Bioactive meroterpenoids and alkaloids from the fungus Eurotium chevalieri

Article abstract of DOI:10.1016/j.tet.2011.05.066

Five new meroterpenoids, chevalones A-D (1-4), aszonapyrone B (8), and a new sequiterpene alkaloid, eurochevalierine (5), together with four known compounds, sequiterpene (6), terpenoid pyrrolobenzoxazine named CJ-12662 (7), meroterpenoid, aszonapyrone A (9), and ergosterol were isolated from the fungus Eurotium chevalieri. The structures were established on the basis of spectroscopic evidence. The configurations of 1 and 5 were determined by X-ray analysis. The biosynthetic pathway of 1-3, 8, and 9 were proposed. Chemical transformation of aszonapyrone A (9) was also studied. Compounds 4, 5, and 7 exhibited antimalarial activity against Plasmodium falciparum, while 3, 5, and 7 showed antimycobacterial activity against Mycobacterium tuberculosis. In addition, compounds 2-7 showed cytotoxicity against cancer cell lines.

Full text of DOI:10.1016/j.tet.2011.05.066

Tetrahedron 67 (2011) 5461e5468
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Bioactive meroterpenoids and alkaloids from the fungus Eurotium chevalieri
,
a
b
Kwanjai Kanokmedhakul a, Somdej Kanokmedhakul a , Ruchiruttikorn Suwannatrai , Kasem Soytong ,
*
,
,d
Samran Prabpai c d, Palangpon Kongsaeree c
a Natural Products Research Unit, Department of Chemistry, and Center for Innovation in Chemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand
b Department of Plant Pest Management, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Ladkrabang, Bangkok 10520, Thailand
c Department of Chemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
d Center for Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 March 2011
Received in revised form 28 April 2011
Accepted 16 May 2011
Available online 25 May 2011
Five new meroterpenoids, chevalones AeD (1e4), aszonapyrone B (8), and a new sequiterpene alkaloid,
eurochevalierine (5), together with four known compounds, sequiterpene (6), terpenoid pyrrolo-
benzoxazine named CJ-12662 (7), meroterpenoid, aszonapyrone A (9), and ergosterol were isolated from
the fungus Eurotium chevalieri. The structures were established on the basis of spectroscopic evidence.
The configurations of 1 and 5 were determined by X-ray analysis. The biosynthetic pathway of 1e3, 8,
and 9 were proposed. Chemical transformation of aszonapyrone A (9) was also studied. Compounds 4, 5,
and 7 exhibited antimalarial activity against Plasmodium falciparum, while 3, 5, and 7 showed anti-
mycobacterial activity against Mycobacterium tuberculosis. In addition, compounds 2e7 showed cyto-
toxicity against cancer cell lines.
Keywords:
Chevalones
Aszonapyrone
Eurochevalierine
Meroterpenoids
Antimalarial
Ó 2011 Elsevier Ltd. All rights reserved.
Antimycobacterial
Anticancer
Eurotium chevalieri
1. Introduction
KB. Investigation of the extracts of dried fungal biomass of E. che-
valieri cultured in two different periods of time have led to the
Eurotium chevalieri Mangin imperfect stage of Aspergillus che-
valieri (Mangin) Thom and Church belongs to Phylum Ascomycota.1
Colonies reach 7.00 cm diameter on potato dextrose agar in 10 days
at a temperature of 30 ꢀC, with gray in the central areas and asco-
mata abundant on medium in a continuous yellow layer. There are
eight ascospores per ascus, ascospores lens-shaped, roughened,
isolation of six new compounds (1e5, and 8) and four known
compounds, 6, 7, 9, and ergosterol (Fig. 1). We report herein the
isolation, characterization, chemical transformation of 9 and pro-
posed biosynthesis of 1e3, 8, and 9, as well as bioactivity of these
compounds.
and with equatorial crests 0.8
m
m apart, 4.5e4.8ꢁ3.2e3.4
mm.
These characteristics are similar to those reported by Blaser (1976).2
Previous investigation on secondary metabolites from the fungus
genus Eurotium species3e10 including E. chevalieri11ꢂ13 resulted in
the isolation of numerous types of compounds, such as alkaloids,
indole derivatives, phenolic compounds, anthraquinones, and
diketopiperazines. In our continuing search for bioactive constitu-
ents from fungi, we noted that hexane, EtOAc, and MeOH extracts of
the fungus E. chevalieri showed antimycobacterial activity against
Mycobacterium tuberculosis with MIC values of 12.5, 3.13, and
2. Results and discussion
The fungus E. chevalieri was stationary cultured at 30 ꢀC for 40
days in potato dextrose broth (PDB). The hexane, EtOAc, and MeOH
extracts from the air-dried fungal biomass was separated on silica
gel column chromatography and preparative TLC to yield com-
pounds 1e7. The PDB sample cultured for 30 days produced three
compounds, 8, 9, and ergosterol. Structures of the known com-
pounds were identified by physical and spectroscopic data mea-
surements (1H and 13C NMR, 2D NMR, and MS) and by comparing
the data obtained with published values, as diterpene (6),14 ter-
penoid pyrrolobenzoxazine (7),14 and aszonapyrone A (9) [mp
240e242 ꢀC (lit.,15 242e244 ꢀC)].
6.26 mg/mL, respectively. In addition, the EtOAc extract of the strain
also exhibited cytotoxicity against cancer cell lines, NCI-H187 and
Compound 1 was obtained as colorless crystals, and its molec-
ular formula C26H38O4, was deduced from HRMS m/z 437.2669
* Corresponding author. Tel.: þ66 43 202222x12243; fax: þ66 43 202373;
e-mail address: somdej@kku.ac.th (S. Kanokmedhakul).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.05.066

5462
K. Kanokmedhakul et al. / Tetrahedron 67 (2011) 5461e5468
Fig. 1. Structures of compounds 1e9.
