New mycotoxins from the scale insect fungus Aschersonia coffeae Henn. BCC 28712

Article abstract of DOI:10.1016/j.tet.2012.07.059

Nine new mycotoxins; five xanthones 1-5, hydroxanthone 6, and three anthraquinones 7-9, together with nine known compounds; sterigmatocystin (10), demethylsterigmatocystin (11), dihydrodemethylsterigmatocystin (12), sterigmatin (13), austocystin F (14), averufin (15), aflatoxin B1, paeciloquinone A, and zeorin, were isolated from the scale insect fungus Aschersonia coffeae Henn. BCC 28712. The structures of these compounds were elucidated using NMR spectroscopic and MS spectrometric analyses. Compounds 1e3 and 6e9 displayed cytotoxic activity while the xanthone 2 and anthraquinones 8 and 9 also showed antimalarial activity.

Full text of DOI:10.1016/j.tet.2012.07.059

Tetrahedron 68 (2012) 8480e8486
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
New mycotoxins from the scale insect fungus Aschersonia coffeae Henn. BCC 28712
Jittra Kornsakulkarn, Siriporn Saepua, Kitlada Srichomthong, Sumalee Supothina,
*
Chawanee Thongpanchang
National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road, Klong Luang, Pathumthani 12120, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 May 2012
Received in revised form 19 June 2012
Accepted 17 July 2012
Available online 21 July 2012
Nine new mycotoxins; five xanthones 1e5, hydroxanthone 6, and three anthraquinones 7e9, together
with nine known compounds; sterigmatocystin (10), demethylsterigmatocystin (11), dihydrodemethyl-
sterigmatocystin (12), sterigmatin (13), austocystin F (14), averufin (15), aflatoxin B1, paeciloquinone A,
and zeorin, were isolated from the scale insect fungus Aschersonia coffeae Henn. BCC 28712. The struc-
tures of these compounds were elucidated using NMR spectroscopic and MS spectrometric analyses.
Compounds 1e3 and 6e9 displayed cytotoxic activity while the xanthone 2 and anthraquinones 8 and 9
also showed antimalarial activity.
Keywords:
Mycotoxins
Bioactive secondary metabolites
Scale insect fungi
Aschersonia coffeae
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
compounds, sterigmatocystin (10),9 demethylsterigmatocystin
(11),10 dihydrodemethylsterigmatocystin (12),11 sterigmatin (13),10
Aschersonia is a member of entomogenic fungi that attacks scale
insects and whitefly and has been studied for the use in bio-
control.1e3 As a consequence, the study of their bioactive com-
pounds is of great interest. Several biologically active secondary
metabolites from this genus have been reported. Examples are;
austocystin F (14),12 averufin (15),13,14 aflatoxin B1,15 paeciloqui-
none A,16 and zeorin.17 Many xanthones and anthraquinones were
recognized as mycotoxins and were mostly found in many species
of fungi in the genus Aspergillus,18,19 interestingly, they have now
been reported for the first time from fungus in the genus Ascher-
sonia. The biological activities of these compounds were also
evaluated.
dustanin and the 3
BCC 1785 exhibited antimycobacterial activity,4 ascherxanthone A
from Aschersonia sp. BCC 8401, 17(21)-hopane-6 ,12 -diol, and
b-acetoxyl derivative from Aschersonia tubulata
a
b
aschernaphthopyrone A from Aschersonia apraphysata BCC 11964
showed antiplasmodial activity,5,6 ascherxanthone B from Ascher-
sonia luteola BCC 8774 revealed antifungal activity,7 and destruxins
A4 and A5 from an Aschersonia species exhibited insecticidal ac-
tivity.8 As part of a research program on the discovery of novel
bioactive compounds from Thai microorganisms, we investigated
the constituents of the insect pathogenic fungus Aschersonia coffeae
Henn. BCC 28712, guided by its antimalarial (Plasmidium falcipa-
2. Results and discussion
Compounds 4, 5, 8, 9, averufin (15), and aflatoxin B1 were
obtained from both culture broth and mycelia extract of BCC 28712.
Compounds 1, 2, 3, 7, sterigmatocystin (10), demethylster-
igmatocystin (11), sterigmatin (13), austocystin F (14), paeciloqui-
none A, and zeorin were obtained from the crude extract of the
mycelia, while compounds
6
and dihydrodemethylster-
rum) activity (IC50¼4.39
m
g/mL) and cytotoxic activity against hu-
g/mL), oral human
g/mL), and human small-
g/mL) cell lines. The study
igmatocystin (12) were present only in the culture broth extract.
Compound 1 was obtained as a yellow solid. The molecular
formula was established by HRMS (ESITOF), in combination with
13C NMR spectroscopy as C18H12O6. The IR spectrum showed an
absorption band at 3369 cmꢀ1 for a hydroxyl group and the
man breast cancer (MCF-7, IC50¼10.39
m
epidermoid carcinoma (KB, IC50¼9.42
cell lung cancer (NCI-H187, IC50¼2.89
m
m
led to the isolation and structural elucidation of nine new com-
pounds, which are five xanthones (1e5), one hydroxanthone (6)
and three anthraquinones (7e9), together with nine known
characteristic absorption bands of a
g-pyrone unit at 1652 and
1631 cmꢀ1. The 1H NMR spectroscopic data were closely related to
that of sterigmatocystin (10).9 The spectrum showed signals for
one chelated phenolic proton, three protons of
a 1,2,3-
trisubstitued benzene ring, one singlet aromatic proton, one cis-
double bond, two vicinal methine protons, and one methoxyl
* Corresponding
(C. Thongpanchang).
author.
E-mail
address:
chawanee@biotec.or.th
0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.07.059

J. Kornsakulkarn et al. / Tetrahedron 68 (2012) 8480e8486
8481
group. Correlations from H-5 to C-8a/C-10a, from H-6 to -10a, from
H-7 to C-8a/C-9, and from H-2 to C-9a in HMBC spectrum revealed
the xanthone moiety. The presence of methoxyl group at C-8 was
indicated by an HMBC correlation between the methoxyl protons
and their corresponding carbon. The NOESY correlation between
the methoxyl protons and H-7 also supported the position of
methoxyl group at C-8 of the xanthone unit. The hydroxyl group
was assigned to C-1 on the basis of its chemical shift. The bishy-
drofuran part was established on the COSY correlations from H-10
to H-20, H-20 to H-30, and H-30 to H-40 together with HMBC cor-
relations from H-10 to C-20/C-30/C-40, from H-20 to C-10/C-30/C-40,
from H-30 to C-10/C-20/C-40/C-3/C-4, and from H-40 to C-10/C-30/C-
3/C-4. The cross peak between H-30 and H-40 in NOESY spectrum
indicated a cis ring fusion of the bishydrofuran unit. The attach-
ment of the bishydrofuran part to C-3 and C-4 of the xanthone
unit was confirmed by the correlations between H-30 and C-4a in
the HMBC spectrum and the correlation between H-2 and hy-
droxyl proton in the NOESY spectrum. Therefore, compound 1 was
determined as a new 8-O-methyldemethylsterigmatocystin. For
confirmation, methyl ether derivatives of compound 1 and ster-
igmatocystin (10) were prepared, which showed identical 1H NMR
spectra. Their optical rotations were almost identical to that of the
reported monomethyl ether derivative of sterigmatocystin (16),20
which indicated the same absolute configuration of compound 1
and sterigmatocystin.21
Compound 2 was obtained as a yellow solid, possessing the
molecular formula C18H12O7 as deduced from HRMS (ESITOF), in
combination with 13C NMR spectroscopy. The 1H NMR spectrum
was similar to that of sterigmatocystin (10)9 except for the presence
of an additional hydroxyl signal at
d 8.28 and a different pattern in
the aromatic region. Compound 2 had two ortho-coupled protons
together with one singlet proton, whereas sterigmatocystin
showed signals for a 1,2,3-trisubstituted benzene ring in addition to
a similar singlet proton. The HMBC correlations from OH-8 to C-8/
C-7/C8a supported the location of the chelated hydroxyl group at C-
8. The assignment of an additional hydroxyl group at C-5 was
established on the basis of its chemical shift. The cross peak be-
tween methoxyl protons and H-2 in the NOESY spectrum con-
firmed the attachment of the bishydrofuran moiety at C-3 and C-4
of the xanthone unit. Compound 2 was therefore assigned as a new
5-hydroxysterigmatocystin. The almost identical rotations of
compound 2 and sterigmatocystin9 indicated the same configura-
tion of these two compounds.
