Dihydronaphthalenones from endophytic fungus Fusarium sp. BCC14842

Article abstract of DOI:10.1016/j.tet.2011.07.078

Eleven new dihydronaphthalenones 1-11, together with five known compounds; 5-hydroxydihydrofusarubin C (12), javanicin, bostrycoidin, anhydrofusarubin, and 3-O-methylfusarubin, were isolated from the endophytic fungus Fusarium sp. BCC14842. Structures were elucidated by analyses of the NMR spectroscopic and mass spectrometry data. Javanicin, 3-O-methylfusarubin, compounds 3 and 7 exhibited antimycobacterial and cytotoxic activities. Javanicin also displayed antifungal activity with IC₅₀ of 6.16 μg/mL, while compounds 2, 4, 5, 8, and 12 showed only cytotoxic activity.

Full text of DOI:10.1016/j.tet.2011.07.078

Tetrahedron 67 (2011) 7540e7547
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Dihydronaphthalenones from endophytic fungus Fusarium sp. BCC14842
Jittra Kornsakulkarn a, Kulvadee Dolsophon b, Nattawut Boonyuen a, Tanapong Boonruangprapa a,
,
,
,
*
Pranee Rachtawee a, Samran Prabpai b c, Palangpon Kongsaeree b c, Chawanee Thongpanchang a
a National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road,
Klong Luang, Pathumthani 12120, Thailand
b Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand
c Center for Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
Eleven new dihydronaphthalenones 1e11, together with five known compounds; 5-hydroxydihydrofusarubin
C (12), javanicin, bostrycoidin, anhydrofusarubin, and 3-O-methylfusarubin, were isolated from the en-
dophytic fungus Fusarium sp. BCC14842. Structures were elucidated by analyses of the NMR spectro-
scopic and mass spectrometry data. Javanicin, 3-O-methylfusarubin, compounds 3 and 7 exhibited
antimycobacterial and cytotoxic activities. Javanicin also displayed antifungal activity with IC50 of
Received 30 March 2011
Received in revised form 24 June 2011
Accepted 26 July 2011
Available online 31 July 2011
6.16
m
g/mL, while compounds 2, 4, 5, 8, and 12 showed only cytotoxic activity.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Dihydronaphthalenones
Bioactive secondary metabolites
Endophytic fungi
Fusarium sp.
1. Introduction
and mycelia extract of BCC14842. Anhydrofusarubin, compounds 1,
and 5e11 were obtained from the culture broth extract, while
bostrycoidin was solely isolated from an n-hexane extract of the
mycelium.
Endophytic fungi in the genus Fusarium are reported to produce
a diversity of bioactive secondary metabolites including naph-
thoquinones, e.g., javanicin, fusarubin, solaniol, marticin, and
nectraiafurone.1e3 This class of compounds is of interest due to the
broad spectrum of their biological activities, such as antibacterial,4e6
antifungal,6,7 phytotoxic,1 insecticidal,8 and cytotoxic9 properties. As
part of our research program on bioactive compounds from fungi, we
investigated the constituents of endophytic fungus Fusarium sp.
