Bioactive polyketides from the fungus Astrocystis sp. BCC 22166

Article abstract of DOI:10.1016/j.tet.2014.02.004

Five new compounds, phthalide 1, dihydroisocoumarin 2, and 3, pyrone 4, and benzophenone 5, together with nine known compounds, 3,4-dihydro-4,5,8- trihydroxy-3-methylisocoumarin, sclerotinin A, methyl-8-hydroxy-6- methylxanthone-1-carboxylate, sydowinin A, conioxanthone A, 1,3,8-trihydroxy-6- methylxanthone, 1,8-dihydroxy-3-methoxy-6-methylxanthone, coniochaetone B, and xylaranol B, were isolated from the fungus Astrocystis sp. BCC 22166. Structures of these compounds were elucidated using NMR spectroscopic and MS spectrometric analyses. Compound 1 exhibited antibacterial activity against Bacillus cereus (IC₅₀=12.5 μg/mL) while compound 2 showed cytotoxicity to KB and Vero cells (IC₅₀=22.6 and 48.2 μg/mL).

Full text of DOI:10.1016/j.tet.2014.02.004

Tetrahedron 70 (2014) 2129e2133
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Bioactive polyketides from the fungus Astrocystis sp. BCC 22166
Jittra Kornsakulkarn, Siriporn Saepua, Somjit Komwijit, Pranee Rachtawee,
*
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:
Five new compounds, phthalide 1, dihydroisocoumarin 2, and 3, pyrone 4, and benzophenone 5, together
with nine known compounds, 3,4-dihydro-4,5,8-trihydroxy-3-methylisocoumarin, sclerotinin A, methyl-
8-hydroxy-6-methylxanthone-1-carboxylate, sydowinin A, conioxanthone A, 1,3,8-trihydroxy-6-
methylxanthone, 1,8-dihydroxy-3-methoxy-6-methylxanthone, coniochaetone B, and xylaranol B, were
isolated from the fungus Astrocystis sp. BCC 22166. Structures of these compounds were elucidated using
NMR spectroscopic and MS spectrometric analyses. Compound 1 exhibited antibacterial activity against
Received 3 December 2013
Received in revised form 23 January 2014
Accepted 3 February 2014
Available online 7 February 2014
Keywords:
Polyketides
Bioactive secondary metabolites
Astrocystis sp.
Bacillus cereus (IC50¼12.5
m
g/mL) while compound 2 showed cytotoxicity to KB and Vero cells (IC50¼22.6
and 48.2 g/mL).
m
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
were obtained from both culture broth and mycelia extracts of
BCC 22166.
Astrocystis is a genus of fungi in the family Xylariaceae, which is
known to be a potential source for bioactive compounds.1,2 How-
ever, to our knowledge, the study of secondary metabolites of the
genus Astrocystis has never been reported. As part of our continuing
research into bioactive compounds from Thai microorganisms, we
investigated the constituents of the fungus Astrocystis sp. BCC
22166, of which the crude extract exhibited antibacterial activity
Compounds 1, 3, 4, 5, 1,8-dihydroxy-3-methoxy-6-methyl
xanthone,
3,4-dihydro-4,5,8-trihydroxy-3-methylisocoumarin,
sclerotinin A, and xylaranol B were obtained from the culture broth
extract while 1,3,8-trihydroxy-6-methylxanthone was isolated
solely from an n-hexane extract of the mycelium.
Compound 1 was obtained as a white solid, possessing the mo-
lecular formula of C12H12O6 as deduced from ESIMS, in combination
with 13C NMR spectroscopy. The IR spectrum showed major absorp-
tion bands at 3372,1752, and 1680 cmꢀ1 corresponding, respectively,
to a hydroxyl, a carbonyl, and H-bonding carbonyl group. The 1H and
13C NMR spectra revealed the presence of one methyl group, one
methoxy group, two oxymethine protons, 1,2,3,4-tetrasubstitued
benzene, two hydroxyl groups (D2O exchangeable), and two car-
boxyl carbons (Table 1). The HMBC correlations from H-3 to C-1/C-3a/
C-4, H-5 to C-3a/C-7, and H-6 to C-4/C-7a established the iso-
benzofuranone ring. The hydroxyl group was assigned at C-7 on the
basis of its chemical shift. The HMBC correlations from the methoxy
protons to C-10 and from H-5 to C-10 established the methyl-
carboxylate group at C-4. The cross peaks from H-3 to H-8 and from
H-8 to the methyl protons (H3-9) and OH-8 in the COSY spectrum and
the correlations from methyl protons to C-3/C-8, OH-8to C-8, and H-3
to C-8/C-9 in the HMBC spectrum indicated the 1-hydroxyethyl side
chain at C-3. This assignment was also supported by the NOESY
correlations from the methoxy protons to H-8 and OH-8. Therefore,
compound 1 was named as astrophthalide (Fig. 1).