Compound 3 with the molecular formula C23H20O10 from HRMS
(ESITOF) was obtained as a yellow solid. The 1H NMR spectrum
indicated that compound 3 was closely related to sterigmatocystin
(10).9 Analysis of the 13C and 2D NMR spectra revealed the similar
structural features of these two compounds except for the re-
placement of the methoxyl group at C-1 with a sugar unit. The
correlations from the anomeric proton H-100 to H-200, H-200 to H-300,
2'
1'
H
H
3'
O
4' O
R3
10
O
4
5
H
H
O
O
O
O
3
9
7
1
OR2
OR1
OR2
O
OR1
1, R1 = R3 = H, R2 = Me
2, R1 = Me, R2 = H, R3 = OH
4, R1 = H, R2 = Me
12, R1 = R2 = H, Dihydrodemethylsterigmatocystin
O
3, R2 = R3 = H, R1 =
HO
HO
OCH3
10, R1 = Me, R2 = R3 = H, Sterigmatocystin
11, R1 = R2 = R3 = H, Demethylsterigmatocystin
16, R1 = R2 = Me, R3 = H, O-Methylsterigmatocystin
H
10
5
H
H
4
O
O
O
HO
O
O
O
O
2'
1'
7
4'
9
1
R
OH
OH
5, R = OMe
13, R = H, Sterigmatin
14, R = OH, Austocystin F
O HO
OH
OH
6
6'
Me
O
O
H
5
4
R1O
O
O
O
R1O
10
5'
O
7
9
R2
1
3'
1'
H
OH
OH
OH
OH
OH
OH
O
O
R2
7, R1 = HO
, R2 = H
9, R1 =
OCH3 , R2 = OH
OH
OCH3
OH
HO
O
O
O
OH
8, R1 =
, R2 = OH
15, R1 = R2 = H, Averufin
HO
OCH3
OH
18, R1 = R2 = OH, nidurufin
17, R1 = H, Vericolorin B, R2 = H

8482
J. Kornsakulkarn et al. / Tetrahedron 68 (2012) 8480e8486
H-300 to H-400, and H-400 to H-500 in the COSY spectrum established
the sugar moiety. The equatorial position of H-400 was deduced on
the basis of small coupling constants of both axial Ha-500 and
equatorial Hb-500 with H-400 (J¼3.0 and 3.7 Hz). The large coupling
constant between H-300 and H-200 (J¼7.2 Hz) and small coupling
constant between H-200 and H-100 (J¼4.1 Hz) indicated the axial
orientations of H-300 and H-200 and the equatorial orientation of H-
100, respectively. The enhancement of Ha-500 when irradiated at H-300
in NOE experiment also supported the axial positions of these two
protons. The locations of the hydroxyl and methoxyl groups on the
sugar ring were confirmed by HMBC correlations of these protons
to their corresponding carbons. These results indicated that the
the xanthone unit. HMBC correlations from the methoxyl protons
to C-8/C-7 and from the hydroxyl proton to C-1/C-2/C-9a estab-
lished the attachment of the methoxyl and hydroxyl groups at C-8
and C-1, respectively. The cross peak between methoxyl protons
and H-7 and the hydroxyl proton and H-2 also supported the as-
signment of these two groups. Compound 4 was, therefore, de-
termined as a new 8-O-methyldihydrodemethylsterigmatocystin.
The same absolute configuration of compound 4 and dihy-
drosterigmatocystin11 was indicated by the almost identical optical
rotations of these two compounds.
Compound 5 was obtained as a yellow solid, and the molecular
formula was deduced as C18H12O7 by HRMS (ESITOF), in combi-
nation with 13C NMR spectroscopy. The 1H NMR spectrum was
similar to that of austocystin F (14)12 except for the presence of an
sugar unit was 4-O-methyl-b-arabinopyranose. The position of at-
tachment of the sugar moiety at C-1 of the xanthone unit was
assigned on the basis of HMBC correlation between the anomeric
proton H-100 and C-1. The correlation between H-2 and C-1 in the
HMBC spectrum also supported the linkage of the bishydrofuran
part at C-3 and C-4 of the xanthone unit. Comparison of the specific
additional methoxyl absorption at
d 3.4 instead of the hydroxyl
signal. Assignment of the methoxyl group at C-40 was established
by the HMBC correlation between the O-methyl protons and their
corresponding carbon (C-40). The cross peak from O-methyl pro-
tons to H-10 and H-30 in NOESY spectrum also supported the po-
sition of the methoxyl group at C-40 as well as the cis ring fusion of
the bishydrofuran ring system. The attachment of the bishy-
drofuran moiety at C-2 and C-3 of the xanthone unit, which
revealed the linear structure of this compound, was deduced by
the HMBC correlation between H-4 and C-4a. Compound 5 was,
therefore, assigned as a new 40-O-methylaustocystin F. The same
absolute configuration of Compound 5 and austocystin F23 was
indicated by the almost identical optical rotations of these two
compounds.
rotation of the aqueous layer of its acid hydrolysate ([
a
]
25 þ137.0, c
D
27
0.05, water) with that of 4-O-methyl-
L-arabinopyranose ([
a]
D
þ132, c 1.0, water)22 established the
L configuration of 4-O-methyl-
b-arabinopyranose. However, other unidentified products were
obtained in the organic layer instead of the aglycone unit, deme-
thylsterigmatocystin. The same result was also observed for the
acid hydrolysis of demethylsterigmatocystin (11) itself. Therefore,
compound 3 was determined as a new demethylsterigmatocystin-
1-(4-O-methyl)-b-L-arabinopyranose. Selected NOESY and HMBC
correlations for compound 3 are presented in Fig. 1.
