported.
aromatic methyl substituents at δ 2.51 (H3-1′) and 2.53 (H3-8′).
Compound 1 was obtained as a white solid, and its molecular
The low-field singlet signal (δ 12.06) was assigned as a chelated
formula, C21H18O7, was deduced from the HRESITOFMS (observed
OH involving the carbonyl of an aldehyde group (δ 10.53) at the
m/z 383.1161 [M + H]+), indicating 13 degrees of unsaturation.
ortho position. The 1H and 13C NMR spectroscopic data of 1 were
The IR spectrum showed the presence of OH (3407 cm-1), carbonyl
comparable to an analogue, mollicellin H (9),18 except for the prenyl
ester (1731 cm-1), aromatic aldehyde (1656 cm-1), R,ꢀ-unsaturated
group at C-8, which was replaced by a 1-oxo-3-methylbut-2-enyl
ketone (1638 cm-1), and aromatic (1594 cm-1) groups. The 13C
1
moiety. This unit was deduced from the H and 13C NMR signals
NMR and DEPT spectra (Table 1) indicated 21 signals attributable
at δH 6.31 (s, H-4′), 2.23 (s, Me-6′), and 2.01 (s, Me-7′) and δC
to 13 sp2 quaternary (including two carbonyl groups), four sp2
196.0 (C-3′), 126.0 (C-4′), 158.3 (H-5′), 21.5 (Me-6′), and 28.1
(Me-7′), and it was located at C-8 on the basis of the HMBC
* To whom correspondence should be addressed. Tel: +66-43-202222-
correlations of H-8′ to C-8, C-9, C-9a, and C-3′, and H-6 to C-5a,
41, ext. 12243. Fax: +66-43-202373. E-mail: somdej@kku.ac.th.
C-7, C-8, C-9a, and C-3′. The HMBC spectrum of 1 also showed
correlations of H-1′ to C-1, C-2, C-11, C-4a, and C-11a; H-2 to
C-3, C-4, C-11a, C-1′, C-4a, and C-11; the OH proton at C-3 to
† Department of Chemistry, Khon Kaen University.
‡ Department of Microbiology, Khon Kaen University.
§ King Mongkut’s Institute of Technology Ladkrabang.
10.1021/np9003189 CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 08/07/2009

1488 Journal of Natural Products, 2009, Vol. 72, No. 8
Khumkomkhet et al.
Table 1. 1H NMR Data (δ, ppm) of Compounds 1-8 in CDCl3
position
1
2
3
4
5
6
7
8
2
6
6.72 (s)
6.69 (s)
2.51 (s)
10.53 (s)
6.31 (s)
2.23 (s)
2.01 (s)
2.53 (s)
12.06 (s)
10.88 (s)
6.71 (s)
6.57 (s)
2.52 (s)
10.60 (s)
6.19 (s)
2.20 (s)
1.93 (s)
2.23 (s)
12.01 (s)
6.72 (s)
6.71 (s)
6.66 (s)
2.52 (s)
10.53 (s)
2.67 (s)
1.41 (s)
1.41 (s)
2.66 (s)
12.03 (s)
6.71 (s)
6.66 (s)
2.60 (s)
10.54 (s)
2.68 (s)
1.41 (s)
1.4 1 (s)
2.67 (s)
12.68 (s)
1′
2.53 (s)
10.85 (s)
2.74 (s)
1.47 (s)
1.47 (s)
2.59 (s)
12.19 (s)
2.51 (s)
10.83 (s)
6.25 (s)
2.23 (s)
1.96 (s)
2.17 (s)
12.17 (s)
2.57 (s)
10.81 (s)
6.24 (s)
2.23 (s)
1.96 (s)
2.16 (s)
12.75 (s)
2.53 (s)
10.78 (s)
2.68 (s)
1.40 (s)
1.40 (s)
2.52 (s)
12.77 (s)
2′
4′
6′
7′
8′
OH-3
OH-7
OMe-7
OMe-8
OH-9
3.76 (s)
3.73 (s)
5.84 (s)
3.72 (s)
5.67 (s)
5.64 (s)
Table 2. 13C NMR Data (δ, ppm) of Compounds 1-8 in CDCl3
position
1
2
3
4
5
6
7
8
1
2
3
4
4a
5a
6
7
8
9
153.4
117.8
165.3
110.7
161.6
153.5
106.8
158.3
122.3
131.1
135.8
163.7
112.5
22.8
154.3
118.2
165.7
111.2