[MþNa]þ, implying eight degrees of unsaturation. The IR spectrum
showed absorption bands of hydroxyl (3467 cmꢂ1) and a conju-
gated carbonyl (1686 cmꢂ1) groups. The 13C NMR and DEPT spectra
revealed 26 signals attributable to six methyl, seven methylene, five
methine (including alkene), and eight quaternary carbons. The 1H
NMR spectrum of 1 (Table 1) showed five singlet signals of methyl
junction. The correlations of H-7 to C-9, C-5, C-8, and C-17; and
H-14 to C-13, C-9, C-8, C-7, C-16, and C-17 extended between two
fragments to include the B/C ring junction. In addition, the HMBC
spectrum showed correlation of methylene protons H-1 to C-3, C-5,
C-9, C-10, and C-18 and H-5 to C-6, C-18, and C-20 to complete the
planar structure of compound 1 (Fig. 2).
groups at
(H-16), an oxymethine proton at
six methylene protons at 0.75e2.05 and three methine protons at
0.75e1.41, providing a most useful indicator for the presence of
a tricyclic terpane.16 The methylene protons at
2.42 (dd, J¼16.8,
4.8 Hz, H-15), a vinyl proton at
5.66 (1H, s, H-50) and a singlet
d
0.77 (H-20), 0.82 (H-18), 0.86 (H-17), 0.95 (H-19), 1.18
The relative stereochemistry was determined by a combination
of coupling constant and analysis of the NOESY spectral data
(Fig. 3). The 1,3-diaxial NOESY correlation was observed between
H-5 and H-3. The J values of 4.4 and 11.2 Hz for the coupling of H-3
to H-2 also supported the axial position of H-3. There was no cor-
relation between H-5 and the methyl group at C-10 (H-18) sug-
gesting the trans A/B ring junction. A 1,3-diaxial correlation was
observed for H-14 and H-9, and H-14 and H-12, while the corre-
lation between H-14 and H-16 was not observed, indicating the
trans C/D ring junction. Finally, the relative configuration of 1 was
confirmed by a single-crystal X-ray diffraction analysis (Fig. 4).
From the above spectroscopic analysis it was found that most of the
constitutive structure of 1 was similar to taondiol,16 except for the
structure of ring E. The structure of 1 was established as a new
meroterpenoid type as described by Geris and Simpson,17 and has
been named chevalone A.
d
3.18 (1H, dd, J¼11.2, 4.4 Hz, H-3),
d
d
d
d
signal at d 2.16 (3H), which was assigned to a methyl group adjacent
to an alkene carbon were part of rings D and E. The 13C NMR
(Table 2) showed signals characteristic for two oxygenated olefinic
carbons, at
the other olefinic carbons at C-30 and C-50 appeared at
100.6, respectively. Two oxygenated carbons, C-3 and C-13 showed
resonances at 78.7 and 80.5, respectively. The COSY exhibited
d
163.2 (C-40) and 159.7 (C-60) of an
a-pyrone ring, while
d
97.8 and
d
a cross coupling network between H-1/H-2/H-3, H-5/H-6/H-7, H-9/
H-11/H-12, H-14/H-15, and H-50/H-70 (allylic coupling) allowed
formulation of five fragments (Fig. 2). The HMBC spectrum
exhibited correlation of an olefinic proton H-50 to C-30, C-40, C-60,
and C-70; and H-70 to C-50 and C-60 to complete the assignment of
Compound 2 was obtained as colorless crystals, and its molec-
ular formula, C28H40O5, was deduced from the HRMS m/z 479.2771
[MþNa]þ, implying nine degrees of unsaturation. The IR spectrum
showed the presence of ester (1733 cmꢂ1), conjugate lactone
(1702 cmꢂ1) and alkene (1652 and 1588 cmꢂ1) groups. The 1H and
the a-pyrone ring (E) and its substituents. The cross-peaks of cor-
relations of H-15 to C-8, C-14, C-13, C-20, C-30, and C-40 extended the
connectivity between two fragments, C and E through the D ring

K. Kanokmedhakul et al. / Tetrahedron 67 (2011) 5461e5468
5463
Table 1
1H NMR data of compounds 1e4 and 8 (CDCl3, 400 MHz)
Position
1
1
2
3
4
8
1.72 mb
1.71 mb
1.72 mb
1.73 mb
1.09 mb
1.69 mb
1.67 mb
0.97 m
1.56 mb
0.96 mb
1.06 mb
1.66 mb
1.06 mb
2
1.64 mb
1.63 mb
1.56 mb
1.60 mb
3
5
6
3.18 dd (11.2, 4.4)a
0.75 mb
4.44 dd (11.4, 4.8)
0.88 mb
4.45 dd (11.4, 5.2)
4.48 dd (11.5, 4.7)
0.90 mb
3.14 dd (10.0, 6.4)
0.83 m
0.85 mb
1.55 mb
1.57 mb
1.55 mb
1.60 mb
1.58 mb
1.45 mb
1.47 mb
1.45 mb
1.44 m
7
1.82 tt (13.2, 3.2)
1.83 tt (13.08, 3.08)
1.88 m
1.65 mb
1.87 m
1.42 m
1.07 m
1.61 m
1.29 m
2.24 m
1.92 m
2.48 m
2.58 m
2.42 m
4.81 br s, 4.62 br s
0.74 s
1.02 mb
1.01 mb
1.03 mb
1.05 mb
9
11
0.89 dd (12.2, 2.0)
1.69 mb
0.92 m
0.93 m
1.01 mb
1.69 mb
1.69 mb
1.76 mb
1.33 mb
1.33 m
1.33 m
12
2.05 td, (12.4, 3.0)
2.06 mb
2.09 m
2.15 mb
1.78 mb
2.08 mb
2.50 m
1.59 mb
1.61 mb
1.66 mb
14
15
1.41 mb
1.43 mb
1.51 mb
2.42 dd (16.8, 4.8)
2.42 dd (16.7, 4.6)
2.53 dd (16.4, 4.8)
2.15 dd (16.4, 3.6)
1.27 s
2.14 mb
2.15 mb
16
17
18
19
20
30
1.18 s
0.86 s
0.82 s
0.95 s
1.18 s
1.35 s
0.87 sb
0.85 sb
0.85 sb
0.85 sb
5.91 s
2.35 s
0.87 s
0.87 s
0.84 sb
0.83 sb
0.81 s
0.94 s
0.74 s
0.85 sb
0.85 sb
0.77 s
0.84 sb
0.83 sb
50
5.66 s
2.16 s
5.69 s
2.17 s
2.03 s
5.96 s
2.18 s
2.02 s
5.87 s
2.14 s
70
200
2.05 s
a
Figures in parentheses are coupling constants in Hz.