Compound 6 was obtained as a red brown solid. Its molecular
formula was determined by HRMS (ESITOF) in combination with
13C NMR spectroscopy as C17H14O8. Comparison of the 1H NMR
spectrum with that of austocystin F (14)12 showed a very good
correlation for the bishydrofuran moiety. For the xanthone part, the
spectrum revealed a lack of signals for a 1,2,3-trisubstitued benzene
ring and the presence of additional signals for two methylene
groups, two oxy-methine protons, and two hydroxyl protons. COSY
correlations from H-5 to H-6, H-6 to H-7, and H-7 to H-8 together
with HMBC correlations from H-5 to C-10a/C-8a and H-7 to C-8a
2'
1'
H
3'
4' O
H
10
O
4
5
H
O
3
H
O
H
H
HO
H
H
9
7
6
H
1
H
HO
5
H
OH
O
O
H
H 8
7
O
O
H
H
OH
H
H
1'' O
H
H
H
3
HO
6
3''
8
HO
established the cyclohexene part attached to the g-pyrone ring. The
H
OCH3
positions of the hydroxyl groups at C-6 and C-8 on cyclohexene
were assigned on the basis of their chemical shifts together with
the HMBC correlations between hydroxyl protons and their corre-
sponding carbons. The linear structure of this compound was
supported by the correlation between H-4 and C-4a in the HMBC
spectrum. The large coupling constant between axial Ha-5 and H-6
(J¼8.3 Hz) and the small coupling constant between axial Ha-7 and
H-8 (J¼4.1 Hz) deduced the axial and equatorial orientations of H-6
and H-8, respectively, which indicated the trans relationship be-
tween these two protons. The NOESY correlations from H-6 to OH-8
and from H-30 to OH-40 also supported the trans relationship be-
tween OH-6 and OH-8 and the cis relationship between H-30 and
OH-40, respectively. However, due to the shortage of sample, the
absolute configuration was not determined. Therefore, compound 6
was only identified as a new austocystin J. Selected NOESY corre-
lations for compound 6 are presented in Fig. 1.
H
H
O
H
6''
H
H
HO
O
5
4
O
H3C
O
O
2'
1'
10
9
1''
HO
3''
7
H HO
4'
H
H
H
1
H
OH
H
HO
O
7
H
6'
Me
5'
O
H
6''
H
HO
O
5
4
O
H3C
O
O
10
9
1''
HO
3''
7
H HO
O
H
H
H
1
3'
1'
OH
HO
O
H
OH
9
HMBC (H
NOESY
C)
Compound 7 was obtained as a yellow solid. The HRMS (ESITOF),
in combination with 13C NMR spectroscopy, gave the molecular
formula as C25H24O12. The 1H and 13C NMR spectroscopic data
showed a good correlation to those of versicolorin B (17)24 except
for the presence of an additional sugar unit. The axial orientation of
protons 100e500of the sugar moiety were established on the basis of
Fig. 1. Selected NOESY correlations of compounds 3 and 6e9.
Compound 4 was obtained as a yellow solid. The HRMS (ESI-
TOF), in combination with 13C NMR spectroscopy, gave the mo-
lecular formula as C18H14O6. The similarity of the 1H NMR
spectrum of compound 4 to that of dihydrosterigmatocystin11
suggested that the structures of both compounds were closely
related. Analysis of the 13C NMR and 2D NMR spectroscopic data
revealed differences only at the position of the methoxyl group on
00 00
00 00
large vicinal trans-coupling constants (J1 ,2 ¼7.8 Hz, J2 ,3 ¼8.0 Hz,
00 00
00 00
J3 ,4 ¼9.4 Hz, and J4 ,5 ¼9.4 Hz). The HMBC correlation from O-
methyl protons to C-400 indicated the position of the methoxyl
group at C-400. The sugar unit was then assigned as 4-O-methyl-
b-
glucopyranose. Attachment of the sugar unit at C-6 of the

J. Kornsakulkarn et al. / Tetrahedron 68 (2012) 8480e8486
8483
Table 1
anthaquinone part was deduced by the correlation between the
Biological activities of compounds 1e3 and 5e9
anomeric proton H-100 and C-6 in the HMBC spectrum. Comparison
of the specific rotations of the aqueous layer of its hydrolysate ([aD25
Compound
Anti-malaria, Cytotoxicity, IC50
(
m
g/mL)
IC50 g/mL)
(
m
þ54.0, c 0.02, MeOH) with that of 4-O-methyl-
D
-glucopyranose
KB cells MCF-7 NCI-H187 Vero
([a
]
D
20 þ80, c 1.30, MeOH)25 established the
D configuration of 4-O-
cells
cells
cells
-glucopyranose. The 1H NMR spectroscopic data as well as
Compound 1
Compound 2
Compound 3
Compound 5
Compound 6
Compound 7
Compound 8
Compound 9
Doxorubicinb
Ellipticineb
>10a
8.54
>10
>10a
>10
>10
3.88
1.60
d
>50a
>50
>50
>50a
>50
35.62
37.58
>50
0.50
0.74
d
>50a
>50
>50
>50a
>50
>50
>50
>50
8.57
d
1.17a
12.93
2.86
>50a
20.28
>50
8.06
5.12
0.06
1.31
d
0.34a
1.40
0.33
>50a
>50
> 50
>50
>50
d
methyl-
b
optical rotation of the aglycone unit, obtained by acid hydrolysis of
compound 7, were in good agreement with those of veriscolorin
B.24 Compound 7 was, therefore, assigned as a new versicolorin B-
6-(4-O-methyl)-b-D-glucopyranose. Selected NOESY and HMBC
correlations for compound 7 are presented in Fig. 1.
Compound 8 with the molecular formula C25H24O13 from HRMS
(ESITOF), in combination with 13C NMR spectroscopy, was obtained
as a yellow solid. The 1H NMR spectrum was similar to that of
compound 7 except for the presence of an additional hydroxyl
d
1.29
d
Dihydroartemisininc 0.0005
d
a
Partially soluble in 100% DMSO.
Cytotoxicity controls.
Antimalarial control.
signal at
d
5.67, one methine proton at
d 4.44 and the absence of two
b
c
methylene protons at
d
2.18. Assignment of an additional hydroxyl
group at C-10 was established on the basis of its chemical shift. The
2D NMR spectroscopic data of the sugar moiety were in good
agreement with those of the sugar unit of compound 7. The at-
tachment of the sugar unit at C-6 was confirmed by the correlation
between the anomeric proton H-100 and C-6 in the HMBC spectrum.
However, due to the small quantity available, acid hydrolysis of
compound 8 was not performed. Compound 8 was, consequently,
3. Conclusion
In conclusion, nine new mycotoxins; five xanthones 1e5,
hydroxanthone 6, and three anthraquinones 7e9, together with
nine known compounds; sterigmatocystin (10), demethylster-
igmatocystin (11), dihydrodemethylsterigmatocystin (12), ster-
assigned as a new 10-hydroxyversicolorin B-6-(4-O-methyl)-
b-
glucopyranose. Selected NOESY and HMBC correlations for com-
pound 8 are presented in Fig. 1.
igmatin (13), austocystin
F (14), averufin (15), alfatoxin B1,
paeciloquinone A, and zeorin, were isolated from the insect fungus
A. coffeae Henn. BCC 28712. Compounds 2, 8, and 9 exhibited an-
timalarial and cytotoxic activities. Compounds 1, 3, 6, and 7 dis-
played only cytotoxic activity, while compound 5 was inactive in all
biological tests.