162.3
151.0
101.6
153.8
129.4
131.8
137.1
165.2
113.3
22.8
193.3
194.6
126.0
158.1
21.6
28.5
13.5
56.8
150.3
121.1
161.0
110.6
161.2
154.7
107.5
158.6
117.2
134.4
136.5
161.3
114.0
19.7
153.2
117.9
165.3
111.0
163.7
137.5
122.6
115.8
146.2
142.0
135.4
161.5
112.6
22.1
153.6
117.9
165.2
110.7
161.5
154.7
107.5
158.6
117.0
134.3
136.7
163.5
112.5
22.2
153.1
117.7
165.2
110.9
161.4
140.2
134.0
117.5
141.2
139.2
138.7
164.5
112.7
22.1
149.8
121.3
161.2
110.8
160.9
140.0
134.2
117.4
141.2
139.3
138.8
162.2
114.2
19.5
149.8
121.1
161.0
110.9
161.3
137.3
122.6
115.9
146.2
142.0
135.4
161.4
114.1
19.5
9a
11
11a
1′
2′
192.6
196.0
126.0
158.6
21.5
192.5
192.5
50.1
79.5
26.3
195.4
192.0
50.4
80.9
26.4
192.6
192.5
50.1
79.3
14.2
193.4
195.4
125.2
159.1
21.1
193.3
195.2
125.1
159.3
21.2
195.1
191.9
50.4
81.1
26.4
3′
4′
5′
6′
7′
28.1
16.3
26.3
14.2
26.4
13.1
14.2
26.3
28.0
12.1
28.0
12.1
26.4
13.0
8′
OMe-7
OMe-863.1
63.1
63.1
C-2, C-3, and C-4; H-6 to C-5a, C-7, C-8, and C-9a; the aldehyde
proton (H-2′) to C-3 and C-4; the OH proton at C-7 to C-6, C-7,
and C-8; H-4′ to C-3′, C-6′, and C-7′; H-6′ to C-4′, C-5′, C-7′, and
C-3′; and H-7′ to C-4′, C-5′, and C-6′ to confirm the connectivity
H-6, H-6, and the OCH3 protons at C-7 to support the structure of
2. Thus, 2 was defined as a new depsidone and has been named
mollicellin L.
Compound 3 was obtained as a white solid, C21H17ClO7, as
deduced from HRESITOFMS (observed m/z 439.0561[M + Na]+
and its 37Cl isotope m/z 441.0601 [M + 2 + Na]+), implying 13
degrees of unsaturation. The IR spectrum of 3 showed character-
istics of OH (3454 cm-1), ester carbonyl (1736 cm-1), aromatic
aldehyde (1688 cm-1), conjugated ketone (1652 cm-1), and aromatic
(1600 cm-1) groups. The 13C NMR and DEPT spectra revealed 21
signals attributable to 13 sp2 quaternary (including two carbonyl
groups), one sp3 quaternary, two sp2 methine (including an aldehyde
group), one sp3 methylene, and four methyl carbons. From these
data, 3 contained two aromatic rings and a conjugated carbonyl
3
in the molecule (Figure 1). Surprisingly, the J correlation of the
aldehyde proton (H-2′) to C-4a was not observed in the HMBC
experiment. Thus, the chemical shift of C-4a was then identified
by comparison with those reported for the analogues mollicellins I
and J18 and also from the J correlations of H-2 and H-1′ in the
4
HMBC spectrum. In addition, the NOESY spectrum of 1 demon-
strated correlations between aldehyde proton H-2′ and H-6, between
H-1′ and H-2, and from H-4′ and H-8′ to H-7′ (Figure 1). Chemical
transformation of 1 by cyclization with MeOH in the presence of
p-toluenesulfonic acid yielded the product that was identical (mp,
IR, NMR, and behavior on TLC) to natural mollicellin B (5) (Tables
1 and 2). On the basis of the above data, the structure of 1 was
defined as a new depsidone and has been named mollicellin K.