Overlapping with other protons.
b
Table 2
13C NMR data of compounds 1e4 and 8 (CDCl3, 100 MHz)
Position
1
2
3
4
8
1
2
3
4
5
6
7
8
38.3 ta
27.2 t
78.7 d
38.8 s
55.3 d
17.8 t
40.9 t
37.2 s
60.3 d
37.1 s
18.6 t
40.2 t
80.5 s
51.9 d
16.8 t
20.5 q
16.3 q
16.0 q
27.9 q
15.2 q
38.0 t
23.5 t
80.6 d
37.7 s
55.4 d
17.7 t
40.8 t
37.2 s
60.2 d
37.0 s
18.6 t
40.2 t
80.4 s
51.9 d
16.8 t
20.5 q
16.0 q
16.3 q
27.9 q
16.4 q
37.9 t
23.5 t
80.5 d
37.7 s
55.4 d
17.7 t
40.9 t
37.2 s
60.1 d
37.0 s
18.6 t
40.0 t
84.1 s
52.2 d
15.3 t
20.4 q
16.0 q
16.4 q
27.9 q
16.3 q
37.9 t
23.4 t
80.4 d
37.7 s
55.4 d
17.5 t
39.9 t
37.1 s
59.8 d
36.9 s
18.6 t
40.4 t
80.3 s
51.8 d
32.8 t
21.9 q
15.5 q
16.4 q
27.9 q
16.3 q
38.6 t
26.7 t
78.4 d
38.7 s
55.3 d
18.5 t
40.0 t
39.8 s
60.0 d
37.4 s
23.3 t
38.1 t
148.9 s
53.4 d
18.7 t
105.7
14.6 q
15.8 q
27.5 q
14.9 q
9
10
11
12
13
14
15
16
17
18
19
20
10
Fig. 2. COSY (bold line) and selected HMBC (arrow line) correlations of 1.
195.5 s
154.0 s
108.9 d
198.4 s
31.8 q
20
165.3 s
97.8 s
163.2 s
100.6 d
159.7 s
19.6 q
165.3 s
97.7 s
162.5 s
98.4 s
167.3 s
102.6 s
166.1 s
105.5 d
159.6 s
18.8 q
30
40
163.2 s
100.6 d
159.7 s
19.6 q
170.9 s
21.2 q
180.5 s
111.8 d
160.4 s
19.2 q
170.9 s
21.2 q
50
60
70
100
200
170.9 s
21.2 q
Fig. 3. NOESY correlations of 1.
a
Multiplicities were determined by analysis of the DEPT and HSQC spectra.
Compound 3 was obtained as a white solid, and its molecular
formula, C28H40O5, was deduced from the HRMS m/z 479.2773
[MþNa]þ, implying nine degrees of unsaturation. The IR spectrum
showed the presence of ester (1734 cmꢂ1), conjugate ketone
(1669 cmꢂ1), and alkene (1637 and 1596 cmꢂ1) groups. The 1H and
13C NMR spectra of 3 (Tables 1 and 2) were similar to those of 2,
13C NMR spectra of 2 (Tables 1 and 2) were similar to those of 1,
except for an appearance of an acetoxy group at C-3 (dH 2.03, dC 21.2
and 170.9). The relative stereochemistry was determined by
a combination of coupling constant and analysis of the NOESY
spectrum, which was similar to those of 1. Thus, 2 was identified as
an acetyl derivative of 1 and was named chevalone B.
except for the
a-pyrone, which was displaced by g-pyrone, dH 5.96
(s, H-50), 2.18 (H-70) and dC 162.5 (C-20), 98.4 (C-30), 111.8 (C-50),

5464
K. Kanokmedhakul et al. / Tetrahedron 67 (2011) 5461e5468
Table 3
NMR data of compound 5 (CDCl3, 400 MHz)
No
dH
dC
COSY
HMBC
NOESY
1
2
3
170.2 sb
47.6 d
3.80 dd (17.7, 3.8) 40.4 t
3.60 dd (17.7, 3.7)
199.8 s
5.02 td (7.6, 3.8)a
H-3, 11
H-2
C-1, 3, 4, 12 H-3
C-1, 2, 4
H-2, 5
4
4a
5
116.1 s
131.8 d H-6, 7
7.66 d (8.1)
C-4, 4a, 8a,
C-7
H-3, 6
6
7
8
8a
9
10
11
12
10
6.56 t (7.6)
7.38 t (7.7)
6.66 d (8.7)
114.4 d H-5, 7
136.3 d H-5, 6, 8
111.7 d H-7
152.5 s
C-5, 8, 4a
C-5, 8a
C-4, 4a, 6
H-5
H-8
H-7, 10
Fig. 4. ORTEP plot of the crystal structure of 1.
8.65 (NH) br q
2.90 d (5.0)
6.70 (NH) d (7.4)
8.17 s
H-10
C-8, 4a, 10
C-8, 8a
C-12
H-10
29.3 q
d
H-9
H-2, 12
H-9, 8
H-12
H-11, 10
H-120
160.8 d H-11
C-2
160.4 (C-60), 180.5 (C-40), and 19.2 (C-70). The HMBC spectrum
clearly demonstrated correlations of H-50 to C-30, C-40, C-60, and C-
70; methyl protons at C-60 (H-70) to C-50 and C-60; as well as
methylene protons (H-15) to C-14, C-13, C-20, C-30, and C-40, con-
5.41 br s
74.9 d
72.6 d
129.4 s
H-20
C-1, 20, 30,
C-80, 40a
C-10, 30, 40,
20
5.14 br s
H-10, 40,
H-40a, 110 C-90, 110, 80a
H-110
30
40
firming the formation of a
g-pyrone ring. The relative stereo-
5.32 br s
2.27 m
2.30 m
127.5 d H-20, 110, C-110, 80a
H-110,
H-150
H-60,
OH-80a
d
H-40a
chemistry of 3 was assigned by a combination of coupling constant
and analysis of the NOESY spectral data, which was similar to those
of 1 and 2. On the basis of the above evidence, 3 was a new mer-
oterpenoid, which has been named chevalone C.