Compound 9 with the molecular formula C27H28O13 from HRMS
(ESITOF) was obtained as an orange solid. The 1H, 13C, and 2D NMR
spectroscopic data revealed the closely related structure of com-
pound 9 and nidurufin (18)14 except for the presence of a sugar unit
instead of one hydroxyl group. For the sugar moiety, the large
00 00
00 00
vicinal trans-coupling constants (J1 ,2 ¼8.1 Hz, J2 ,3 ¼8.5 Hz,
4. Experimental
00 00
00 00
J3 ,4 ¼9.2 Hz, and J4 ,5 ¼9.2 Hz) proved the axial orientation of
protons 100e500. Assignment of the methoxyl group on the sugar unit
was deduced by an HMBC correlation of the O-methyl protons to C-
4.1. General procedures
400. These results indicated that the sugar unit was 4-O-methyl-
b-
Melting points were measured using an electrothermal IA9100
digital melting point apparatus and are uncorrected. Optical rota-
tion measurements were obtained using a JASCO P-1030 digital
polarimeter. UV and FT-IR spectra were recorded on a Varian Cary
1E UVevis spectrophotometer and a Bruker Alpha spectrometer.
NMR spectra were recorded on Bruker AV400 and Bruker AV500D
spectrometers. ESITOF MS data were obtained on Micromass LCT
and Bruker micrOTOF mass spectrometers. Preparative thin layer
chromatography was performed on silica gel 60 GF254 (Merck).
Column chromatography was performed on silica gel 60
(70e230 mesh ASTM, Merck).
glucopyranose. The position of attachment of the sugar unit at C-6
was established on the basis of an HMBC correlation between the
anomeric proton and C-6. The
D-configuration of 4-O-methyl-b-
glucopyranose was established by comparing the specific rotations
25
of the aqueous layer of its hydrolysate ([
a
]
þ58.2, c 0.10, MeOH)
20
D
with that of 4-O-methyl-
D
-glucopyranose ([
a
]
D
þ80,
c 1.30,
MeOH).25 The 1H NMR spectroscopic data and the optical rotation
of the aglycone unit, obtained by acid hydrolysis of compound 9,
were identical to those of nidurufin.13,14 Therefore, compound 9
was determined as a new nidurufin-6-(4-O-methyl)-b-D-glucopyr-
anose. Selected NOESY and HMBC correlations for compound 9 are
presented in Fig. 1.
4.2. Fungal material
The structures of nine known compounds were elucidated on
the basis of their HRMS and NMR spectroscopic data, which were
identical in all respects to those of sterigmatocystin (10),9 deme-
thylsterigmatocystin (11),10 dihydrodemethylsterigmatocystin
(12),11 sterigmatin (13),10 austocystin F (14),12 averufin (15),13,14
aflatoxin B1,15 paeciloquinone A,16 and zeorin.17
Eight new compounds, 1e3, and 5e9, were subjected to bi-
ological assays for antimalarial (P. falciparum K1) activity and cy-
totoxicity against KB, MCF-7, NCI-H187, and Vero cells (Table 1).
Compound 4 was not tested due to the sample shortage. Com-
pounds 2, 8, and 9 exhibited moderate antimalarial activity (IC50
The fungus A. coffeae Henn. was isolated from a Homoptera scale
insect, collected from the underside of dicotyledonous leaf at Phu
Khiao Wildlife sanctuary, Chaiyaphoom Province, Thailand. The
specimen was identified by Mrs. Suchada Mongkolsamrit, BIOTEC.
This fungus was deposited at the BIOTEC Culture Collection Labo-
ratory (BCC) as BCC 28712 on December 3, 2007.
4.3. Fermentation and isolation
1.60e8.54
erate cytotoxic activity against both NCI-H187 and Vero cells (IC50
0.34e12.93 g/mL), while compounds 6 and 9 were active against
only NCI-H187 cells (IC50 20.28 and 5.12 g/mL, respectively). Only
compounds 7 and 8 exhibited weak cytotoxicity to KB cells (IC50
35.62 and 37.58 g/mL, respectively).
m
g/mL). Compounds 1, 2, and 3 showed strong to mod-
A. coffeae Henn. BCC 28712 was maintained on potato dextrose
agar at 25 ꢁC, the agar was cut into pieces (1ꢂ1 cm) and inoculated
into 4ꢂ250 mL Erlenmeyer flasks containing 25 mL of potato
dextrose broth (PDB, potato starch 4.0 g, dextrose 20.0 g/L). After
incubation at 25 ꢁC for 7 days on a rotary shaker (200 rpm), each
m
m
m
primary culture was transferred into 1 L Erlenmeyer flask

8484
J. Kornsakulkarn et al. / Tetrahedron 68 (2012) 8480e8486
containing 250 mL of the same liquid medium (PDB) and incubated
under the same conditions for 4 days. Each 25 mL portion of the
secondary culture was transferred into 40ꢂ1 L Erlenmeyer flasks
containing 250 mL of an M102 medium (composition: sucrose
(30.0 g), malt extract (20.0 g), bacto-peptone (2.0 g), yeast extract
(1.0 g), KCl (0.5 g), MgSO4$7H2O (0.5 g), and KH2PO4 (0.5 g), in
1000 mL of distilled water), and fermentation was carried out un-
der shaking conditions at 250 rpm, 25 ꢁC for 15 days.
After filtration of the culture, the cells were macerated in MeOH
(1 L) for 3 days and then in CH2Cl2 (1 L) for 3 days. The MeOH and
the CH2Cl2 extracts were combined and evaporated under reduce
pressure. Water (200 mL) was added, and the mixture was
extracted with n-hexane (3ꢂ200 mL), followed by EtOAc
(3ꢂ200 mL). The dark brown gum (4.38 g), obtained from EtOAc
extraction of the mycelium, was fractionated using silica gel col-
umn (5ꢂ20 cm), step gradient elution with 20e100% EtOAc/n-
hexane, to provide 7 fractions (1ꢀ7). After further purification by
silica gel column chromatography, sterigmatin (1.7 mg) and com-
pound 5 (0.305 g) were obtained from fractions 1 and 2, re-
4a), 157.8 (C-10a), 161.1 (C-8), 165.0 (C-3), 165.2 (C-1), 181.5 (C-9);
HRMS (ESITOF) m/z 325.0705 [MþH]þ (calcd for: C18H13O6,
325.0707).