BCC14842, of which the crude extract exhibited strong cytotoxic
Compound 1 was obtained as a red solid. The molecular formula
was established by HRMS (ESITOF), in combination with 13C NMR
spectroscopy, as C14H16O6. The IR spectrum showed major ab-
sorption bands at 3415, 1712, and 1625 cmꢀ1 corresponding,
respectively, to a hydroxyl, a carbonyl and a conjugated carbonyl
group. The unusually low IR absorption frequency of the conjugated
carbonyl is brought about by the H-bonding between the
activity against human breast cancer (MCF-7, IC50 value of 8.14
mg/
unsaturated ketone and b
-OH group. The 1H NMR spectrum
mL). The study led to the isolation of eleven new compounds, 4-
hydroxydihydronorjavanicin (1), dihydronaphthalenone 2 and its
diastereomer 3, 5-hydroxydihydrofusarubins A, B, and D (4e6), and
the methyl ether derivatives 7e11, together with five known
showed signals for one chelated hydroxy proton, one aromatic
proton, two hydroxy protons, two methine protons, one methoxy,
one methyl, and two methylene groups. The cross peak between
H-2 and H-3, H-3, and H-4 in COSY spectrum and the correlations
from H-2 to C-1/C-4/C-8a, from H-3 to C-1/C-4a, from H-4 to
C-2/C-4a/C-5/C-8a, and from H-7 to C-1, C-8a in HMBC spectrum
established a dihydronaphthalenone nucleus. The location of OH-5,
OH-8 and methoxy group (H3-12) at C-5, C-8 and C-6 of the
benzene ring, respectively, was indicated by HMBC correlations
between these protons and their corresponding carbons. The ace-
tonyl side chain at C-3 was determined by the COSY correlation
between H-3 and H-9 and the HMBC correlations from H3-11 to
C-9/C-10 and from H-9 to C-2/C-3/C-4/C-10. The remaining
OH-group was indicated at C-4 on the basis of its chemical shift,
together with HMBC correlation from this proton (OH-4) to the
compounds, 5-hydroxydihydrofusarubin
C
(12),10 javanicin,4
bostrycoidin,11 anhydrofusarubin,12 and 3-O-methylfusarubin.9
Biological activities of these compounds were also evaluated.
2. Results and discussion
Javanicin, 3-O-methylfusarubin, 5-hydroxydihydrofusarubin C
(12), and compounds 2e4 were obtained from both culture broth
* Corresponding author. E-mail address: chawanee@biotec.or.th (C. Thongpanchang).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.07.078

J. Kornsakulkarn et al. / Tetrahedron 67 (2011) 7540e7547
7541
corresponding carbon. The relative configuration of compound 1
was deduced on the basis of the coupling constants and NOESY
data. The cross peak from axial Ha-2 to H-4 in NOESY spectrum
established the axial orientation of H-4 and the small coupling
constant between H-3 and H-4 (4.7 Hz) indicated the cis relation-
ship between H-3 and H-4. Compound 1 was therefore assigned as
4-hydroxydihydronorjavanicin.
1624 cmꢀ1 for an unsaturated carbonyl group of which its IR ab-
sorption frequency is unusually low due to the H-bonding between
the unsaturated ketone and b
-OH group. The 1H and 13C NMR data
as well as the optical rotation of compound 12 were identical to
those of 5-hydroxydihydrofusarubin,10 however, the configura-
tional assignment of the stereocenters has not clearly been repor-
ted. The relative configuration of compound 12 has now been
Compound 2 was obtained as a pale orange solid, possessing the
molecular formula C15H16O6 as deduced from HRMS (ESITOF), in
combination with 13C NMR spectroscopy. The 1H NMR data of
compound 2 were closely related to those of compound 1. Analysis
of 13C and 2D NMR spectra revealed the similar structures of these
two compounds except for the presence of an additional oxy-
methylene group at C-2 and the replacement of hydroxyl group at
C-4 with alkoxy group. The COSY correlation between H-2 and H-13
and the HMBC correlations from H-13 to C-1/C-2/C-3/C-4 estab-
lished an oxa-bicyclic ring system. The relative configuration of
compound 2 was assigned on the basis of NOESY spectroscopic
data, which the cross peak from H-3 to Hb-13 suggested the trans
relationship between H-2 and H-3, and between H-3 and H-4. The
absolute configurations at C-2, C-3, and C-4 were addressed by the
X-ray diffraction analysis of 6, 8-bis-O-(3-bromobenzoyl) derivative
13 (Fig. 1), which were assigned as R, S, and R, respectively.