40a
50
39.4 d
40.7 d
25.7 t
30.1 t
H-20, 40,
H-110
H-70, 140,
H-150
Compound 4 was obtained as a white solid and its molecular
formula, C27H40O5, was deduced from the HRMS m/z 467.2773
[MþNa]þ, implying eight degrees of unsaturation. The IR spectrum
showed the presence of ester carbonyl (1729 cmꢂ1), conjugated ke-
60
1.49 m
1.28 m
1.52 m
1.42 m
1.95 m
H-40a
70
H-50, 80
C-50, 80, 80a
H-120
80
30.6 d
72.3 s
169.8 s
20.7 q
19.7 q
H-70, 120
H-60, 70, 120
tone (1702 cmꢂ1), and alkene (1653 and 1594 cmꢂ1) groups. The 13
C
80a
90
NMR and DEPT spectrum showed twenty seven carbon signals at-
tributable to seven methyl (including acetoxy group), seven methy-
lene, five methine (including alkene carbon) and eight quaternary
(including three carbonyl function) carbons. The 1H and 13C NMR
spectra of 4 (Tables 1 and 2) were similar to those of 2, except that the
100
110
2.09 s
1.62 s
C-90
H-40, 40a, C-20, 30, 40
H-20
H-80
H-20, 40
H-10, 70, 80
H-150
120
130
140
0.93 d (6.6)
14.8 q
146.9 s
110.9 t
C-80, 80a
H-50, 150
H-50, 140
C-50, 150
a-pyrone ring was replaced by the a,b unsaturated diketones, dH 5.91
4.82 br s
4.60 br s
1.24 s
(H-30) and 2.35 (H-50), dC 198.4 (C-40), 195.5 (C-10),154.0 (C-20),108.9
(C-30), and 31.8 (C-50). The HMBC spectrum showed correlation of
avinyl proton, H-30 to C-10, C-20, and C-50; methylene protons, H-15 to
C-13, C-10, and C-20; methyl protons (H-50) to C-30 and C-40 to confirm
the formation of ring D and its substituents. The relative stereo-
chemistry was assigned by a combination of coupling constant and
analysisof theNOESY spectraldataandbycomparisontothoseof1, 2,
and3. Onthebasisoftheaboveevidence, 4 wasanewmeroterpenoid
and we named it chevalone D.
150
21.4 q
C-50, 130, 140 H-40, 140
C-80, 80a H-40a
OH-8a 2.39 s
a
Figures in parentheses are coupling constants in Hz.
Multiplicities were determined by analysis of the DEPT and HSQC spectra.
b
a proton in the substituents at N-9 of the secondary amine, re-
spectively. This was checked by mixing a sample of 5 with D2O,
wherein the signal at
showed as a singlet. The triplet of doublet signals at
3.8 Hz), which coupled to methylene protons at
3.80 (dd, J¼17.7,
3.8 Hz) and 3.60 (dd, J¼17.7, 3.7 Hz) at C-3, and a proton in the
substituents at N-11 of secondary amide
6.70 (d, J¼7.4 Hz) was
d
8.65 disappeared, while the signal at
d 2.90
5.02 (J¼7.6,
Compound 5 was obtained as yellow needles, and its molecular
formula, C29H38N2O7, was deduced from the HRMS m/z 527.2842
[MþH]þ, implying twelve degrees of unsaturation. The IR spectrum
showed the presence of hydroxyl (3331 cmꢂ1), NH (3440 cmꢂ1),
ester carbonyls (1754 and 1725 cmꢂ1), aromatic ketone
(1670 cmꢂ1), amide (1644 cmꢂ1), and aromatic (1569 cmꢂ1) groups.
The 13C NMR and DEPT spectra showed twenty nine signals at-
tributable to five methyl, four methylene (including terminal al-
kene), twelve methine (including aromatic, alkene, and
formamide), and eight quaternary carbons. The four carbonyl
groups were confirmed by 13C NMR spectrum showing signals at
d
d
d
assigned to a methine proton at C-2. The COSY spectrum demon-
strated correlation between H-3/H-2/H-11/H-12, H-5/H-6/H-7/H-8
of four aromatic protons, and also H-9/H-10 supported the partial
units for the alkaloid structure of 5 (Table 3). The HMBC spectrum
demonstrated correlation of the methyl group at N-9 (H-10) to C-8a
and C-8; H-9 to C-8, C-10, and C-4a confirmed the methyl amino
group located at C-8a. Complete HMBC correlations of 5 are shown
in Fig. 5 and Table 3. Finally, the X-ray structure of 5 (Fig. 6) proved
the presence of all protons in space clearly supporting the NOESY
spectral data (Table 3). The X-ray structure of 5 showed stereo-
chemistry of the terpenoid unit similar to those reported for
compound 6, which has been proven to be the absolute configu-
ration.14 Therefore, it can be concluded that compound 5, especially
the terpenoid unit, has absolute configuration similar to that of 6. It
also implies the S absolute configuration at C-2 in the alkaloid unit.
From the above evidence, compound 5 is a new sesquiterpene al-
kaloid, which has been named eurochevalierine.
d
199.8, 170.2, 169.8, and 160.8. The 1H and 13C NMR spectral data of
5 (Table 3) indicated a structure relating to a known terpenoid
pyrrolobenzoxazine (7) especially, a terpenoid unit.14 The 1H NMR
spectrum of 5 displayed a singlet signal at
to a carbon at dC 160.8 (H-12) in the HSQC spectrum revealing
a formamide unit.18 Four resonance signals at
d 8.17, which correlated
d
7.66 (d, J¼8.1 Hz, H-
5), 6.56 (t, J¼7.6 Hz, H-6), 7.38 (t, J¼7.7 Hz, H-7), and 6.66 (d,
J¼8.7 Hz, H-8) indicated the presence of a 1,2-disubstituted aro-
matic group. A sharp doublet signal at
d
2.90 (J¼5.0 Hz) and a broad
quartet signal at
d 8.65 were assigned to a methyl group and

K. Kanokmedhakul et al. / Tetrahedron 67 (2011) 5461e5468
5465
chloro-derivative compound 7, could be employed for this experi-
ment by ring-opening with CH2Cl2 in the presence of p-toluene-
sulfonic acid. However, the attempt was not satisfied due to the
decomposition and limitation of the sample. Therefore, the fungus
was re-cultured for 30 days, and only aszonapyrone A (9), aszo-
napyrone B (8) and ergosterol have been isolated.
Compound 8 was obtained as colorless crystals, and its molecular
formula C26H38O4, was deduced from HRMS m/z 415.2849 [MþH]þ,
implying nine degrees of unsaturation. The 1H and 13C NMR spectral
data of 8 (Tables 1 and 2) were similar to those of aszonapyrone A (9),
which has been reported from the fungus Aspergillus zonatus,15 ex-
cept for the acetoxy group at C-3, which was displaced by a hydroxyl
group. A terminal alkene appeared at dH 4.81 (s) and 4.62 (s) and dC
105.7 and 148.9. To prove that 8 was a new isolated compound not
derived from 9 during the isolation process, compound 9 was stirred
in various solvents (CH2Cl2, MeOH, EtOAc, and hexane) with or
without silica gel for 3 days, and no hydrolysis product 8 occurred.
Thus, the new compound 8 was named aszonapyrone B.
Fig. 5. Selected HMBC (H/C) of 5.