against Bacillus cereus (IC50¼4.79
m
g/mL) and cytotoxic activity to
oral human epidermoid carcinoma (KB, IC50¼48.09
mg/mL) cells.
The study led to the isolation and structure elucidation of five new
compounds, phthalide 1, dihydroisocoumarin 2, and 3, pyrone 4,
and benzophenone 5, together with nine known compounds, 3,4-
dihydro-4,5,8-trihydroxy-3-methylisocoumarin (6),3 sclerotinin A
(7),4 methyl-8-hydroxy-6-methylxanthone-1-carboxylate (8),5
sydowinin A (9),6 conioxanthone A (10),7 1,3,8-trihydroxy-6-
methylxanthone (11),8 1,8-dihydroxy-3-methoxy-6-methylxan-
thone (12),9 coniochaetone B,10 and xylaranol B.11 Biological activ-
ities of these compounds were also evaluated.
2. Results and discussion
Compound 2, methyl-8-hydroxy-6-methylxanthone-1-car
boxylate, conioxanthone A, sydowinin A, and coniochaetone B
The cross peak between H-3 and H-8 in the NOESY spectrum and
the small coupling constant between these two protons (1.9 Hz)
* Corresponding author. Tel.: þ66 2 564 6700x3559; fax: þ66 2 564 6632; e-mail
address: chawanee@biotec.or.th (C. Thongpanchang).
0040-4020/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2014.02.004

2130
J. Kornsakulkarn et al. / Tetrahedron 70 (2014) 2129e2133
Table 1
showed signals for two non-chelated phenolic protons, one aro-
matic proton, one oxymethine proton, one methoxy, one meth-
ylene, and one methyl groups (Table 2). Analysis of 13C and 2D
NMR spectroscopic data revealed the structural feature of dihy-
droisocoumarin derivative. The cross peak between the methyl
protons and H-3 in the COSY spectrum together with the corre-
lations from the methyl protons to C-3/C-4 in the HMBC spec-
trum established the methyl group at C-3. The positions of the
substituents on the aromatic ring were established on the basis
of their chemical shifts together with HMBC correlations from H-
4 to C-5, methoxy protons to C-8, and H-6 to C-4a/C-5/C-7/C-8.
Cross peaks between H-6 and the two hydroxyl protons in the
NOESY spectrum also supported this assignment. Compound 2
was, therefore, assigned as 5,7-dihydroxy-O-methylmellein. The
almost identical CD spectrum and optical rotations of compound
2 and those of (3R)-5,6-dihydroxymellein15 indicated the R con-
figuration at C-3 as depicted in Fig 1.
1H (400 MHz) and 13C (100 MHz) NMR data for 1 in acetone-d6
Position
1
dC
dH mult. (J in Hz)
HMBC
1
3
3a
4
5
6
7
7a
8
9
10
7-OH
8-OH
11-OMe
169.1
85.8
150.7
119.0
131.7
120.1
167.0
108.6
68.7
15.5
169.6
d
d
d
5.88 d (1.9)
d
1, 3a, 4, 8, 9
d
d
d
7.93 d (8.5)
7.20 d (8.5)
d
3a, 7, 10
4, 7, 7a
d
d
d
4.39 m
0.76 d (6.0)
d
d
3, 8
d
11.5 s
4.39 m
4.08 s
d
d
8
10
52.7
Fig. 1. Structures of compounds 1e12.
suggested the cis relationship between H-3 and H-8. The absolute
configuration at C-8 was addressed by application of the modified
Mosher method.12 Compound 1 was treated with (R)-MTPACl and
(S)-MTPACl to afford (S)- and (R)-MTPA esters 1a and b, respectively.