Compound 3 with the molecular formula C15H16O6 from HRMS
(ESITOF) was obtained as a pale orange solid. The UV and IR spectra
of compound 3 were almost identical to those of compound 2. Their
1H NMR spectra were also similar. Analysis of 2D NMR data
revealed the same structure for these two compounds except for
the stereogenic centers at C-2 and C-4. The NOESY correlation
between H-9 and Hb-13 indicated cis relationship between H-2,
H-3 and H-4. The X-ray diffraction analysis of 6, 8- bis-O-
(4-bromobenzoyl) derivative 14 (Fig. 2) established the S, S, and S
configurations at C-2, C-3, and C-4, respectively.13
Fig. 1. X-ray crystal structure of compound 13.
deduced on the basis of the coupling constants and NOESY data.
The large coupling constant between axial Ha-1 and H-10a (9.5 Hz)
and the small coupling constant between H-10a and H-4a (3.8 Hz)
established the axial and equatorial orientation of H-10a and H-4a,
respectively, which indicated the cis ring junction through H-10a
and H-4a. The cross peak between H-10a and axial Ha-4 in NOESY
spectrum also supported the axial position of H-10a. The cis re-
lationship between H-4a and H-5 was deduced from the cross peak
Compound 12 was obtained as an orange solid, possessing the
molecular formula C15H18O7 as deduced from HRMS (ESITOF), in
combination with 13C NMR spectroscopy. The IR spectrum showed
absorption bands at 3274 cmꢀ1 for a hydroxyl group and at

7542
J. Kornsakulkarn et al. / Tetrahedron 67 (2011) 7540e7547
Fig. 4. Dd-Values (dS dR) of (S)- and (R)-THENA esters 15a and 15b.
ꢀ
Compound 7 with the molecular formula C16H20O7 from HRMS
(ESITOF) was obtained as a red solid. The 1H NMR spectrum was
similar to that of compound 4 except for the presence of an addi-
tional methoxy signal at dH 3.18. The HMBC correlation from the
additional methoxy protons (H3-13) to C-3 revealed the re-
placement of hydroxyl group in compound 4 with methoxy group
in compound 7 at C-3. Analysis of NOESY spectrum and the cou-
pling constants suggested that the relative configurations at C-3,
C-4a, C-5, and C-10a are the same as those in compound 4. The
almost identical optical rotation and CD spectrum of compound 4
and compound 7 indicated the same absolute configuration of
these two compounds. Therefore, the absolute configurations at
C-3, C-4a, C-5, and C-10a of compound 7 were assigned as R, S, R,
and R, respectively, and compound 7 was determined as 5-hydroxy-
3-methoxydihydrofusarubin A.
Fig. 2. X-ray crystal structure of compound 14.
between H-4a and H-5 in NOESY spectrum. The NOESY correlation
between axial Ha-4 and CH3-3 indicated the equatorial orientation
of CH3 group at C-3, which established cis relationship between
CH3 group and H-10a (Fig. 3). Compound 12 was therefore de-
termined as 5-hydroxydihydrofusarubin C.
Compound 5 with the molecular formula C15H18O7 from HRMS
(ESITOF) was obtained as a pale orange solid. The UV and IR
spectra were identical to those of compound 4. Their 1H NMR
spectra were also similar. The coupling constants and 2D NMR
data analysis revealed that the structures of these two com-
pounds were the same except for the relative configuration at C-
10a. The large coupling constant (13.2 Hz) between axial Ha-4
and H-4a and the small coupling constant (3.2 Hz) between ax-
ial Ha-1 and H-10a established the axial and equatorial orienta-
tion of H-4a and H-10a, respectively, which indicated the cis ring
junction through H-4a and H-10a. The cross peak between
equatorial Hb-4 and H-5 in NOESY spectrum deduced the trans
relationships between H-4a and H-5 (Fig. 3). Therefore, com-
pound 5 was the (10aS)-epimer of 5-hydroxydihydrofusarubin A
and was assigned as 5-hydroxydihydrofusarubin B.