With
8 and 9 in hand, the biosynthetic pathway of
meroterpenoids 1e3 and aszonapyrones 8 and 9 drew our atten-
tion. According to Gonzales et al., the biosynthesis of taondiol, the
closest compound to 1, was proposed to occur by a synchronous
cyclization of the terpene unit.16 However, the finding of com-
pounds 8 and 9 has led us to believe that 1 and other related
compounds, 2 and 3 occurred by cyclization of 8 or 9. Biosynthesis
of 8 might be proposed via the cyclization of terpene units to form
tricyclic rings AeC similar pathway to breviane skeleton,20 followed
by deprotonation. Cyclization of 8 should provide 1, which trans-
forms to 2 by acetylation. Acetylation of 8 should give 9, which
could transform to 2 and 3 by cyclization (Fig. 7).
To prove the structures of 2 and 3, chemical transformation of 9
was performed by reaction in CHCl3 in the presence of p-toluene-
sulfonic acid at room temperature overnight, yielding compounds 2
(8.7%), 3 (39.5%), and a deacetylated product, compound 12 (3.2%).
These products were identical (mp, IR, NMR, and behavior on TLC)
Fig. 6. ORTEP plot of the crystal structure of 5.
We assumed that 5 should be derived from the ring opening of
a terpenoid pyrrolobenzoxazine, CJ-12663, which has previously
been isolated from Aspergillus fischeri var. thermomutatus ATCC
18618.14,19 Since CJ-12663 could not be isolated from E. chevalieri, its
to the isolated compounds 2 and 3. The occurrence of
derivative 2 was formed by cyclization of the intermediate 10. The
-pyrone derivative was proposed to be formed from
a-pyrone
g
3
Fig. 7. Proposed biosynthesis for 1e3, 8 and 9.

5466
K. Kanokmedhakul et al. / Tetrahedron 67 (2011) 5461e5468
tautomerization of
a
-pyrone 9 in acid conditions to form an in-
Plus 400 spectrometer, using residual CHCl3 as an internal standard.
HRMS spectra were obtained using a Micromass LCT mass spec-
trometer, and the lock mass calibration was applied for the de-
termination of accurate masses. Column chromatography and
preparative TLC were carried out on silica gel 60 (230e400 mesh)
and PF254, respectively.
termediate 11, followed by bond rotation and then cyclization to
give 3. Also some of 3 was hydrolyzed to yield compound 12 (Fig. 8).
The results of bioactivity tests on the isolated compounds are
shown in Table 4. Compounds 4, 5, and 7 exhibited antimalarial
activity against Plasmodium falciparum with IC50 values of 3.1, 3.4,
and 6.5
mycobacterial activity against M. tuberculosis with MIC values of
6.3, 50.0, and 12.5 g/mL, respectively. Compounds 3, 4, 5, and 7
had respective IC50 values against the BC1 cell line of 8.7, 7.8, 5.9,
and 7.6 g/mL. In addition, compounds 2 and 5 exhibited cytotox-
icity against two cancer cell lines, KB and NCI-H187 with IC50 values
of 2.9 and 3.9 and 9.8 and 9.2 g/mL, respectively. Among these,
compound 5 was active against all tests. Its related structure 7 was
also active against malaria, TB and BC1. However, 6, which is part of
5 and 7, was not active for all tests.
mg/mL, respectively, while 3, 5, and 7 showed anti-
3.2. Fungal material
m
The fungus was collected from rhizosphere soil of para rubber
tree, 40 cm soil depth at Surathani Province, Thailand, in 2007 and
was identified by one of the authors (K. S.). A voucher specimen
(EuC01) was deposited at the Department of Plant Pest Manage-
ment, King Mongkut’s Institute of Technology Ladkrabang, Bang-
kok, Thailand. The fungus was cultivated in Potato Dextrose Broth
by standing at 25e28 ꢀC for 30 to 40 days.
m
m
Fig. 8. Proposed mechanism for the formation of 2, 3, and 12 from 9.
Table 4
3.3. Extraction and isolation
Biological activities of isolated compounds 1e7
Compound
Antimalarial anti-TB
(IC50, mg/mL) (MIC, g/mL)
Cytotoxicity (IC50
BC1a KBb
Inactive Inactive Inactive
,
m
g/mL)
3.3.1. Cultivation for 40 days. Dried fungal biomass of E. chevalieri
(140 g) cultured for 40 days was ground into powder and
then extracted successively with hexane (3ꢁ700 mL), EtOAc
(3ꢁ700 mL), and MeOH (3ꢁ700 mL). Removal of solvents under
reduced pressure gave crude hexane (13.9 g, 9.9%), EtOAc (6.8 g,
4.8%), and MeOH (6.3 g, 4.5%) extracts, respectively.
The hexane extract (13.9 g) was subjected to silica gel column
chromatography (CC), eluted with a gradient system of hex-
aneeEtOAc and EtOAceMeOH, to give ten combined fractions,
HF1eHF10. Fraction HF5 was dissolved in MeOH, the solid pre-
cipitate was filtered out and recrystallized from EtOAcehexane to
yield colorless crystals of 2 (10.0 mg). Further separation of the
residue by silica gel flash column chromatography (FCC), eluted
with a gradient system of hexaneeEtOAc and EtOAceMeOH, yiel-
ded eight combined fractions, HF5.1eHF5.8. Fraction HF5.3 was fur-
m
NCI-H187c
1
2
3
4
5
6
7
Inactive
Inactive
Inactive
3.1
Inactive
Inactive
6.3
Inactive
50.0
Inactive 2.9 9.8
8.7
7.8
5.9
Inactive Inactive
Inactive Inactive
3.4
3.9
9.2
Inactive
6.5
Inactive
12.5
Inactive Inactive Inactive
7.6
Inactive Inactive
Dihydroartemisinin 0.001
Isoniazid
Kanamycin sulfate
Ellipticine
0.05
2.5
0.36
0.26
0.32
a
Human breast cancer cells.
Human epidermoid carcinoma in the mouth.