4.3.2. Compound 2. Yellow solid; [
(EtOH) lmax (log
a
]
24 ꢀ360.5 (c 0.32, acetone); UV
D
3
) 247 (4.06), 277 (3.66), 328 (3.73) nm; IR (ATR)
nmax 3321, 3107, 2947, 1653, 1627, 1586, 1489, 1296, 1232, 1129,
1096 cmꢀ1; 1H NMR (500 MHz, acetone-d6)
d 3.96 (3H, s, OCH3-1),
4.97 (1H, dt, J¼2.4, 7.1 Hz, H-30), 5.56 (1H, t, J¼2.4 Hz, H-20), 6.58
(1H, d, J¼8.8 Hz, H-7), 6.60 (1H, s, H-2), 6.63 (1H, t, J¼2.4 Hz, H-10),
6.92 (1H, d, J¼7.1 Hz, H-40), 7.22 (1H, d, J¼8.8 Hz, H-6), 8.28 (1H, s,
OH-5), 12.67 (1H, s, OH-8); 13C NMR (125 MHz, acetone-d6)
d 47.9
(C-30), 56.1 (OCH3-1), 90.6 (C-2), 102.9 (C-20), 105.6 (C-9a), 107.0 (C-
4), 109.1 (C-8a), 109.6 (C-7), 113.6 (C-40), 123.0 (C-6), 136.2 (C-5),
143.0 (C-10a), 145.0 (C-10), 153.9 (C-4a), 154.4 (C-8), 163.5 (C-1),
164.7 (C-3), 180.8 (C-9); HRMS (ESITOF) m/z 363.0484 [MþNa]þ
(calcd for: C18H12O7Na, 363.0475).
4.3.3. Compound 3. Pale yellow solid; [
UV (EtOH) lmax (log
a
]
26 ꢀ183.6 (c 0.44, CHCl3);
D
spectively. Fraction
3
was subjected to silica gel column
3
) 231 (4.03), 241 (4.09), 324 (3.75) nm; IR
chromatography (4ꢂ18 cm), using 10% MeOH/CH2Cl2 as an eluent,
to furnish compound 5 (0.106 g), sterigmatocystin (67.2 mg),
averufin (53.1 mg), and zeorin (0.36 mg). Trituration of fraction 4
with MeOH followed by filtration afforded more of ster-
igmatocystin (0.568 g) as a yellow solid. Further purification of
fraction 5 by preparative HPLC using reverse phase column (SunFire
(ATR) nmax 3390, 2929, 1649, 1626, 1612, 1584, 1484, 1460, 1270,
1230,1044 cmꢀ1; 1H NMR (400 MHz, CDCl3) 3.33 (3H, s, OCH3-400),
d
3.49 (1H, dd, J¼3.7, 10.6 Hz, Ha-500), 3.54 (1H, dd, J¼3.0, 10.6 Hz, Hb-
500), 3.80 (1H, d, J¼8.7 Hz, OH-300), 4.06 (1H, ddd, J¼2.2, 7.2, 8.7 Hz,
H-300), 4.29 (2H, obs, H-200, H-400), 4.44 (1H, d, J¼10.1 Hz, OH-200),
4.75 (1H, d, J¼7.1 Hz, H-30), 5.36 (1H, t, J¼2.4 Hz, H-20), 5.73 (1H, d,
J¼4.1 Hz, H-100), 6.44 (1H, t, J¼2.4 Hz, H-10), 6.68 (1H, s, H-2), 6.69
(1H, d, J¼8.4 Hz, H-7), 6.77 (1H, d, J¼7.1 Hz, H-40), 6.78 (1H, d,
J¼8.4 Hz, H-5), 7.46 (1H, t, J¼8.4 Hz, H-6), 12.74 (1H, s, OH-8); 13C
C18 OBD, 5 mm, 19ꢂ150
mm, step gradient elution with 45e60%
MeCN/H2O) yielded compounds 2 (18.7 mg), 3 (39.4 mg), ster-
igmatocystin (32.9 mg), demethylsterigmatocystin (32.0 mg), and
austocystin F (9.0 mg). Aflatoxin B1 (94.4 mg) and additional
amount of compounds 3 (41.9 mg) and demethylsterigmatocystin
(31.9 mg) were obtained from fraction 6 after further purification
by preparative HPLC (step gradient elution with 45e70% MeCN/
H2O). Fraction 7 was subjected to silica gel chromatography
(4.0ꢂ18 cm), using 10% MeOH/CH2Cl2 as an eluent, to give 4 frac-
tions (7-1e7-4). More of compound 3 (12.9 mg) and demethyl-
sterigmatocystin (6.9 mg) were obtained from fractions 7-1 and 7-
2, respectively. Fractions 7-3 and 7-4 were further purified by
preparative HPLC (step gradient elution with 20e50% MeCN/H2O)
to afford compounds 7 (6.1 mg), 8 (1.4 mg), 9 (15.7 mg), austocystin
F (0.9 mg), and paeciloquinone A (6.4 mg).
NMR (100 MHz, CDCl3)
d
47.9 (C-30), 59.5 (OCH3-400), 70.7 (C-300),
72.4 (C-400), 72.5 (C-500), 86.4 (C-200), 94.6 (C-2), 102.3 (C-20), 102.5
(C-100),106.1 (C-5),106.6 (C-9a),108.3 (C-4),108.8 (C-8a),111.5 (C-7),
113.3 (C-40), 136.3 (C-6), 145.6 (C-10), 153.5 (C-4a), 155.1 (C-10a),
159.6 (C-1), 162.2 (C-8), 164.7 (C-3), 181.8 (C-9); HRMS (ESITOF) m/z
457.1138 [MþH]þ (calcd for: C23H21O10, 457.1129).
26
4.3.4. Compound 4. Pale yellow solid; [
UV (EtOH) lmax (log
(ATR) nmax 3350, 2920, 2852, 1656, 1605, 1480, 1310, 1262, 1246,
a
]
ꢀ219.2 (c 0.17, CHCl3);
D
3
) 247 (3.97), 257 (3.94), 325 (3.82) nm; IR
1167, 1109, 1086 cmꢀ1; 1H NMR (500 MHz, CDCl3)
d 2.27e2.30 (2H,
m, H-20), 3.67e3.72 (1H, m, Ha-10), 4.04 (3H, s, OCH3-8), 4.16 (2H,
obs, Hb-10, H-30), 6.27 (1H, s, H-2), 6.46 (1H, d, J¼5.6 Hz, H-40), 6.83
(1H, d, J¼8.2 Hz, H-7), 7.04 (1H, d, J¼8.2 Hz, H-5), 7.62 (1H, t,
Purification of the crude n-hexane extract (0.162 g) by silica gel
column chromatography (4.0ꢂ18 cm), step gradient elution with
0e5% MeOH/CH2Cl2, followed by preparative thin layer chroma-
tography provided compounds 1 (1.7 mg), 4 (1.7 mg), 5 (12.7 mg),
sterigmatocystin (6.8 mg), demethylsterigmatocystin (7.3 mg), and
zeorin (28.5 mg).
The culture broth was extracted with EtOAc (3ꢂ10 L) then
evaporated to dryness, leaving a dark brown gum (0.807 g). The
crude extract was fractionated using Sephadex LH 20 (3.5ꢂ50 cm),
eluted with 100% MeOH, and then further purified by preparative
HPLC (step gradient elution with 20e60% MeCN/H2O) to give
compounds 4 (4.2 mg), 5 (2.0 mg), 6 (9.3 mg), 8 (2.0 mg), 9
(13.3 mg), dihydrodemethylsterigmatocystin (14.5 mg), alfatoxin
B1 (18.0 mg), and averufin (1.0 mg).