The differences in chemical shift values (DdSeR, Fig. 2) were consis-
tent with the 8R configuration. Considering the relative configura-
tion deduced by NOESY and coupling constant data, the absolute
configuration at C-3 was assigned as R. The CD spectra of compound
1, which showed the same chirality as those of spirolaxine13 and
pestaphthalide B,14 also supported the R configuration at C-3.
Table 2
1H and 13C data for 2 and 3
Position
2a (In acetone-d6)
3b (In CDCl3)
dC
dH mult. (J in Hz)
dC
dH mult. (J in Hz)
1
3
4
161.9
74.5
28.6
d
169.9
75.6
28.8
d
4.50 m
2.47 dd (11.4, 16.5)
3.05 dd (2.8, 16.5)
d
4.63 m
2.68 dd (11.5, 16.7)
3.08 dd (3.2, 16.7)
d
4a
5
6
7
8
8a
9
117.9
149.5
108.1
150.2
142.4
119.0
20.3
d
131.4
137.8
159.6
99.0
160.8
100.2
20.8
60.9
55.9
d
d
d
6.73 s
d
d
6.42 s
d
d
d
d
1.42 d (6.3)
8.08 s
d
1.53 d (6.3)
3.74 s
3.90 s
d
5-OMe/OH
6-OMe
7-OH
d
d
8.54 s
3.77 s
8-OMe/OH
61.1
d
11.14 s
a
Recorded at 400 (1H) and 100 (13C) MHz.
Recorded at 500 (1H) and 125 (13C) MHz.
b
Compound 3 was obtained as a pale yellow solid, and the mo-
Fig. 2. Dd-Values (
d
SedR) of bis-(S)- and (R)-MTPA esters 1a and b.
lecular formula was deduced as C12H14O5 by ESIMS in combination
with 13C NMR spectroscopy. The 1H NMR spectrum was similar to
that of 5,6-dihydroxymellein15 except for the presence of two ad-
ditional methoxy groups (Table 2). The hydroxyl group at C-8 was
Compound 2 with the molecular formula C11H12O5 from
ESIMS was obtained as a pale yellow solid. The 1H NMR spectrum

J. Kornsakulkarn et al. / Tetrahedron 70 (2014) 2129e2133
2131
assigned on the basis of its chemical shift and the correlations from
the hydroxyl proton to C-7/C-8/C-8a in the HMBC spectrum. The
attachment of two additional methoxy groups at C-5 and C-6 was
established by HMBC correlations from these protons to their cor-
responding carbons, from H-4 to C-5, and from H-7 to C-5/C-8a.
Thus, the structure of compound 3 was determined as 5,6-
dimethoxymellein, which was known as a synthetic product16
but is now isolated for the first time as a natural product. The
potent than that of any of the compounds isolated. This observation
could result from the amount of the active compounds being too
small to be detected during the separation process.
3. Conclusion
In conclusion, five new compounds, phthalide 1, dihy-
droisocoumarin 2, naturally new dihydroisocoumarin 3, pyrone 4,
and synthetically known benzophenone 5, together with nine known
same absolute configuration of compound
3 with (3R)-5,6-
dihydroxymellein15 was indicated by the almost identical optical
rotations and CD spectra of these two compounds.
compounds,
3,4-dihydro-4,5,8-trihydroxy-3-methylisocoumarin,
sclerotinin A, methyl-8-hydroxy-6-methylxanthone-1-carboxylate,
sydowinin A, conioxanthone A, 1,3,8-trihydroxy-6-methylxanthone,
1,8-dihydroxy-3-methoxy-6-methylxanthone, coniochaetone B, and
xylaranol B, were isolated from the fungus Astrocystis sp. BCC 22166.
Compounds 1 and 3 showed weak antibacterial and cytotoxic activ-
ities, respectively.
Compound 4 was assigned the molecular formula C13H18O5 on
the basis of ESIMS, in combination with 13C NMR spectroscopy. The
1H NMR spectrum indicated the presence of three methyl groups,
one methoxy group, two oxymethine protons, two vinyl protons,
and two hydroxyl protons. The HMBC correlations from H3-12 to C-
2/C-3/C-4, from the methoxy protons (H3-13) to C-4, and from H-5
to C-3/C-4/C-6 established the
a
-pyrone substituted by the methyl
4. Experimental
and methoxy group at C-3 and C-4, respectively. The side chain at C-
6 was deduced by the COSY correlations from H3-14 to H-8, H-8 to
H-9, H-9 to H-10, and H-10 to H3-11 together with HMBC correla-
tions from H-5 to C-7, H-8 to C-6/C-14, and H3-14 to C-6/C-7/C-8.