Compound 8 with the molecular formula C16H20O7 from HRMS
(ESITOF) was obtained as a yellow solid. The 1H NMR spectrum
indicated that compound 8 was closely related to compound 5
except for the presence of an additional methoxy group at dH 3.36.
The position of methoxy group at C-5 was determined by the HMBC
correlation from these protons to C-5. The relative configurations at
C-3, C-4a, C-5, and C-10a of compound 8 were assigned to be the
same as those of compound 5 by the analysis of NMR data of these
Fig. 3. Selected NOESY correlations of compounds 4e6 and 12.
Compound 4 was obtained as a pale orange solid. The HRMS
(ESITOF) spectrum indicated the molecular formula as C15H18O7.
The UV and IR spectra were identical to those of compound 12.
Their 1H NMR spectrum was also closely related. Analysis of 2D
NMR data as well as coupling constants revealed the same struc-
tural features for both compounds with the difference being in the
relative configuration at C-4a. The trans diaxial arrangement be-
tween H-4a and H-10a and between H-4a and H-5 were assigned
on the basis of large coupling constant of 13.1 Hz and 9.8 Hz, re-
spectively. The cross peak between H-10a and H-5 in NOESY
spectrum also supported the cis relationship of these two protons.
The absolute configuration at C-5 was addressed by application of
THENA (Tetrahydro-1,4-epoxynaphthalene-1-carboxylic acid)
method.14 Compound 4 was treated with MeI (2.5 equiv) in K2CO3/
acetone to afford dimethylated derivative, which was further con-
verted into (S)- and (R)-THENA esters 15a and 15b, respectively. The
differences in chemical shift values (DdSeR, Fig. 4) were consistent
with the 5R configuration.15 Therefore, compound 4 was de-
termined as (3R, 4aS, 5R, 10aR)-5-hydroxydihydrofusarubin A.
two compounds. Compound
5-methoxydihydrofusarubin B.
8 was therefore determined as
Compound 9 was obtained as a red solid. The molecular formula
was deduced from HRMS (ESITOF), in combination with 13C NMR
data as C17H22O7. The 1H NMR spectrum was similar to that of
compound 8 except for the presence of an additional methoxy
signal at dH 3.19. The HMBC correlation from the additional
methoxy protons to C-3 revealed the replacement of hydroxyl
group in compound 8 with methoxy group in compound 9 at C-3.
The analysis of NOESY spectrum and the coupling constants sug-
gested that the relative configurations at C-3, C-4a, C-5, and C-10a
were the same as those in compound 8. Therefore, compound 9 was
determined as 3,5-dimethoxydihydrofusarubin B.
Compound 6 was obtained as an orange solid. The HRMS (ESI-
TOF) spectrum indicated the molecular formula as C15H18O7. The
UV and IR spectra were identical to those of compound 4. Their 1H

J. Kornsakulkarn et al. / Tetrahedron 67 (2011) 7540e7547
7543
NMR spectrum was also closely related. The analysis of 2D NMR
data as well as coupling constants revealed the same structure for
both compounds with the difference in relative configuration at
C-5. The trans diaxial arrangement between H-4a and H-10a and cis
relationship between H-4a and H-5 were assigned on the basis of
large coupling constant of 12.0 Hz and small coupling constant of
3.1 Hz, respectively. The cross peak between equatorial Ha-4 and
H-5 in NOESY spectrum also supported cis relationship of H-4a and
H-5. Application of modified Mosher method16 with compound 6,
by methylation and conversion into bis-(S)- and bis-(R)-MTPA
esters 16a and 16b, established (5S) configuration (Fig. 5). Com-
pound 6 was (5S)-epimer of compound 4 and was therefore
assigned as (3R, 4aS, 5S, 10aR)-5-hydroxydihydrofusarubin D.