J¼8.2 Hz, H-6),13.53 (1H, s, OH-1); 13C NMR (125 MHz, CDCl3)
d 31.6
(C-20), 43.8 (C-30), 56.6 (OCH3-8), 67.7 (C-10), 93.3 (C-2), 103.2 (C-4),
104.5 (C-9a), 106.1 (C-7), 109.6 (C-5), 111.0 (C-8a), 113.1 (C-40), 135.2
(C-6), 151.7 (C-4a), 157.6 (C-10a), 160.8 (C-8), 165.0 (C-1), 166.5 (C-
3), 181.2 (C-9); HRMS (ESITOF) m/z 327.0865 [MþH]þ (calcd for:
C18H15O6, 327.0863).
4.3.5. Compound 5. Pale yellow solid; [
UV (EtOH) lmax (log
a
]
25 ꢀ235.5 (c 0.50, CHCl3);
D
3
) 225 (3.69), 250 (3.87), 266 (3.74), 325
(3.58) nm; IR (ATR) nmax 3330, 2925, 2852, 1665, 1636, 1612, 1478,
1454, 1375, 1269, 1233, 1132, 1090 cmꢀ1; 1H NMR (500 MHz, CDCl3)
d
3.40 (3H, s, OCH3-40), 5.68 (1H, d, J¼2.8 Hz, H-10), 6.46 (1H, s, H-4),
6.48 (1H, s, H-30), 6.61 (1H, d, J¼2.8 Hz, H-20), 6.78 (1H, dd, J¼0.7,
8.3 Hz, H-7), 6.87 (1H, dd, J¼0.7, 8.3 Hz, H-5), 7.57 (1H, t, J¼8.3 Hz,
H-6), 11.77 (1H, s, OH-8), 12.44 (1H, s, OH-1); 13C NMR (125 MHz,
26
4.3.1. Compound 1. Yellow solid; [
(EtOH) lmax (log
nmax 3369, 2924, 2853, 1652, 1631, 1603, 1479, 1261, 1229,
a
]
ꢀ301.4 (c 0.21, CHCl3); UV
D
3
) 238 (4.39), 256 (4.21), 324 (4.11) nm; IR (ATR)
CDCl3) d
52.7 (OCH3-40), 90.8 (C-4), 95.5 (C-40), 103.6 (C-2), 104.0 (C-
1104 cmꢀ1; 1H NMR (400 MHz, CDCl3)
d
4.05 (3H, s, OCH3-8), 4.77
10), 107.0 (C-5), 107.4 (C-8a), 108.9 (C-9a), 111.2 (C-7), 113.7 (C-30),
137.0 (C-6), 149.2 (C-20), 156.0 (C-10a), 158.3 (C-1), 158.9 (C-4a),
161.3 (C-8), 167.2 (C-3), 185.0 (C-9); HRMS (ESITOF) m/z 339.0508
[MꢀH]ꢀ (calcd for: C18H11O7, 339.0510).
(1H, d, J¼7.0 Hz, H-30), 5.43 (1H, s, H-20), 6.36 (1H, s, H-2), 6.50
(1H, s, H-10), 6.80 (1H, d, J¼7.0 Hz, H-40), 6.84 (1H, d, J¼8.4 Hz, H-
7), 7.05 (1H, d, J¼8.4 Hz, H-5), 7.63 (1H, t, J¼8.4 Hz, H-6), 13.51 (1H,
s, OH-1); 13C NMR (100 MHz, CDCl3)
d
47.8 (C-30), 56.7 (OCH3-8),
94.4 (C-2), 102.8 (C-20), 104.4 (C-4), 104.8 (C-9a), 106.3 (C-7), 109.9
(C-5), 111.2 (C-8a), 113.2 (C-40), 135.6 (C-6), 145.6 (C-10), 151.5 (C-
4.3.6. Compound 6. Red brown solid; [
UV (EtOH) lmax (log
a
]
24 ꢀ133.2 (c 0.23, acetone);
D
3
) 239 (4.28), 256 (4.25), 291 (3.91) nm; IR

J. Kornsakulkarn et al. / Tetrahedron 68 (2012) 8480e8486
8485
(ATR) nmax 3369, 2921, 2849, 1665, 1632, 1612, 1478, 1454, 1382,
1195, 1117 cmꢀ1 1H NMR (500 MHz, acetone-d6)
1.81 (1H, ddd,
J¼2.4 Hz, H-5), 12.14 (1H, s, OH-8), 12.43 (1H, s, OH-1); 13C NMR
;
d
(125 MHz, DMSO-d6) d
23.1 (C-30), 27.7 (C-60), 30.6 (C-40), 60.1
J¼4.1, 10.3, 13.1 Hz, Ha-7), 2.10 (1H, ddd, J¼3.1, 3.1, 13.1 Hz, Hb-7),
2.62 (1H, dd, J¼8.3, 17.8 Hz, Ha-5), 3.02 (1H, dd, J¼4.9, 17.8 Hz,
Hb-5), 4.15 (1H, d, J¼3.3 Hz, OH-8), 4.33 (1H, d, J¼3.9 Hz, OH-6),
4.39e4.41 (1H, m, H-6), 5.06 (1H, dd, J¼3.4, 4.1 Hz, H-8), 5.40
(1H, s, OH-40), 5.65 (1H, d, J¼2.8 Hz, H-10), 6.35 (1H, s, H-30), 6.47
(1H, s, H-4), 6.68 (1H, d, J¼2.8 Hz, H-20), 13.21 (1H, s, OH-1); 13C
(OCH3-400), 60.6 (C-600), 63.9 (C-20), 71.2 (C-10), 73.7 (C-200), 76.2
(C-500), 76.4 (C-300), 79.3 (C-400), 100.1 (C-100), 102.1 (C-50), 108.1 (C-4),
109.1 (C-9a), 109.4 (C-5), 109.6 (C-7), 111.1 (C-8a), 115.6 (C-2), 134.0
(C-4a), 135.2 (C-10a), 159.1 (C-1), 160.6 (C-3), 164.1 (C-6), 164.3
(C-8), 181.1 (C-10), 189.7 (C-9); HRMS (ESITOF) m/z 559.1443
[MꢀH]ꢀ (calcd for: C27H27O13, 559.1457).
NMR (125 MHz, acetone-d6) d 37.1 (C-5), 38.9 (C-7), 60.9 (C-8), 62.0
(C-6), 89.1 (C-40), 89.5 (C-4), 105.9 (C-9a), 106.5 (C-10), 112.1 (C-2),
117.8 (C-30), 118.2 (C-8a), 148.2 (C-20), 157.7 (C-1), 158.5 (C-4a), 164.9
(C-3), 165.1 (C-10a), 182.0 (C-9); HRMS (ESITOF) m/z 347.0763
[MþH]þ (calcd for: C17H15O8, 347.0761).