Compound 2 was obtained as a white solid, and its molecular
formula, C22H20O7, was deduced from HRESITOFMS (observed
m/z 397.1286 [M + H]+), indicating 13 degrees of unsaturation.
The IR spectrum showed the presence of OH (3439 cm-1), carbonyl
ester (1729 cm-1), aromatic aldehyde (1677 cm-1), R,ꢀ-unsaturated
ketone (1644 cm-1), and aromatic (1608 cm-1) groups. The 1H and
13C NMR spectra of 2 were similar to those of 1, except for the
OH group at C-7, which was substituted by an OCH3 group (δH
3.76, δC 56.8). The complete assignments of the 1H and 13C NMR
signals of 2 were established from the DEPT, COSY, HSQC,
HMBC, and NOESY data (Tables 1 and 2). The NOESY spectrum
of 2 showed correlations between an aldehyde proton (H-2′) and
1
ester as described in 1. The H NMR spectrum of 3 (Table 1)
showed only one aromatic singlet signal at δ 6.66 (H-6) and two
aromatic methyl substituents at δ 2.60 (H-1′) and 2.68 (H-8′), which
were different from 1. The 1-oxo-3-methylbut-2-enyl moiety at C-8
Figure 1. Key HMBC 2J and 3J correlations (HfC) and 4J
correlations (HfC, dashed arrows) and NOESY correlations of
mollicellin K (1).

Antimalarial Depsidones from Chaetomium brasiliense
Journal of Natural Products, 2009, Vol. 72, No. 8 1489
Table 3. Biological Activities of the Isolated Compounds
antimalarial
anti-TB
antifungal
cytotoxicity (IC50, µg/mL)
BC1b NCI-H187c
6.8
compound
(IC50, µg/mL)
(MIC, µg/mL)
(IC50, µg/mL)
KBa
1.9
33.9
Inactive
25.9
37.1
46.3
nd
1
2
3
4
5
6
7
8
9
10
1.2
3.4
2.9
inactive
4.7
9.1
3.2
inactive
nd
4.9
12.5
1.2
0.35
9.5
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
inactive
49.9
39.7
nd
nd
nd
nd
inactive
nd
nd
inactive
nd
0.68
13.5
14.7
3.1
1.0
37.9
16.6
29.1
nd
nd
13.1
3.9
nd
23.3
artmisinin
0.001
isoniazid
0.05
2.5
kanamycin sulfate
amphotericin B
ellipticine
0.034
0.36
0.26
0.32
a Human epidermoid carcinoma of the mouth. b Human breast cancer. c Human small cell lung cancer. nd ) not determined. Inactive at >50 µg/mL.