Small cell lung cancer.
b
c
3. Experimental section
3.1. General
ther purified by FCC, eluted with
a gradient system of
hexaneeCH2Cl2 and CH2Cl2eEtOAc, to give six fractions,
HF5.3.1eHF5.3.6. Fraction HF5.3.5 was dissolved in EtOAc, the pre-
cipitate was filtered out and was recrystallized from CH2Cl2ehex-
ane to give colorless crystals of 6 (5.0 mg). Fraction HF5.4 was
filtered and a solid was recrystallized from EtOAcehexane to yield
an additional amount of 2 (94.6 mg). The residue was further
separated by FCC, eluted with a gradient system of EtOAceCH2Cl2
and MeOHeEtOAc, to give an additional amount of 2 (105.3 mg),
a bright green solid of 4 (25.9 mg) and colorless crystals of 6
Melting points were determined using a Gallenkamp melting
point apparatus and were uncorrected. Optical rotations were
obtained using a JASCO DIP-1000 digital polarimeter. UV spectra
were measured on an Agilent 8453 UVevis spectrophotometer. IR
spectra were taken on a PerkineElmer Spectrum One spectropho-
tometer. NMR spectra were recorded in CDCl3 on a Varian Mercury

K. Kanokmedhakul et al. / Tetrahedron 67 (2011) 5461e5468
5467
(131.4 mg). Fraction HF5.5 was dissolved in MeOH, the solid pre-
cipitate was filtered out and further recrystallized from
EtOAcehexane to yield an additional amount of 2 (326.6 mg). The
filtrate was further separated by FCC, eluted with a gradient system
of hexaneeEtOAc, to yield an additional amount of 4 (10.4 mg).
Fraction HF5.6 was purified by FCC, eluted with a gradient system of
hexaneeCH2Cl2 (50e100%) and CH2Cl2eEtOAc, to give colorless
needles of 1 (4.7 mg). Fraction HF6 was subjected to silica gel CC,
eluted with a gradient system of hexaneeEtOAc and EtOAceMeOH,
to yield four combined fractions, HF6.1eHF6.4. Fraction HF6.2 was
dissolved in MeOH, the solid precipitate was filtered out and was
recrystallized from EtOAcehexane to give an additional amount of
2 (6.5 mg). Fraction HF6.3 was filtered and a solid was recrystallized
from EtOAc to yield an additional amount of 1 (24.0 mg). The res-
idue was further separated by FCC, eluted with a gradient system of
CH2Cl2eEtOAc, to give colorless crystals of 7 (32.4 mg). Fraction
HF6.4 was subjected to FCC, eluted with a gradient system of
CH2Cl2eEtOAc and EtOAceMeOH, to give five combined fractions,
HF6.4.1eHF6.4.5. Fraction HF6.4.3 was recrystallized from MeOH to
yield yellow needles of 5 (48.6 mg). Fraction HF6.4.4 was separated
by FCC, eluted with a gradient system of hexaneeEtOAc to give an
additional amount of 7 (55.5 mg). Fraction HF8 was recrystallized
from hexaneeCH2Cl2 to yield a white solid of 3 (77.7 mg).
a gradient system of hexaneeEtOAc and EtOAceMeOH to give six
combined fractions, E1eE6. Fraction E2 was recrystallized from
EtOAcehexane to yield ergosterol (59.8 mg). Fraction E3 was dis-
solved in a mixture of MeOHeEtOAc to give a white precipitate of 9
(199.2 mg). Fraction E4 was dissolved in a mixture of MeOHeEtOAc
to give an additional amount of 9 (315.4 mg). The filtrate was fur-
ther purified by FCC, eluted with a gradient system of hex-
aneeEtOAc and EtOAceMeOH to yield seven subfractions, E4.1eE4.7
.
Subfraction E4.3 was recrystallied from MeOHeEtOAc to give an
addition amount of 9 (207.7 mg). Subfraction E4.4 was recrystallied
from MeOHeEtOAc to give colorless needles of 8 (34.2 mg). Fraction
E5 was purified by FCC, eluted with
a gradient system of
CH2Cl2eEtOAc and EtOAceMeOH to yield an additional amount of
colorless needles of 8 (36.2 mg).
3.3.3. Chevalone A (1). Colorless crystals; mp 303e305 ꢀC; Rf (50%
24
EtOAcehexane) 0.43; [
nm (log
a
]
ꢂ121.6 (c 1.0, CHCl3); UV (MeOH) lmax
D
3
): 208 (4.28), 286 (3.83); IR (KBr) nmax: 3467, 2988, 2944,
2871, 2845,1686,1644,1574,1446 cmꢂ1; 1H and 13C NMR see Tables
1 and 2; HRMS (ESI): [MþNa]þ, found 437.2669. C26H38O4 Na re-
quires 437.2668.
3.3.4. Chevalone B (2). Colorless crystals; mp 162e165 ꢀC; Rf (50%
23
The EtOAc extract (6.8 g) was subjected to silica gel FCC, eluted
with a gradient system of hexaneeEtOAc and EtOAceMeOH, to give
five combined fractions, EF1eEF5. Fraction EF2 was separated by
EtOAcehexane) 0.60; [
nm (log
a]
ꢂ50.8 (c 1.0, CHCl3); UV (MeOH) lmax
D
3
): 208 (4.30), 286 (4.06); IR (KBr) nmax: 2943, 2868, 1733,
1702, 1652, 1588, 1447, 1406, 1379, 1239 cmꢂ1; 1H and 13C NMR see
Tables 1 and 2; HRMS (ESI): [MþNa]þ, found 479.2771. C28H40O5Na
requires 479.2773.
FCC, eluted with
a gradient system of hexaneeEtOAc and
EtOAceMeOH, to yield four combined fractions, EF2.1eEF2.4. Frac-
tion EF2.2 was further separated by FCC, eluted with a gradient
system of hexaneeEtOAc and EtOAceMeOH (10e20%) to yield nine
combined fractions, EF2.2.1eEF2.2.9. Fractions EF2.2.4, EF2.2.6 and
EF2.2.8 were recrystallized from EtOAcehexane and gave an addi-
tional amount of 2 (204.7 mg), 4 (5.1 mg) and 6 (10.9 mg), re-
spectively. Fraction EF2.3 was separated by FCC, eluted with
a gradient system of CH2Cl2eEtOAc to yield an additional amount of
1 (17.6 mg). Fraction EF3 was further separated by FCC, eluted with
a gradient system of CH2Cl2eEtOAc, to yield five combined frac-
tions, EF3.1eEF3.5. Fraction EF3.2 gave an additional amount of 1
(14.2 mg). Fraction EF3.3 was separated by FCC, eluted with a gra-
dient system of CH2Cl2eEtOAc to yield four combined fractions,
EF3.3.1eEF3.3.4. Fraction EF3.3.2 yielded an additional amount of 5
(166.5 mg). Fraction EF3.3.4 was recrystallized from MeOH to give an
additional amount of 7 (28.8 mg). Fraction EF4 was separated by
3.3.5. Chevalone C (3). White solid; mp 118e119 ꢀC; Rf (50%
23
EtOAcehexane) 0.15; [
nm (log
a
]
ꢂ120.6 (c 1.0, CHCl3); UV (MeOH) lmax
D
3
): 207 (4.12), 245 (3.87), 259 (3.90); IR (KBr) nmax: 2941,
2860, 1734, 1669, 1637, 1596, 1427, 1386, 1240, 1188 cmꢂ1 1H and
;
13C NMR see Tables 1 and 2; HRMS (ESI): [MþNa]þ, found 479.2773.