4.4. Methylation of compound 1 and sterigmatocystin
Compound 1 (2.1 mg, 6.5 mmol) was methylated with MeI
(1.0 mL, 16.2 mmol) in K2CO3 (2.2 mg, 16.2 mmol) and acetone
(2.0 mL) at room temperature for 18 h. The reaction mixture was
evaporated then diluted with H2O (1.0 mL) and extracted with
EtOAc (1.0 mL). The organic layer was concentrated under reduced
pressure to leave a dark brown solid, which was purified by pre-
parative thin layer chromatography (using 40% EtOAc/n-hexane as
eluent) to furnish the dimethylated product 16 (2.0 mg, 91% yield,
4.3.7. Compound 7. Yellow solid; [
(MeOH) lmax (log
a
]
26 ꢀ133.2 (c 0.03, dioxane); UV
D
3
) 221 (4.18), 265 (4.07), 284 (4.15), 318 (3.71), 444
(3.73) nm; IR (ATR) nmax 3346, 2924, 2853, 1734, 1653, 1627, 1457,
1437, 1291, 1194, 1084 cmꢀ1
d
;
1H NMR (500 MHz, DMSO-d6)
2.14e2.21 (2H, m, H-10), 3.06 (1H, t, J¼9.1 Hz, H-400), 3.29 (1H, ddd,
J¼5.2, 7.8, 8.0 Hz, H-200), 3.42e3.45 (1H, m, H-300), 3.46 (3H, s, OCH3-
400), 3.48e3.53 (3H, m, Ha-20, H-500, Ha-600), 3.64 (1H, dd, J¼4.5,
9.7 Hz, Hb-600), 4.14 (2H, obs, Hb-20, H-40), 4.76 (1H, t, J¼5.4 Hz, OH-
600), 5.14 (1H, d, J¼7.8 Hz, H-100), 5.34 (1H, d, J¼5.5 Hz, OH-300), 5.55
(1H, d, J¼5.0 Hz, OH-200), 6.56 (1H, d, J¼4.82 Hz, H-30), 6.94 (1H, d,
J¼2.4 Hz, H-7), 7.14 (1H, s, H-4), 7.23 (1H, d, J¼2.4 Hz, H-5), 12.2 (2H,
[a
]
25 ꢀ139.2, c 0.10, CHCl3) as a white solid.
D
Methylation of sterigmatocystin (10, 10.4 mg, 32.1 mmol) was
conducted as the same manner to also obtain product 16 (9.0 mg,
83% yield, [
a]
25 ꢀ135.2, c 0.56, CHCl3).
D
The 1H NMR spectra and the optical rotations of these two
dimethylated products were identical to those of the reported
monomethyl ether derivative of sterigmatocystin.20,26
br s, OH-1, OH-8); 13C NMR (125 MHz, DMSO-d6) 30.7 (C-10), 44.0
d
(C-40), 60.1 (OCH3-400), 60.6 (C-600), 67.7 (C-20), 73.7 (C-200), 76.3 (C-
300), 76.4 (C-500), 79.3 (C-400), 100.1 (C-100), 102.1 (C-4), 109.5 (C-5),
109.5 (C-7), 110.9 (C-8a), 111.6 (C-9a), 113.9 (C-30), 120.8 (C-2), 135.1
(C-4a), 136.1 (C-10a), 159.7 (C-1), 164.0 (C-6), 164.4 (C-8), 166.0 (C-
3), 181.2 (C-10), 190.0 (C-9); HRMS (ESITOF) m/z 515.1199 [MꢀH]ꢀ
(calcd for: C25H23O12, 515.1195).
4.5. Hydrolysis of compound 3
Compound 3 (5.0 mg, 10.9 mmol) was hydrolyzed with 3 M
aqueous HCl (0.5 mL) in dioxane (0.1 mL) at 90 ꢁC for 12 h. The
reaction mixture was then diluted with H2O (2.0 mL) and extracted
with EtOAc (2.0 mL). The aqueous layer was concentrated in vacuo
24
4.3.8. Compound 8. Yellow solid; [
(MeOH) lmax (log
442 (3.80) nm; IR (ATR) nmax 3408, 2924, 2854, 1716, 1628, 1611,
a
]
ꢀ76.81 (c 0.04, MeOH); UV
25
D
to yield 4-O-methyl-
L-arabinopyranose (1.2 mg, 67% yield, [a]
D
3
) 222 (4.18), 265 (4.12), 285 (4.18), 318 (3.78),
þ137.0, c 0.05, water).
1466, 1388, 1294, 1193, 1077 cmꢀ1 1H NMR (500 MHz, DMSO-d6)
;
4.6. Hydrolysis of compound 7
d
3.06 (1H, t, J¼9.1 Hz, H-400), 3.29e3.31 (1H, m, H-200), 3.42 (1H,
m, H-300), 3.46 (3H, s, OCH3-400), 3.51e3.53 (2H, m, H-500, Ha-600),
3.57 (1H, dd, J¼1.9, 9.9 Hz, Ha-20), 3.64 (1H, dd, J¼3.4, 9.7 Hz, Hb-
600), 3.90 (1H, d, J¼9.9 Hz, Hb-20), 3.95 (1H, d, J¼5.6 Hz, H-40), 4.44
(1H, br s, H-10), 4.77 (1H, br s, OH-600), 5.14 (1H, d, J¼7.8 Hz, H-100),
5.34 (1H, d, J¼5.2 Hz, OH-300), 5.56 (1H, d, J¼4.5 Hz, OH-200), 5.67
(1H, d, J¼2.9 Hz, OH-10), 6.65 (1H, d, J¼5.6 Hz, H-30), 6.94 (1H, d,
J¼2.1 Hz, H-7), 7.09 (1H, s, H-4), 7.22 (1H, d, J¼2.1 Hz, H-5), 12.14
(1H, br s, OH-1), 12.37 (1H,br s, OH-8); 13C NMR (125 MHz, DMSO-
Compound 7 (2.8 mg, 5.4 mmol) was hydrolyzed by the method
described for compound 3. The aqueous layer was concentrated in
vacuo to yield 4-O-methyl-
D
-glucopyranose (0.5 mg, 52% yield,
25
[a]
þ54.0, c 0.02, MeOH). The organic layer was evaporated to
D
dryness under reduced pressure to obtain the aglycone unit
(1.3 mg, 71% yield, [
spectrum and the optical rotation were identical to those of versi-
25
a
]
ꢀ176.0, c 0.01, dioxane) whose 1H NMR
D
colorin B.24
d6)
d
53.8 (C-40), 60.1 (OCH3-400), 60.6 (C-600), 73.4 (C-10), 73.7 (C-
200), 75.4 (C-20), 76.2 (C-500), 76.4 (C-300), 79.3 (C-400), 100.1 (C-100),
102.3 (C-4), 109.5 (C-5), 109.6 (C-7), 110.9 (C-8a), 111.5 (C-9a),
113.6 (C-30), 118.0 (C-2), 135.1 (C-10a), 136.2 (C-4a), 160.0 (C-1),
164.1 (C-6), 164.4 (C-8), 166.1 (C-3), 181.1 (C-10), 189.9 (C-9);
4.7. Hydrolysis of compound 9
Compound 9 (5.0 mg, 8.9 mmol) was hydrolyzed as mentioned
above. The aqueous layer was concentrated in vacuo to yield 4-O-
HRMS (ESITOF) m/z 531.1144 [MꢀH]ꢀ (calcd for: C25H23O13
,
25
methyl-D-glycopyranose (1.5 mg, 94% yield, [
a
]
þ58.2, c 0.10,
D
531.1145).