Table 4. Biological Activities of the Isolated Compounds
in 1 was replaced by a dihydropyrone ring fused to an aromatic
against Cholangiocarcinoma
ring, as shown by the signals of two geminal methyl groups both
cytotoxicity (IC50, µg/mL)
compound KKU-100a KKU-M139b KKU-M156c KKU-M213d KKU-M214e
at δ 1.41 (H-6′ and H-7′) and one methylene group at δ 2.67 (s,
H-4′). The low-field singlet signal at δ 12.68 was assigned as an
OH chelated to an aldehyde carbonyl group (δ 10.54) at the ortho
position as in 1. The structure of 3 was constructed by a combination
of 2D NMR analyses. The HMBC spectrum demonstrated correla-
tions of H-1′ to C-1, C-2, C-11, C-4a, and C-11a; the OH proton
at C-3 to C-2, C-3, and C-4; H-6 to C-5a, C-7, C-8, and C-9a; the
aldehyde proton (H-2′) to C-3 and C-4; H-4′ to C-8, C-3′, C-5′,
C-6′, and C-7′; H-6′ to C-4′, C-5′, and C-7′; H-7′ to C-4′, C-5′,
and C-6′; and H-8′ to C-7, C-5a, C-8, C-9, C-3′, and C-9a (Figure
1
3
4
5
6
7
8
4.46 ( 0.08 13.94 ( 2.87 6.65 ( 1.15
nd
4.92 ( 0.26
6.28 ( 0.94 8.55 ( 3.20 6.04 ( 0.15 13.19 ( 1.17 4.51 ( 0.18
6.46 ( 1.24 4.34 ( 0.08 2.90 ( 0.07 3.00 ( 0.36 5.05 ( 0.50
4.63 ( 0.01 11.66 ( 2.70 15.66 ( 0.88 6.94 ( 1.02 4.50 ( 0.39
5.12 ( 0.17 2.51 ( 0.36 4.18 ( 0.11 3.2 ( 0.52 3.35 ( 0.04
4.83 ( 0.05 5.03 ( 0.16 5.22 ( 0.08 4.44 ( 0.05 7.81 ( 1.13
5.21 ( 0.04 5.21 ( 0.04 5.36 ( 0.15 4.98 ( 0.05 4.40 ( 0.02
ellipticine 7.11 ( 0.09 1.21 ( 0.03 2.02 ( 0.11 0.30 ( 0.001 0.21 ( 0.04
a Poorly differentiated adenocarcinoma. b Squamous carcinoma. c Mod-
erately differentiated adenocarcinoma. d Adenosquamous carcinoma. e Mod-
erately differentiated denocarcinoma. nd ) not determined.
2). In addition, the Cl-C(2) was determined by comparing its 13
C
NMR spectrum with those reported for mollicellins J,18 K (1), and
B (5), as well as the HMBC correlation of H-1′ to C-2 (δC 121.1).
The NOESY spectrum of 3 also supported the structure via the
correlations between H-2′ and H-6 and between H-4′ and H-6′
and H-7′ (Figure 3) On the basis of the above data, the structure
of 3 was defined as a new depsidone, and it has been named
mollicellin M.
(1644 cm-1), and aromatic (1574 cm-1) groups. The 13C NMR and
DEPT spectra revealed 21 signals attributable to 13 sp2 quaternary
(including two carbonyl groups), one sp3 quaternary, two sp2
methine (including an aldehyde group), one sp3 methylene, and four
methyl carbons. The H and 13C NMR spectra of 4 (Tables 1 and
1
2) showed splitting patterns similar to those of 3 with four singlet
methyl (δH 2.53, 2.59, 1.47, and 1.47), one methylene (δH 2.74),
and two methine groups (δH 6.72 and 10.85). However, groups
substituted on the aromatic ring and chromone units of 4 were
located at different positions than in 3. The HMBC spectrum
exhibited correlations of H-1′ to C-1, C-2, C-4a, C-11, and C-11a;
H-2 to C-1, C-3, C-4, C-4a, and C-11a; the OH group at C-3 to
C-2, C-3, and C-4; the OH group at C-9 to C-8, C-5a, and C-9a;
aldehyde proton H-2′ to C-3 and C-4; H-4′ to C-7, C-3′, C-5′, C-6′,
and C-7′; H-6′ to C-4′, C-5′, and C-7′; and H-7′ to C-4′, C-5′, and
C-6′, confirming the structure of 4 (Figure 3). The NOESY spectrum
of 4 showed correlations between H-2 and H-1′ and between H-4′
and H-6′ and H-7′. Unfortunately, the correlation between aldehyde
proton H-2′ and C3-8′ was not observed and the NOE-difference
data showed the same correlations of protons as in the NOESY
experiment (Figure 3). This suggested that the distance between
the aldehyde proton (H-2′) and C3-8′ is more than 4.2 Å.22 On the
basis of the above evidence, compound 4 was determined to be a
new depsidone, and it was named mollicellin N.