Compound 11 was obtained as a red solid. The molecular for-
mula was deduced from HRMS (ESITOF), in combination with 13C
NMR data as C17H22O7. The 1H NMR spectrum indicated that com-
pound 11 was closely related to compound 10 except for the
presence of an additional methoxy group at dH 3.36. The position of
methoxy group at C-5 was determined by the HMBC correlation
from these protons to C-5. The configurations at C-3, C-4a, C-5, and
C-10a of compound 11 were assigned to be the same as those of
compound 6 by the analysis of NMR data as well as the almost
identical of both optical rotations and CD spectra of these
two compounds. Compound 11 was therefore determined as
3,5-dimethoxydihydrofusarubin D.
Due to the shortage of the sample, the absolute configuration of
compounds 1, 5, 8, 9, and 12 were not determined.
The structure of five known compounds were elucidated on the
basis of HRMS and NMR spectroscopic data, which were identical to
those of 5-hydroxydihydrofusarubin C (12),10 javanicin,4 bos-
trycoidin,11 anhydrofusarubin,12 and 3-O-methylfusarubin.9
Six new compounds, 2e5, 7, and 8, 5-hydroxydihydrofusarubin
A (12), javanicin, and 3-O-methylfusarubin were tested for anti-
fungal, antimycobacterial, and cytotoxic activities (Table 1). Jav-
anicin, 3-O-methylfusarubin, compounds 3 and 7 showed weak to
moderate antimycobacterial activity against Mycobacterium tuber-
culosis. Only javanicin was strongly active against Candida albicans
Fig. 5. Dd-Values (dS dR) of bis-(S)- and bis (R)-MTPA esters 16a and 16b.
ꢀ
with IC50 of 6.16
moderate cytotoxic activity against KB and NCI-H187 cell lines (IC50
1.62e31.69 g/mL). Compounds 2 and 5 were inactive against both
MCF-7 and vero cells, while compounds 4 and 7 were inactive
against only vero cells.
mg/mL. All of these compounds exhibited weak to
Compound 10 with the molecular formula C16H20O7 from HRMS
(ESITOF) was obtained as an orange solid. The 1H NMR spectrum
was similar to that of compound 6 except for the presence of an
additional methoxy signal at dH 3.17. The HMBC correlation from
m
Table 1
Biological activities of javanicin, 3-O-methylfusarubin, 5-hydroxydihydrofusarubin C (12), compounds 2e5, 7, and 8
Compound
Anti-TB, MIC (
m
g/mL)
Antifungal, IC50
(
m
g/mL)
Cytotoxicity, IC50
KB cells
(mg/mL)
MCF-7 cells
NCIeH187 cells
Vero cells
Javanicina
25.0
50.0
>200
>200
25.0
>200
>200
50.0
>200
0.03
d
6.16
>50
>50
>50
>50
>50
>50
>50
>50
d
1.62
9.28
9.34
31.69
31.29
21.25
31.50
25.48
23.59
d
3.40
8.50
16.58
>50
>50
10.99
>50
14.50
23.43
d
1.91
5.38
9.99
23.10
26.88
12.14
11.89
26.03
13.47
d
6.98
12.77
26.76
>50
12.94
>50
>50
>50
28.26
d
3-O-Methyl fusarubina
Compound12a
Compound 2
Compound 3
Compound 4
Compound 5
Compound 7
Compound 8
Isoniazidb
Amphotericin Bc
Doxorubicind
Ellipticined
0.06
d
d
d
d
d
d
0.28
0.32
0.90
d
0.07
1.21
d
d
d
1.72
a
Antimicrobial activities of javanicin, 3-O-methylfurarubin, and 5-hydroxydihydrofusarubin A against other organisms using a different method were previously
reported.6,9,10
b
c
Anti-TB control.
Antifungal control.