C28H40O5Na requires 479.2773.
3.3.6. Chevalone
EtOAcehexane) 0.65; [
D
(4). White solid; mp 227e229 ꢀC; Rf (50%
a
]
22 ꢂ46.0 (c 1.0, CHCl3); UV (MeOH) lmax nm
D
(log 3): 204 (4.05), 298 (4.20); IR (KBr) nmax: 2944, 2846, 1729, 1702,
1653,1594,1369,1258 cmꢂ1;1Hand13CNMRseeTables 1 and 2;HRMS
(ESI): [MþNa]þ, found 467.2773. C27H40O5Na requires 467.2773.
3.3.7. Compound 5. Yellow needles; mp 140e142 ꢀC; Rf (50%
22
FCC, eluted with
a
gradient system of CH2Cl2eEtOAc and
EtOAcehexane) 0.36; [
nm (log
3331, 3078, 2959, 2928, 1754, 1725, 1670, 1644, 1569, 1523, 1427,
a
]
ꢂ171.2 (c 1.0, CHCl3); UV (MeOH) lmax
D
EtOAceMeOH, to yield an additional amount of 3 (38.5 mg).
The crude MeOH extract (6.3 g) was subjected to silica gel FCC,
eluted with a gradient system of CH2Cl2eEtOAc and EtOAceMeOH
to give five combined fractions, MF1eMF5. Fraction MF2 was sep-
arated by FCC, eluted with a gradient system of CH2Cl2eEtOAc and
MeOHeEtOAc (20e40%) to yield seven combined fractions,
MF2.1eMF2.7. Fraction MF2.2 yielded an additional amount of 2
(50.4 mg). Fraction MF2.3 was filtered and the solid was recrystal-
lized from EtOAceCH2Cl2, to give an additional amount of 1
(9.6 mg). The filtrate was purified by FCC, eluted with a gradient
system of CH2Cl2eEtOAc and EtOAceMeOH, to give an additional
amount of 2 (32.7 mg) and 3 (7.4 mg).
3): 203 (4.30), 230 (4.35), 260 (3.83); IR (KBr) nmax: 3440,
1387, 1218, 1161 cmꢂ1 1H and 13C NMR see Table 3; HRMS (ESI):
;
[MþH]þ, found 527.2842. C29H39N2O7 requires 527.2759.
3.3.8. Compound 6. Colorless crystals; mp 160e163 ꢀC (from
CH2Cl2ehexane) (lit.14 171e173 ꢀC, from from Et2O); Rf (50% Et2O/
hexane) 0.21 (lit14 0.22); [
a]
ꢂ51.2 (c 1.0, CHCl3); IR (KBr) nmax
:
;
22
3445, 3325, 2974, 2958, 293D0, 1733, 1641, 1451, 1404, 1369 cmꢂ1
HRMS (ESI): [MþH]þ, found 295.1926. C17H27O4 requires 295.1928.
3.3.9. Compound 7. White solids; mp 121e125 ꢀC; Rf (50%
24
EtOAcehexane) 0.43; [
a
]
ꢂ78.1 (c 0.5, CHCl3); UV (MeOH) lmax
D
3.3.2. Cultivation for 30 days. Dried fungal biomass of E. chevalieri
(300 g) cultured for 30 days was ground into powder and then
extracted successively with hexane (3ꢁ700 mL), EtOAc
(3ꢁ700 mL), and MeOH (3ꢁ700 mL). Removal of solvents under
reduced pressure gave crude hexane (26.8 g, 8.9%), EtOAc (18.4 g,
6.1%), and MeOH (15.5 g, 5.2%) extracts, respectively. The hexane
extract contained only long chain fatty acids while the MeOH ex-
tract contained mostly sugar. The EtOAc extract was subjected to
silica gel flash column chromatography (FCC), eluted with
nm (log 3): 208 (4.32), 248 (3.72), 287 (3.31); IR (KBr) nmax 3436,
2971, 2922, 1745, 1642, 1439, 1372, 1238, 1177 cmꢂ1; HRMS (ESI):
[MþNa]þ, found 583.1836 and 585.2085. C29H37N2O7ClNa requires
583.2186 and 585.2186.
3.3.10. Compound 8. White solids; mp 173e175 ꢀC; Rf (20%
EtOAceCH2Cl2) 0.23; [
(log
a
]
24 ꢂ87.3 (c 0.5, CHCl3); UV (MeOH) lmax nm
D
3
): 207 (4.1), 290 (4.3); IR (KBr) nmax 3290, 2944, 2922, 1677,
1652, 1581, 1449, 1408, 1257, 1248, 1040 cmꢂ1; 1H and 13C NMR see

5468
K. Kanokmedhakul et al. / Tetrahedron 67 (2011) 5461e5468
Tables 1 and 2; HRMS (ESI): [MþH]þ, found 415.2849. C26H39O4
Blue Assay (MABA).25 The standard drugs, isoniazid and kanamycin
sulfate, showed respective MIC values of 0.05 and 2.5 g/mL.
requires 415.2848.
m
3.3.11. Cyclization of 9. To a solution of 9 (31.1 mg) in dry CHCl3
(3 mL) was added p-toluenesulfonic acid (9.4 mg) and the solution
was stirred at room temperature for 12 h. Water was added and the
reaction mixture was extracted with CHCl3 (10 mLꢁ3). The organic
layer was combined, washed with 10% aq NaHCO3 and then water
and dried over anhydrous Na2SO4. The filtrate was evaporated to
dryness and the residue was separated by preparative TLC (50%
EtOAcehexane) to give 2 (2.7 mg, 8.7%, Rf 0.58); 3 (12.3 mg, 39.5%, Rf
0.15) and 12 (0.9 mg, 3.2%, Rf 0.10). IR and NMR spectra of the
products were identical to the compounds isolated 2 and 3 (Tables
1 and 2).
3.5.3. Cytotoxicity assay. Cytotoxicity assays against human breast
cancer (BC1), human epidermoid carcinoma (KB) and human small
cell lung cancer (NCI-H187) cell lines were performed employing
the colorimetric method as described by Skehan et al.26 The ref-
erence substance was ellipticine, which showed IC50 values of 0.26,
0.36, and 0.32 mg/mL, respectively.