Compound 4 was obtained as a white solid, and its molecular
formula, C21H18O8, was deduced from HRESITOFMS (observed
m/z 421.0897 [M + Na]+), implying 13 degrees of unsaturation.
The IR spectrum of 4 indicated OH (3355 cm-1), ester carbonyl
(1738 cm-1), conjugated aldehyde (1688 cm-1), conjugated ketone
Figure 2. Key HMBC 2J and 3J correlations (HfC) and 4J
correlations (HfC, dashed arrows) and NOESY correlations of
mollicellin M (3).
The isolated compounds were tested for their bioactivities at the
Bioassay Research Facility of the National Center for Genetic
Engineering and Biotechnology (BIOTEC), NSTDA, Thailand, and
the results are shown in Table 3. Cytotoxicity tests against
cholangiocarcinoma were performed at the Liver Fluke and
Cholangiocarcinoma Research Center, Faculty of Medicine, Khon
Kaen University, Thailand, and the results are given in Table 4.
Mollicellins K-M (1-3), B (5), C (6), E (7), and J (10) showed
antimalarial activity against Plasmodium falciparum with IC50
ranging from 1.2-9.1 µg/mL. Only 1 showed moderate activity
Figure 3. Key HMBC 2J and 3J correlations (HfC) and 4J
correlations (HfC, dashed arrows) and NOE-difference data of
mollicellin N (4).

1490 Journal of Natural Products, 2009, Vol. 72, No. 8
Khumkomkhet et al.
J (10) (54 mg), an additional amount of mollicellin E (7) (15.3 g), and
mollicellin N (4) (6 mg). Fraction F1/24 was separated by FCC, eluted
with a gradient of hexane-EtOAc, to yield additional mollicellin K
(1) (67 mg) and mollicellin B (5) (30 mg). Fraction F1/25 was subjected
to FCC, eluted with a gradient of hexane-EtOAc, to give mollicellin
C (6) (13.2 mg), mollicellin B (5) (10 mg), mollicellin M (3) (7.8 mg),
and mollicellin N (4) (16 mg). Fraction F1/26 was separated by silica
gel FCC, eluted with a gradient of hexane-EtOAc, to give three
subfractions, F1/26.1-F1/26.3. Subfraction F1/26.1 was rechromato-
graphed by FCC, eluted with 40% EtOAc-hexane, to afford additional
mollicellin L (2) (6.3 mg). Subfraction F1/26.2 was rechromatographed
by FCC, eluted with 50% EtOAc-hexane, to yield mollicellin H (9)
(14 mg). Fraction F1/27 was further purified by silica gel FCC, eluted
with a gradient of hexane-EtOAc, to give three subfractions, F1/
27.1-F1/27.3. Subfraction F1/27.1 was further purified by preparative TLC
using 20% EtOAc-hexane as eluent to yield additional mollicellin B
(5) (15 mg). Subfraction F1/27.2 was rechromatographed by FCC, eluted
with 50% EtOAc-hexane, to give mollicellin F (8) (21 mg).
The MeOH extract (20.6 g) was subjected to silica gel FCC, eluted
with a gradient of hexane-EtOAc and EtOAc-MeOH to yield fractions
F′′′1-F′′′4. Fraction F′′′2 yielded additional mollicellin C (6) (7 mg).
Fraction F′′′3 afforded additional mollicellin E (7) (11.4 mg).