The attachment of the hydroxyl groups at C-9 and C-10 were in-
dicated by their chemical shifts. The cross peak between H-9 and
H3-14 in the NOESY spectrum led to the proposal of E-configuration
at the 7,8-double bond. The magnitude of the allylic coupling be-
tween H-8 and H3-14 (J4¼1.1 Hz), which was the same value as that
reported for pestalopyrone,17 also supported this assignment.
Compound 4 was, thus, assigned to be astropyrone (Fig. 1). How-
ever, the absolute configurations at C-9 and C-10 were not de-
termined due to the shortage of the sample.
Compound 5 was obtained as a pale yellow solid. The ESIMS, in
combination with 13C NMR spectroscopy, gave the molecular for-
mula as C16H14O7. 1H NMR spectrum was similar to that of mon-
iliphenone3 except the methyl signal at dH 2.19 of moniliphenone
was replaced by oxymethylene signal at dH 4.52. 1H and 13C NMR
assignments were secured by the analyses of 2D NMR spectroscopic
data. The HMBC spectrum showed correlations from H-3 to C-1/C-
5/C-8, H-4 to C-2/C-6, H-5 to C-1/C-3, methoxy protons to C-8, H-30
to C-10/C-50/C-70, H-50 to C-10/C-30/C-70 and H-70 to C-30/C-50. The
attachment of hydroxyl groups at C-6, C-20, and C-60 were indicated
on the basis of their chemical shifts. Therefore, compound 5, named
as astrophenone, was depicted as shown (Fig. 1). The benzophe-
none derivatives are proposed to be the intermediates in the bio-
synthesis of fungal xanthones.18,19 Astrophenone, thus, was
identified as a precursor in the biosynthesis of the isolated sydo-
winin A (9). Although compound 5 is commercially available,20 it is
first reported to be isolated from natural source.
4.1. General procedures
Melting points were measured using a BUCHI M565 melting
point apparatus and are uncorrected. Optical rotation measure-
ments were obtained using a JASCO P-1030 polarimeter. The CD
spectrum was recorded on a JASCO J-180 spectropolarimeter. UV
and FT-IR spectra were recorded on a Spekol 1200, Analytic Jena,
spectrophotometer and
a Bruker Alpha spectrometer. NMR
spectra were recorded on Bruker AV400 and Bruker AV500D
spectrometers. ESITOF MS data were obtained on a Bruker
MicrOTOF mass spectrometer. Preparative thin layer chroma-
tography was performed on silica gel 60 GF254 (Merck). Column
chromatography was performed on silica gel 60 (70e230 mesh
ASTM, Merck). HPLC were performed using Dionex-Ultimate
3000 series equipped with a binary pump, an autosampler, and
a diode array detector.
4.2. Fungal material
The fungus Astrocystis sp. was isolated from a mangrove palm,
Nypa, at Hat Khanom-Mu Ko Thale Tai National Park, Nakhon Si
Thammarat Province, Thailand. The specimen was identified by Mr.
Jones E.B.G., BIOTEC. This fungus was deposited at the BIOTEC
Culture Collection Laboratory (BCC) as BCC 22166.
4.3. Fermentation and isolation
Astrocystis sp. BCC 22166 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
primary culture was transferred into 1 L Erlenmeyer flask con-
taining 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 a PDB liquid medium and fermentation was
carried out under shaking conditions at 200 rpm, 25 ꢁC for 20
days.