Cytotoxicity controls.
d
the additional methoxy protons (H3-13) to C-3 revealed the re-
placement of hydroxyl group in compound 6 with methoxy group
in compound 10 at C-3. The analysis of NOESY spectrum and the
coupling constants suggested that the relative configurations at C-
3, C-4a, C-5, and C-10a are the same as those in compound 6. The
almost identical optical rotation and CD spectrum of compound 6
and compound 10 indicated the same absolute configuration of
these two compounds. Therefore, the absolute configurations at C-
3, C-4a, C-5, and C-10a of compound 10 were assigned as R, S, S, and
R, respectively, and compound 10 was determined as 5-hydroxy-3-
methoxydihydrofusarubin D.
3. Conclusion
In conclusion,11 newcompounds, 4-hydroxydihydronorjavanicin
(1), dihydronaphthalenone
2
and its diastereomer 3, 5-
hydroxydihydrofusarubins A, B, and D (4e6), and the methyl ether
derivatives 7e11, together with five known compounds; 5-
hydroxydihydrofusarubin C (12), javanicin, bostrycoidin, anhy-
drofusarubin, and 3-O-methylfusarubin, were isolated from the en-
dophytic fungus Fusarium sp. BCC14842. Javanicin, 3-O-
methylfusarubin, compounds 3 and 7 exhibited antimycobacterial
and cytotoxic activities. Only javanicin displayed antifungal activity

7544
J. Kornsakulkarn et al. / Tetrahedron 67 (2011) 7540e7547
with IC50 of 6.16
mg/mL, while compounds 2, 4, 5, 8, and 12 showed
(6.7 mg). More of compound 4 (29.7 mg) was obtained from
only cytotoxic activity.
fractions 4 and 5 after further purification by preparative HPLC
(step gradient elution with 20e60% MeCN/H2O) and silica gel
column chromatography, respectively.
4. Experimental
The cells were macerated in MeOH for 3 days and then in CH2Cl2
for 3 days. The MeOH and CH2Cl2 extracts were combined and
evaporated under reduced pressure. Water (200 mL) was added,
and the mixture was extracted with n-hexane (3ꢂ200 mL), fol-
lowed by EtOAc (3ꢂ200 mL). The dark brown solid (0.31 g),
obtained from n-hexane extraction of the mycelium, was subjected
to preparative HPLC (step gradient elution with 15e60% MeCN/
H2O) to furnish bostrycoidin (3.2 mg), javanicin (5.7 mg), 3-O-
methylfusarubin (1.1 mg), and compound 3 (1.4 mg). Purification of
the crude EtOAc extract (0.57 g) by preparative HPLC (step gradient
elution with 15e50% MeCN/H2O) provided an additional amount of
javanicin (2.5 mg), compounds 2 (11.4 mg), 3 (19.9 mg), 4 (10.1 mg),
and 5-hydroxydihydrofusarubin C (12, 2.1 mg).
4.1. General procedures
Melting points were measured using an electrothermali 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 VECTOR 22 spec-
trometer. The CD spectra were recorded on a JASCO J-180 spec-
tropolarimeter. NMR spectra were recorded on Bruker DRX400 and
Bruker AV500D spectrometers. ESITOF MS data were obtained on
Micromass LCT and Bruker micrOTOF mass spectrometers.
4.2. Fungal material
4.3.1. Compound 1. Red solid; ½a D25
ꢀ57.7 (c 0.02, acetone); UV
ꢃ
The fungus used in this study was isolated from Bamboo leaf by
Mr. Prasert Srikitikulchai, collected in the Bamboo forest at Nam
Nao National Park, Phetchabun Province, Thailand. This fungus was
deposited as BCC14842 at the BIOTEC Culture Collection Laboratory
(BCC) on January 5, 2004. It was identified as Fusarium sp. on the
basis of the sequence data of 18S rDNA, 28S rDNA, and internal
transcribed spacer (ITS) genes by Mr. Nattawut Boonyuen.