Mollicelline K (1): white solid; mp 178-181 °C; UV (MeOH) λmax
(log ε) 203 (4.47), 264 (4.52) nm; IR (KBr) νmax 3407, 2977, 2360,
1731, 1656, 1638, 1594, 1573 cm-1; 1H and 13C NMR data, see Tables
1 and 2; HRESITOFMS m/z 383.1161 [M + H]+ (calcd for C21H18O7
+ H, 383.1131).
against Mycobacterium tuberculosis (MIC 12.5 µg/mL) and potent
activity against Candida albicans (1.2 µg/mL), as well as cytotox-
icity against KB cells (1.9 µg/mL). However, compounds 1, 3, 6,
7, and 9 exhibited significant cytotoxicity against NCI-H187 cell
lines with IC50 values of 0.35, 0.68, 3.1, 1.0, and 3.9 µg/mL,
respectively (Table 3). In addition, compounds 1 and 3-8 exhibited
significant cytotoxicity against five cholangiocarcinoma cell lines
(KKU-100, KKU-M139, KKU-M156, KKU-M213, and KKU-
M214) with IC50 values ranging from 2.5 to 15.7 µg/mL. It should
be noted that all compounds exhibited IC50 values against KKU-
100 ranging from 4.5 to 6.5 µg/mL and were more cytotoxic than
the control drug ellipticine (Table 4).
The antimalarial and antimycobacterial activities of the isolated
compounds corresponded to the preliminary screening tests for the
crude extracts. Among the seven depsidones that exhibited anti-
malarial activity, mollicelline K (1) was more active than its crude
extracts. In addition, mollicelline K (1) was the only one that
exhibited antimycobacterial activity and was also more potent than
crude extracts. Moreover, of the compounds that were tested for
cytotoxicity against several cancer cell lines, most of them showed
significant cytotoxicity, especially against cholangiocarcinoma cells.
Experimental Section
General Experimental Procedures. Melting points were determined
using a Gallenkamp melting point apparatus and were uncorrected. UV
spectra were measured on an Agilent 8453 UV-visible spectropho-
tometer. IR spectra were taken on a Perkin-Elmer Spectrum One
spectrophotometer. NMR spectra were recorded in CDCl3 on a Varian
Mercury Plus 400 spectrometer, using residual CHCl3 as an internal
standard. HRESITOFMS were recorded on a Micromass Q-TOF-2
spectrometer. Column chromatography and preparative TLC were
carried out on silica gel 60 (230-400 mesh) and PF254, respectively.
Fungal Material. The fungus C. brasiliense was collected from Doi
Inthanon, Jomtong District, Chiangmai Province, Thailand, in June 2006
and was identified by K.S. A voucher specimen (no. Chbr01) was
deposited at the Department of Plant Pest Management, King Mongkut’s
Institute of Technology Ladkrabang, Bangkok, Thailand. The fungus
was cultured in conical flasks (1 L, 65 flasks) with potato dextrose
broth (PDB) (200 mL/flask) and incubated in standing condition at
25-28 °C for 4 weeks. The culture broth was filtered to give a wet
mycelial mat and then air-dried at room temperature.
Extraction and Isolation. The air-dried mycelial mat (300 g) was
ground and extracted successively at room temperature with hexane
(700 mL × 3), EtOAc (700 mL × 3), and MeOH (700 mL × 3) to
give crude hexane (6.8 g), EtOAc (17.8 g), and MeOH (20.6 g) extracts.