After filtration of the mycelium, the culture broth was extrac-
ted with EtOAc (3ꢂ10 L) and evaporated to dryness leaving a dark
brown gum (2.464 g). The crude extract was fractionated using
Sephadex LH 20 (3.5ꢂ50 cm), eluted with 100% MeOH, to provide
four fractions (1e4). Fraction 2 was subjected to silica gel column
chromatography (5.5ꢂ18 cm), step gradient elution with 1e20%
MeOH/CH2Cl2, to give eight fractions (2-1e2-8). Compound 3
(3.0 mg), methyl-8-hydroxy-6-methylxanthone-1-carboxylate
The structure of nine known compounds were elucidated on the
basis of HRMS and NMR spectroscopic data, which were identical in
all respects to those of 3,4-dihydro-4,5,8-trihydroxy-3-
methylisocoumarin (6),3 sclerotinin A (7),4 methyl-8-hydroxy-6-
methylxanthone-1-carboxylate (8),5 sydowinin
A
(9),6 conio-
xanthone A (10),7 1,3,8-trihydroxy-6-methylxanthone (11),8 1,8-
dihydroxy-3-methoxy-6-methylxanthone (12),9 coniochaetone
B,10 and xylaranol B.11
All isolated compounds were subjected to biological assays for
antibacterial activity against B. cereus and cytotoxicity to three
cancer cell lines, KB, MCF-7, and NCI-H187 and non-cancer Vero
cells. Compound 1 was weakly active to B. cereus (IC50 12.5
Compound 3 exhibited cytotoxic activity against KB and Vero cells
with IC50 22.6 and 48.2 g/mL, respectively. 1,3,8-Trihydroxy-6-
mg/mL).
m
methylxanthone displayed both antibacterial, against B. cereus,
and cytotoxic activities, against MCF-7, BC, and Vero cells, with IC50
12.5, 44.76, 14.026 and 88.3
mg/mL, respectively. It would be noted
that the bioactivity of the crude extract against B. cereus was more

2132
J. Kornsakulkarn et al. / Tetrahedron 70 (2014) 2129e2133
(0.5 mg), and 3,4-dihydro-4,5,8-trihydroxy-3-methylisocoumarin
(12.4 mg) were obtained from fractions 2-2, 2-3, and 2-8, re-
spectively. Purification of fraction 2-4 by silica gel column chro-
matography, using 30% EtOAc/n-hexane as eluent, followed by
preparative HPLC using reverse phase column (SunFire C18 OBD,
spectroscopic data in CDCl3, Table 2; HRMS (ESITOF) m/z 239.0919
[MþH]þ (calcd for: C12H15O5, 239.0914).
25
4.3.4. Compound 4. White solid; [
(MeOH) lmax (log
2930, 2872, 1681, 1617, 1554, 1459, 1384, 1359, 1246, 1169,
1071 cmꢀ1; 1H NMR (400 MHz, acetone-d6)
a
]
þ6.6 (c 0.34, MeOH); UV
D
3
) 227 (3.69), 324 (3.43) nm; IR (ATR) nmax 3377,
5
m
m, 19ꢂ150 mm, step gradient elution with 45e60% MeCN/H2O)
furnished compound 1 (1.6 mg), sclerotinin A (4.0 mg), and con-
iochaetone B (40.1 mg). Fraction 2-5 was subjected to silica gel
column chromatography (4.0ꢂ18 cm), using 45% EtOAc/n-hexane
as eluent, then was further purified by preparative HPLC (step
gradient elution with 20e60% MeCN/H2O) to yielded compounds
1 (7.5 mg) and 2 (14.8 mg). Compound 4 (4.6 mg), 1,8-dihydroxy-
d
1.10 (3H, d, J¼6.0 Hz,
CH3-11), 1.84 (3H, s, CH3-12), 2.0 (3H, d, J¼1.1 Hz, CH3-14),
3.78e3.81 (2H, m, H-10, OH-10), 3.96 (1H, s, OH-9), 4.0 (3H, s,
OCH3-13), 4.34 (1H, dd, J¼4.9, 8.7 Hz, H-9), 6.51 (1H, dd, J¼0.9,
8.7 Hz, H-8), 6.53 (1H, s, H-5); 13C NMR (100 MHz, acetone-d6)
d 8.2
(C-12), 12.5 (C-14), 18.3 (C-11), 56.3 (OCH3-13), 70.4 (C-10), 72.5 (C-
9), 93.4 (C-5), 101.6 (C-3), 128.6 (C-7), 133.9 (C-8), 159.5 (C-6), 163.7
(C-2), 166.1 (C-4); HRMS (ESITOF) m/z 277.1045 [MþNa]þ (calcd for:
3-methoxy-6-methylxanthone (2.0 mg), and sydowinin
A
(4.2 mg) were provided from fraction 2-6 after consecutive puri-
fication by silica gel column chromatography (using 60% EtOAc/n-
hexane as eluent) and preparative HPLC (step gradient elution
with 10e40% MeCN/H2O). Fractions 2-7 was further purified by
preparative HPLC (step gradient elution with 20e50% MeCN/H2O)
to afford compound 5 (4.2 mg) and xylaranol B (34.5 mg). Fraction
3 was subjected to silica gel column chromatography, using 5%
MeOH/CH2Cl2 as an eluent, to give six fractions (3-1e3-6). After
further purification by preparative HPLC (step gradient elution
with 20e40% MeCN/H2O), coniochaetone B (6.5 mg) and con-
ioxanthone A (7.6 mg) were obtained from fractions 3-2 and 3-4,
respectively.