(MeOH) lmax (log ) 214 (4.00), 242 (4.07), 282 (3.95), 357 (3.85)
3
nm; IR (KBr) nmax 3415, 2925, 2853, 1712, 1625, 1493, 1440, 1356,
1297, 1277, 1226, 1205, 1163, 1095, 1079, 1036 cmꢀ1 1H NMR
(500 MHz, acetone-d6)
2.09 (3H, s, 11-CH3), 2.36 (1H, dd, J¼5.7,
;
d
17.2 Hz, Ha-2), 2.45 (1H, dd, J¼8.1, 17.7 Hz, Ha-9), 2.65 (1H, dd,
J¼5.5, 17.7 Hz, Hb-9), 2.78 (1H, m, H-3), 3.03 (1H, dd, J¼4.7, 17.2 Hz,
Hb-2), 3.93 (3H, s, 12-OCH3), 4.74 (1H, d, J¼4.3 Hz, OH-4), 5.04 (1H,
dd, J¼4.3, 4.7 Hz, H-4), 6.45 (1H, s, ArH-7), 7.92 (1H, s, OH-5), 12.63
4.3. Fermentation and isolation
(1H, s, OH-8); 13C NMR (125 MHz, CDCl3)
d 29.2 (C-11), 36.3 (C-3),
38.4 (C-2), 44.6 (C-9), 55.7 (C-12), 66.1 (C-4), 98.9 (C-7), 108.1 (C-
8a), 126.6 (C-4a), 137.4 (C-5), 155.6 (C-6), 158.6 (C-8), 202.0 (C-1),
206.0 (C-10); HRMS (ESITOF) m/z 303.0837 [MþNa]þ (calcd for:
C14H16O6Na, 303.0839).
Fusarium sp. BCC14842 was maintained on potato dextrose agar
at 25 ꢁC, and 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 liquid medium (PDB). The fungus was cul-
tivated under shaking conditions at 250 rpm, 25 ꢁC for 20 days.
After filtration of the mycelium, the culture broth was
extracted with (3ꢂ10 L) EtOAc and evaporated to dryness leaving
a dark brown solid (2.45 g). The crude extract was fractionated
using a silica gel column (4ꢂ20 cm), eluted with 2% MeOH/
CH2Cl2, to provide five fractions. Fraction 1 was subjected to silica
gel column chromatography (3ꢂ15 cm), using 30% EtOAc/n-hex-
ane as eluent, to give seven fractions (1e1e1e7). Javanicin
(24.6 mg) and compound 3 (100.2 mg) were obtained from
fractions 1e3 and 1e7, respectively. Fraction 1e2 was subjected to
preparative HPLC using a reverse phase column (SunFire C18 OBD,
4.3.2. Compound 2. Pale orange solid; ½a D24
ꢀ78.22 (c 0.12, ace-
ꢃ
tone); UV (MeOH) lmax (log ) 215 (4.10), 244 (4.12), 284 (4.03), 355
3
(3.94) nm; IR (KBr) nmax 3443, 2923, 1713, 1632, 1486, 1441, 1386,
1292, 1201, 1162, 1106 cmꢀ1; 1H NMR (500 MHz, CDCl3)
d
2.04 (3H,
s, 11-CH3), 2.41 (2H, d, J¼6.9 Hz, H-9), 2.98 (1H, m, H-3), 3.20 (1H, t,
J¼4.9, H-2), 3.80 (1H, d, J¼9.0 Hz, Ha-13), 3.81 (3H, s,12-OCH3), 4.26
(1H, dd, J¼4.9, 9.0 Hz, Hb-13), 5.62 (1H, d, J¼5.1 Hz, H-4), 5.94 (1H,
s, OH-5), 6.29 (1H, s, ArH-7),11.74 (1H, s, OH-8); 13C NMR (125 MHz,
CDCl3)
d 30.2 (C-11), 40.4 (C-9), 43.1 (C-3), 52.9 (C-2), 56.3 (C-12),
68.3 (C-13), 72.4 (C-4), 99.0 (C-7), 108.1 (C-8a), 125.0 (C-4a), 135.7
(C-5), 155.0 (C-6), 158.5 (C-8), 202.3 (C-1), 205.8 (C-10); HRMS
(ESITOF) m/z 315.0843 [MþNa]þ (calcd for: C15H16O6Na, 315.0839).