CH2Cl2 (35 mL)-hexane (300 mL) was added to the hexane extract to
give a solid (95 mg), which was recrystallized from EtOAc-hexane
to give mollicellin B (5) (34 mg). The filtrate was evaporated to yield
a residue, which was subjected to silica gel flash column chromatog-
raphy (FCC), eluted with a gradient system of hexane-EtOAc to give
six fractions, F1-F6. The solid in fraction F2 was recrystallized from
EtOAc-hexane to give 24(R)-5R,8R-epidioxyergosta-6,22-diene-3ꢀ-
ol (74 mg). The filtrate was evaporated to yield a residue, which was
further subjected to silica gel FCC eluted with a gradient system of
hexane-EtOAc to give ergosterol (104 mg). Fraction F3 was purified
by preparative TLC using 20% EtOAc-hexane to give mollicellin E
(7) (24 mg). Fraction F4 was rechromatographed by FCC, eluted with
20% EtOAc-hexane, to afford additional mollicellin B (5) (26.3 mg)
and mollicellin K (1) (43 mg). Fraction F5 was purified by silica gel
FCC, eluted with a gradient of hexane-EtOAc to give four subfractions,
F5.1-F5.4. Subfraction F5.2 was subjected to silica gel FCC, eluted with
a gradient of hexane-EtOAc, to give mollicellin L (2) (18 mg). Fraction
F6 was purified by silica gel FCC, eluted with a gradient of
hexane-EtOAc, to give subfractions F6.1-F6.4. Subfraction F6.1 was
rechromatographed by FCC, eluted with 20% EtOAc-hexane, to yield
additional amounts of mollicellin B (5) (26.3 mg) and mollicellin K
(1) (45 mg). Fraction F6.3 was rechromatographed by FCC, eluted with
40% EtOAc-hexane to give additional mollicellin E (7) (31.1 mg).
The EtOAc extract (17.8 g) was initially subjected to silica gel FCC,
eluted with the same gradient system as the hexane extract above to
give 10 fractions, F1/21-F1/210. Fraction F1/21 was subjected to silica
gel FCC, eluted with a gradient of hexane-EtOAc, to give mollicellin
Mollicelline L (2): white solid; mp 215-219 °C; UV (MeOH) λmax
(log ε) 203 (4.27), 264 (4.47) nm; IR (KBr) νmax 3439, 3028, 2983,
2931, 1729, 1677, 1644, 1608, 1568 cm-1; 1H and 13C NMR data, see
Tables 1 and 2; HRESITOFMS m/z 397.1286 [M + H]+ (calcd for
C22H20O7 + H, 397.1287).
Mollicelline M (3): white solid; mp 247-250 °C; UV (MeOH) λmax
(log ε) 201 (4.01), 261 (3.95) nm; IR (KBr) νmax 3454, 2979, 2917,
1736, 1688, 1652, 1600, 1562 cm-1; 1H and 13C NMR data, see Tables
1 and 2; HRESITOFMS m/z 439.0561 [M + Na]+ (calcd for
C21H17ClO7 + Na, 439.0561).
Mollicelline N (4): white solid; 251-253 °C; UV (MeOH) λmax (log
ε) 201 (3.67), 267 (3.89) nm; IR (KBr) νmax 3355, 2979, 2932, 1738,
1
1688, 1644, 1574 cm-1; H and 13C NMR data, see Tables 1 and 2;
HRESITOFMS m/z 421.0897 [M + Na]+ (calcd for C21H18O8 + Na,
421.0899).
Mollicelline B (5): white solid; mp 203-205 °C; UV (MeOH) λmax
(log ε) 201 (4.05), 261 (3.95) nm; IR (KBr) νmax 3461, 3089, 2979,
1
2929, 2854, 1739, 1686, 1651, 1602, 1574 cm-1; H and 13C NMR
data, see Tables 1 and 2; HRESITOFMS m/z 405.0953 [M + Na]+
(calcd for C21H18O7 + Na, 405.0950).
Mollicelline C (6): white solid; mp 200-202 °C; UV (MeOH) λmax
(log ε) 206 (4.21), 267 (3.97) nm; IR (KBr) νmax 3389, 2928, 2358,
1
1734, 1645, 1559 cm-1; H and 13C NMR data, see Tables 1 and 2;
ESITOFMS m/z 435.0648 [M + Na]+ (calcd for C22H20O8 + Na,
435.1055).