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 reduced pressure. Water (200 mL)
was added, and the mixture was extracted with n-hexane
(3ꢂ200 mL), followed by EtOAc (3ꢂ200 mL). Purification of the
crude EtOAc extracted (0.585 g) by silica gel column chromatog-
raphy (4.0ꢂ18 cm), step gradient elution with 65% EtOAc/n-
hexanee10% MeOH/EtOAc, followed by preparative thin layer
C13H18O5Na, 277.1046).
4.3.5. Compound 5. Pale yellow solid; UV (MeOH) lmax (log
(4.08), 284 (3.93), 355 (3.39) nm; IR (ATR) nmax 3241, 2930, 2875,
1714, 1633, 1599, 1492, 1433, 1375, 1298, 1021 cmꢀ1 1H NMR
3
) 218
;
(400 MHz, acetone-d6) d
3.69 (3H, s, OCH3-8), 4.52 (2H, s, H-70), 6.38
(2H, s, H-30, H-50), 7.12 (1H, d, J¼7.9 Hz, H-5), 7.28 (1H, t, J¼7.9 Hz, H-
4), 7.47 (1H, d, J¼7.9 Hz, H-3); 13C NMR (100 MHz, acetone-d6)
d 51.5
(OCH3-8), 63.4 (C-70), 104.9 (C-30, C-50), 110.4 (C-10), 120.1 (C-5),
120.8 (C-3), 128.5 (C-2), 128.8 (C-4), 133.7 (C-1), 152.2 (C-40), 153.7
(C-6),162.3 (C-20, C-60),166.2 (C-8), 200.4 (C-7); HRMS (ESITOF) m/z
341.0631 [MþNa]þ (calcd for: C16H14O7Na, 341.0632).
4.4. Preparation of the bis-MTPA ester 1a and 1b
Compound 1 (2.8 mg) was treated with (R)-MTPACl (20 mL) in
CH2Cl2 (0.2 mL) and pyridine (0.2 mL) at room temperature for 16 h.
The mixture was diluted with EtOAc and washed with H2O, and the
organic layer was evaporated under reduced pressure. The residue
was purified by preparative thin layer chromatography (45% EtOAc/
n-hexane) to obtained bis-(S)-MTPA ester 1a (4.2 mg). Similarly,
bis-(R)-MTPA ester 1b was prepared from compound 1 (2.1 mg) and
chromatography provided compound
2 (2.6 mg), methyl-8-
hydroxy-6-methylxanthone-1-carboxylate (2.5 mg), sydowinin A
(2.4 mg), and conioxanthone A (8.6 mg). The dark brown oil
(0.489 g), obtained from n-hexane extraction of the mycelium, was
subjected to silica gel column chromatography step gradient elu-
tion with 10% EtOAc/n-hexanee100% EtOAc, then was further pu-
rified by preparative thin layer chromatography to yield methyl-8-
hydroxy-6-methylxanthone-1-carboxylate (20.9 mg), 1,3,8-
(S)-MTPACl (20 mL) and was obtained in 3.2 mg after purification by
preparative thin layer chromatography.
4.4.1. Bis-(S)-MTPA ester 1a. 1H NMR (400 MHz, acetone-d6)
d 1.04
(3H, d, J¼6.6 Hz, H-9), 3.60 (3H, s, OCH3 of MTPA), 3.76 (3H, s, OCH3
of MTPA), 3.93 (3H, s, OCH3-11), 5.91 (1H, dq, J¼2.0, 6.6 Hz, H-8),
6.17 (1H, d, J¼2.0 Hz, H-3), 7.50 (3H, m, ArH of MTPA), 7.60 (5H, m,
ArH of MTPA), 7.64 (1H, d, J¼8.3 Hz, H-6), 7.75 (2H, m, ArH of
MTPA), 8.19 (1H, d, J¼8.3 Hz, H-5); HRMS (ESITOF) m/z 707.1324
[MþNa]þ (calcd for: C32H26O10F6Na, 707.1322).
trihydroxy-6-methylxanthone (2.0 mg), and coniochaetone
(4.3 mg).