MeOH). The organic layer was evaporated to dryness under reduced
24
24
pressure to obtain the aglycone (3.0 mg, 88% yield, [
a
]
þ113.0, c
D
4.3.9. Compound 9. Orange solid; [
(MeOH) lmax (log
(3.80) nm; IR (ATR) nmax 3374, 2929, 1630, 1597, 1572, 1469, 1408,
1300, 1203, 1168, 1079 cmꢀ1 1H NMR (500 MHz, DMSO-d6)
1.54
a
]
ꢀ62.0 (c 0.07, MeOH); UV
D
0.01, MeOH) whose 1H NMR spectrum as well as optical rotation
3
) 223 (4.18), 265 (4.11), 288 (4.14), 319 (3.73), 447
were in good agreement with those of nidurufin.13,14
;
d
(2H, m, H-30), 1.55 (3H, s, H-60), 1.83 (1H, d, J¼13.1 Hz, Ha-40), 2.17
(1H, ddd, J¼6.7, 13.1, 13.1 Hz, Hb-40), 3.06 (1H, t, J¼9.1 Hz, H-400),
3.29 (1H, ddd, J¼5.2, 8.1, 8.5 Hz, H-200), 3.43e3.45 (1H, m, H-300),
3.46 (3H, s, OCH3-400), 3.51e3.53 (2H, m, H-500, Ha-600), 3.64 (1H, dd,
J¼4.9,10.2 Hz, Hb-600), 3.76e3.78 (1H, m, H-20), 4.77 (1H, t, J¼5.6 Hz,
OH-600), 5.06 (1H, s, H-10), 5.15 (1H, d, J¼8.1 Hz, H-100), 5.34 (1H, d,
J¼5.5 Hz, OH-300), 5.41 (1H, d, J¼4.1 Hz, OH-20), 5.55 (1H, d, J¼5.1 Hz,
OH-200), 6.95 (1H, d, J¼2.4 Hz, H-7), 7.06 (1H, s, H-4), 7.25 (1H, d,
4.8. Biological assays
Assay for activity against P. falciparum (K1, multidrug resistant
strain) was performed using the microculture radioisotope tech-
nique.27 Cytotoxicity to Vero cells (African green monkey kidney
fibroblasts) was performed using the green fluorescent protein
(GFP)-based method.28 Anticancer activities against KB cells (oral
human epidermoid carcinoma), MCF-7 cells (human breast cancer),

8486
J. Kornsakulkarn et al. / Tetrahedron 68 (2012) 8480e8486
8. Krasnoff, S. B.; Gibson, D. M.; Belofsky, G. N.; Gloer, K. B.; Gloer, J. B. J. Nat. Prod.
1996, 59, 485e489.
9. Bullock, E.; Roberts, J. C.; Underwood, J. G. J. Chem. Soc. 1962, 4179e4183.
10. Hamasaki, T.; Matsui, K.; Isono, K.; Hatsuda, Y. Agric. Biol. Chem. 1973, 37,
1769e1770.
11. Hatsuda, Y.; Hamasaki, T.; Ishida, M.; Matsui, K.; Hara, S. Agric. Biol. Chem. 1972,
36, 521e522.
12. Steyn, P. S.; Vleggar, R. J. Chem. Soc., Perkin Trans. 1 1974, 2250e2256.
13. Lee, Y. M.; Li, H.; Hong, J.; Cho, H. Y.; Bae, K. S.; Kim, M. A.; Kim, D. K.; Jung, J. H.
Arch. Pharm. Res. 2010, 33, 231e235.
and NCI-H187 cells (human small-cell lung cancer) were evaluated
using the resazurin microplate assay.29
Acknowledgements
Financial support from the Bioresources Research Network,
National Center for Genetic Engineering and Biotechnology (BIO-
TEC), is gratefully acknowledged.
14. Murphy, R. A. J.; Cava, M. P. J. Chem. Soc. 1984, 106, 7630e7632.
€
15. Asao, T.; BUchi, G.; Abdel-Kader, M. M.; Change, S. B.; Wick, E. L.; Wogan, G. N. J.
Am. Chem. Soc. 1963, 85, 1706e1707.
Supplementary data
16. Fredenhagen, A.; Hug, P.; Sauter, H.; Peter, H. H. J. Antibiot. 1995, 48, 199e204.
17. Tsuda, Y.; Isobe, K.; Fukushima, S.; Ageta, H.; Iwata, K. Tetrahedron Lett. 1967,
23e28.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tet.2012.07.059.
€
18. Brase, S.; Encinas, A.; Keck, J.; Nising, C. F. Chem. Rev. 2009, 109, 3903e3990.
19. Cole, R. J.; Jarvis, B. B.; Schweikert, M. A. In Hanbook of Secondary Fungal
Metabolites; Academic: Sandiago, 2003; Vol. 1, pp 545e595.
20. Birkenshaw, J. H.; Hammady, I. M. M. Biochem. J. 1957, 65, 162e166.
21. Fukuyama, K.; Tsukihara, T.; Katsube, Y.; Hamasaki, T.; Hatsuda, Y.; Tanaka, N.;
Ashida, T.; Kakudo, M. Bull. Chem. Soc. Jpn. 1975, 48, 1980e1983.
22. Siddiqui, I. R.; Bishop, C. T. Can. J. Chem. 1962, 40, 233e235.
23. Ayer, W.; Macaulay, J. B. Can. J. Chem. 1987, 65, 7e14.
24. Hamasaki, T.; Hatsuda, Y.; Terashima, N.; Renbutsu, M. Agric. Biol. Chem. 1965,
29.
References and notes
1. Ferron, P. Ann. Rev. Entomol. 1978, 23, 409e442.
2. Meekes, E. T. M.; van Voorst, S.; Joosten, N. N.; Fransen, J. J.; van Lenteren, J. C.
Mycol. Res. 2000, 1234e1240.
3. Ramakers, P. M. J.; Samson, R. A. J. Appl. Entomol. 1984, 97, 1e8.
4. Boonphong, S.; Kittakoop, P.; Isaka, M.; Palittapongarnpim, P.; Jaturapat, A.;
Canwisetkanjana, K.; Tantichareon, M.; Thebtaranonth, Y. Planta Med. 2001, 67,
279e281.
5. Isaka, M.; Palasarn, S.; Kocharin, K.; Saenboonrueng, J. J. Nat. Prod. 2005, 68,
945e946.
25. Smith, F. J. Chem. Soc. 1951, 2646e2652.
26. Casillas, L. K.; Townsend, C. A. J. Org. Chem. 1999, 64, 4050e4059.
27. Desjardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chulay, J. D. Antimicrob. Agents
Chemother. 1979, 16, 710e718.
28. Changsen, C.; Franzblau, S. G.; Palittapongarnpim, P. Antimicrob. Agents Che-
mother. 2003, 47, 3682e3687.
6. Isaka, M.; Yangchum, A.; Rachtawee, P.; Komwijit, S.; Lutthisungneon, A. J. Nat.
Prod. 2010, 73, 688e692.
29. O’Brien, J.; Wilson, I.; Orton, T.; Pongnan, F. Eur. J. Biochem. 2000, 267,
5421e5426.
7. Chutrakul, C.; Boonruangprapa, T.; Suvannakad, R.; Isaka, M.; Toojinda, T.;
Kirtikara, K. J. Appl. Microbiol. 2009, 107, 1624e1631.