Mollicelline E (7): white solid; mp 169-170 °C; UV (MeOH) λmax
(log ε) 205 (4.18), 267 (3.80) nm; IR (KBr) νmax 3382, 2921, 2850,
1741, 1651, 1624, 1566 cm-1; 1H and 13C NMR data, see Tables 1 and
2; ESITOFMS m/z 469.0427 [M + Na]+ (calcd for C22H19ClO8 + Na,
469.0665).
Mollicelline F (8): white solid; 256-257 °C; UV (MeOH) λmax (log
ε) 201 (3.71), 267 (3.86), 224 (4.05) nm; IR (KBr) νmax 3400, 2980,
2924, 1741, 1688, 1644, 1565 cm-1; 1H and 13C NMR data, see Tables
1 and 2; ESITOFMS m/z 455.0318 [M + Na]+ (calcd for C21H17ClO8
+ Na, 455.0406).
Cyclization of 1. To a solution of 1 (32.1 mg) in MeOH (5 mL)
was added p-toluenesulfonic acid (9.4 mg), and the solution was stirred
at 50 °C for 4 h. Cooled water was added to the reaction mixture, and
it was extracted with EtOAc (10 mL × 3). The organic layer was
combined, washed with water and brine, and dried over anhydrous
Na2SO4. The filtrate was evaporated to dryness, and the residue was
separated by preparative TLC (10% EtOAc-hexane) to give 5 (29.2
mg, 91%); mp 202-204 °C; IR and NMR spectra were identical to
those of mollicellin B (5) (Tables 1 and 2).
Antimalarial Assay. Antimalarial activity was evaluated against the
parasite Plasmodium falciparum (K1, multidrug-resistant strain), using
the method of Trager and Jensen.23 Quantitative assessment of activity
in vitro was determined by means of the microculture radioisotope

Antimalarial Depsidones from Chaetomium brasiliense
Journal of Natural Products, 2009, Vol. 72, No. 8 1491
technique based upon the method described by Desjardins et al.24 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. The standard compound was
artemisinin (Table 3).
Antimycobacterial Assay. Antimycobacterial activity was assessed
against Mycobacterium tuberculosis H37Ra using the microplate
Alamar Blue assay (MABA).25 The standard drugs isoniazid and
kanamycin sulfate were used as the reference compounds (Table 3).
Antifungal Assay. An antifungal assay was performed against
clinical isolated Candida albicans using a method modified from the
soluble formazan assay described by Scudiero and co-workers.26 The
number of living cells was determined by measuring the absorbance
of XTT formazan at 450 nm. The reference substance was amphotericin
B (Table 3).
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Cytotoxicity Assay. Cytotoxic assays against human epidermoid
carcinoma (KB), human breast cancer (BC1), human small cell lung
cancer (NCI-H187), and cholangiocarcinoma cell lines were performed
employing the colorimetric method as described by Skehan and co-
workers.27 The reference substance was ellipticine (Tables 3 and 4).
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Acknowledgment. We are grateful to the Commission on Higher
Education, Thailand, for support by funding under the program Strategic
Scholarships for Frontier Research for Networks for the Ph.D. Program
Thai Doctoral Degree and the Center for Innovation in Chemistry
(PERCH-CIC) for P.K. We acknowledge financial support from the
Bioresources Research Network (BRN), National Center for Genetic
Engineering and Biotechnology (grant number BRN 001 G-51) for S.K.
We thank Assoc. Prof. Dr. B. Sripa for providing cholangiocarcinoma
cell lines.
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Acta 2008, 91, 124–129.
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Supporting Information Available: 1H NMR, 13C NMR, and
NOESY spectra for mollicellins K-N (1-4) and B (5). This material
is available free of charge via the Internet at http://pubs.acs.org.
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