B
4.3.1. Compound 1. White solid; mp 149.5e151.5 ꢁC; [
0.14, MeOH); UV (MeOH) lmax (log
(3.40) nm; CD (MeOH)
(260), þ1.31 (273), ꢀ0.51 (299); IR (ATR) nmax 3372, 2921, 2851,
1752, 1680, 1616, 1593, 1446, 1353, 1307, 1250, 1220, 1133,
1090 cmꢀ1; 1H and 13C NMR spectroscopic data in acetone-d6, Table
1; HRMS (ESITOF) m/z 275.0529 [MþNa]þ (calcd for: C12H12O6Na,
275.0526).
a
]
26 þ127.6 (c
D
3
) 227 (4.10), 250 (3.79), 305
(nm) ꢀ3.79 (228), þ5.05 (242), ꢀ3.36
D
3
4.4.2. Bis-(S)-MTPA ester 1b. 1H NMR (400 MHz, acetone-d6)
d 0.93
(3H, d, J¼6.6 Hz, H-9), 3.66 (3H, s, OCH3 of MTPA), 3.76 (3H, s, OCH3
of MTPA), 3.92 (3H, s, OCH3-11), 5.91 (1H, dq, J¼2.0, 6.6 Hz, H-8),
6.34 (1H, d, J¼2.0 Hz, H-3), 7.50 (3H, m, ArH of MTPA), 7.59 (5H, m,
ArH of MTPA), 7.61 (1H, d, J¼8.3 Hz, H-6), 7.75 (2H, m, ArH of
MTPA), 8.21 (1H, d, J¼8.3 Hz, H-5); HRMS (ESITOF) m/z 707.1320
[MþNa]þ (calcd for: C32H26O10F6Na, 707.1322).
26
4.3.2. Compound 2. Pale yellow solid; mp 110.0e112.0 ꢁC; [
ꢀ132.9 (c 0.19, CHCl3); UV (MeOH) lmax (log
(3.64), 335 (3.50) nm; CD (EtOH)
(nm) ꢀ10.77 (212), þ1.02 (244),
ꢀ4.10 (267), þ0.27 (292); IR (ATR) nmax 3431, 3222, 2924, 2851,
1681,1597,1498,1459,1342,1274,1241,1193,1050 cmꢀ1; 1H and 13
a]
D
3 ) 223 (3.77), 257
D
3
4.5. Biological assays
C
Cytotoxicity to Vero cells (African green monkey kidney fibro-
blasts) were performed using the green fluorescent protein (GFP)-
based method21 and the standard compound, ellipticine, showed
NMR spectroscopic data in acetone-d6, Table 2; HRMS (ESITOF) m/z
247.0579 [MþNa]þ (calcd for: C11H12O5Na, 247.0577).
IC50 value of 0.73 mg/mL. Anticancer activities against KB cells (oral
26
4.3.3. Compound 3. Pale yellow solid; mp 131.5e132.5 ꢁC; [
ꢀ35.2 (c 0.11, CHCl3); UV (MeOH) lmax (log
312 (3.38) nm; CD (EtOH)
a
]
human epidermoid carcinoma), MCF-7 cells (human breast cancer),
and NCI-H187 cells (human small-cell lung cancer) and antibacte-
rial activity to B. cereus were evaluated using the resazurin micro-
plate assay.22 The IC50 values of standard compound, doxorubicin,
D
3
) 227 (3.65), 264 (3.61),
D
3
(nm) ꢀ4.39 (238), þ3.13 (254), ꢀ2.15
(270), þ1.25 (289); IR (ATR) nmax 2926, 2853, 1667, 1625, 1494, 1438,
1376,1338,1311,1283,1249,1204,1168,1098 cmꢀ1; 1H and 13C NMR
against KB, MCF-7, and NCI-H187 cells were 0.50, 8.09, and 0.16 mg/

J. Kornsakulkarn et al. / Tetrahedron 70 (2014) 2129e2133
2133
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