Acknowledgements
Financial support from the Bioresources Research Network
(BRN) of the National Center for Genetic Engineering and Bio-
technology (BIOTEC) (grant number BRN 001 G-51) and the Higher
Education Research Promotion and National Research University
Project of Thailand, Office of the Higher Education Commission
through Khon Kaen University are acknowledged for S. K. We are
grateful to the Center for Innovation in Chemistry (PERCH-CIC) for
a scholarship to R.S. and P.K. acknowledges the support from the
Office of the Higher Education Commission and Mahidol University
under the National Research Universities Initiative.
3.4. X-ray crystallographic analyses of 1 and 5
Colorless crystals of 1 and 5 were obtained in the mixture of
EtOAc/hexane by slow evaporation. X-ray diffraction data were
measured on
graphite monochromated Mo K
a BrukereNonius kappaCCD diffractomer with
ꢀ
a
radiation (l¼0.71073 A) at
298(2) K. The structure was solved by direct methods by SIR97,21
and refined with full-matrix least-squares calculations on F2 us-
ing SHELXL-97.22
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tet.2011.05.066.
3.4.1. X-ray data of 1. C26H38O4, MW¼414.59, orthorhombic, di-
mensions: 0.25ꢁ0.15ꢁ0.10 mm3, D¼1.218 g/cm3, space group
ꢀ
P212121, Z¼4, a¼7.6809(5), b¼11.5023(8), c¼25.594(2) A,
References and notes
3
ꢀ
V¼2261.2(3) A , reflections collected/unique: 14,182/1924
1. Raper, K. B.; Fennell, D. J. The Aspergillus; Williams and Wilkins: Baltimore,
1965; 165.
2. Blaser, P. Sydowia 1976, 28, 1e49.
3. Domsch, K. H.; Gams, W.; Anderson, T. H. Compendium of Soil Fungi; Academic:
London, 1993; Vol. 2; 289e295.
4. Al-Julaifi, M. Z.; Ochratoxin, A. Kuwait J. Sci. Eng. 2003, 30, 59e66.
5. Saiz-Jimenez, C.; Haider, K. An. Edafol. Agrobiol. 1975, 34, 931e941.
6. Saiz-Jimenez, C.; Haider, K.; Martin, J. P. Soil Sci. Soc. Am. Proc. 1975, 39, 649e653.
7. Podojil, M.; Sedmera, P.; Vokoun, J.; Betina, V.; Barathova, H.; Durackova, Z.;
Horakova, K.; Nemec, P. Folia Microbiol. 1978, 23, 438e443.
8. Smetanina, O. F.; Kalinovskii, A. I.; Khudyakova, Y. V.; Slinkina, N. N.; Pivkin, M.
V.; Kuznetsova, T. A. Chem. Nat. Compd. 2007, 43, 395e398.
9. Kimoto, K.; Aoki, T.; Shibata, Y.; Kamisuki, S.; Sugawara, F.; Kuramochi, K.;
Nakazaki, A.; Kobayashi, S.; Kuroiwa, K.; Watanabe, N.; Arai, T. J. Antibiot. 2007,
60, 614e621.
(Rint¼0.0382), number of observations [>2
[I>2
(I)]: R1¼0.0466, wR2¼0.1179.
s(I)] 1728, final R indices
s
3.4.2. X-ray data of 5. C29H38N2O7, MW¼526.63, orthorhombic,
dimensions: 0.15ꢁ0.10ꢁ0.10 mm3, D¼1.247 g/cm3, space group
ꢀ
P212121, Z¼4, a¼9.97890(10), b¼11.2446(2), c¼24.9984(4) A,
3
ꢀ
V¼2805.04(7) A , reflections collected/unique: 14,818/7140
(Rint¼0.034), number of observations [>2
s(I)] 6132, final R indices
[I>2s
(I)]: R1¼0.0545, wR2¼0.1533.
Crystallographic data of compounds 1 and 5 have been de-
posited at the Cambridge Crystallographic Data Center under the
reference numbers CCDC 812972 and 812973. Copies of the data
can be obtained, free of charge, on application to the Director,
CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (e-mail:
deposit@ccdc.cam.ac.uk).
10. Ohtsuki, T. Kobunkazai no Kagaku 1990, 35, 28e34.
11. Ishikawa, Y.; Morimoto, K. J. Am. Oil Chem. Soc. 1984, 61, 1864e1868.
12. Stipanovic, R. D.; Schroeder, H. W. Trans. Br. Mycol. Soc. 1976, 66, 178e179.
13. Hamasaki, T.; Nagayama, K.; Hatsuda, Y. Agric. Biol. Chem. 1976, 40, 203e205.
14. Didier, C.; Critcher, D. J.; Walshe, N. D.; Kojima, Y.; Yamauchi, Y.; Barrett, A. G. M.
J. Org. Chem. 2004, 69, 7875e7879.
15. Kimura, Y.; Hamasaki, T.; Isogai, A.; Nakajima, H. Agric. Biol. Chem. 1982, 46,
1963e1965.
3.5. Bioassay
16. Gonzales, A. G.; Darias, J.; Martin, J. D.; Pascual, C. Tetrahedron 1973, 29,
1605e1609.
17. Geris, R.; Simpson, T. J. Nat. Prod. Rep. 2009, 26, 1063e1094.
18. Pavia, D. L.; Lampman, G. M.; Kriz, G. S.; Vyvyan, J. R. Introduction to Spectros-
copy, 4th ed.; Books/Cole, Cengage Leaning: Belmont, CA, 2009; 178e179.
19. Perry, D. A.; Maeda, H.; Tone, J. UK Patent Appl. 2240 100, 1991.
20. Macıas, F. A.; Varela, R. M.; Simonet, A. M.; Cutler, H. G.; Cutler, S. J.; Duganand,
F. M.; Hill, R. A. J. Org. Chem. 2000, 65, 9039e9046.
3.5.1. Antimalarial assay. Antimalarial activity was evaluated
against the parasite P. falciparum (K1, multidrug resistant strain),
using the method of Trager and Jensen.23 Quantitative assessment of
malarial activity in vitro was determined by means of the micro-
culture radioisotope technique based upon the method described by
Desjardins et al.24 The inhibitory concentration (IC50) represents the
concentration, which causes 50% reduction in parasite growth as
indicated by the in vitro uptake of [3H]-hypoxanthine by P. falcipa-
rum. The standard compound, dihydroartemisinin, exhibited an IC50
value of 1.0 ng/mL.
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3.5.2. Antimycobacterial assay. Antimycobacterial activity was
assessed against M. tuberculosis H37Ra using the Microplate Alamar