4.3.3. Compound 3. Pale orange solid; ½a D26
ꢀ39.9 (c 0.12, acetone);
ꢃ
UV (MeOH) lmax (log ) 214 (3.68), 243 (3.73), 282 (3.61), 352 (3.54)
3
nm; IR (CHCl3) nmax 3453, 2922, 1713, 1633, 1487, 1441, 1389, 1362,
1286, 1225, 1201, 1166, 1108 cmꢀ1; 1H NMR (500 MHz, CDCl3)
d
2.19
5
m
m, 19,150 mm, step gradient elution with 20e60% MeCN/H2O,
(3H, s, 11-CH3), 2.49 (1H, dd, J¼5.8, 18.0 Hz, Ha-9), 2.76 (1H, dd,
J¼7.9, 18.0 Hz, Hb-9), 2.87 (1H, dd, J¼5.8, 7.9 Hz, H-3), 2.99 (1H, d,
J¼5.6, H-2), 3.77 (1H, d, J¼9.3 Hz, Ha-13), 3.91 (3H, s, 12-OCH3), 4.19
(1H, dd, J¼5.6, 9.3 Hz, Hb-13), 5.39 (1H, s, H-4), 5.41 (1H, s, OH-5),
6.38 (1H, s, ArH-7), 11.66 (1H, s, OH-8); 13C NMR (125 MHz, CDCl3)
flow rate 10 mL/min) to furnish compounds 8 (4.7 mg), 9
(3.2 mg), and javanicin (1.4 mg). After further purification by silica
gel chromatography, anhydrofusarubin (5.5 mg), compounds 2
(43.9 mg), 5-hydroxydihydrofusarubin C (12, 9.1 mg), and an
additional amount of javanicin (14.7 mg) were obtained from
fraction 1e1, 1e4, 1e5, and 1e6, respectively. Trituration of frac-
tion 2 with MeOH followed by filtration provided the insoluble
compound 7 (330 mg) as a red solid. The filtrate was subjected to
preparative HPLC (step gradient elution with 15e60% MeCN/H2O,
flow rate 10 mL/min) to afford 3-O-methylfusarubin (11.6 mg),
compounds 4 (67.9 mg), 11 (5.0 mg), 2 (14.4 mg), 3 (14.1 mg), and
7 (123.1 mg). Further purification of fraction 3 by preparative
HPLC (step gradient elution with 10e50% MeCN/H2O) yielded
compounds 1 (4.2 mg), 5 (9.1 mg), 6 (3.2 mg), 10 (5.0 mg) and an
additional amount of compounds 2 (2.6 mg), 4 (35.6 mg), and 8
d
30.3 (C-11), 44.0 (C-3, C-9), 54.1 (C-2), 56.4 (C-12), 66.4 (C-13),
74.6 (C-4), 99.0 (C-7), 107.3 (C-8a), 127.6 (C-4a), 134.5 (C-5), 154.2
(C-6), 158.8 (C-8), 203.5 (C-1), 205.7 (C-10); HRMS (ESITOF) m/z
315.0844 [MþNa]þ (calcd for: C15H16O6Na, 315.0839).
4.3.4. Compound 4. Pale orange solid; ½a D26
ꢀ123.2 (c 0.03, ace-
ꢃ
tone); UV (MeOH) lmax (log ) 217 (3.94), 243 (4.06), 282 (3.97), 354
3
(3.87) nm; CD (MeOH)
D
3
(nm) þ16.36 (243), ꢀ1.56 (264), þ12.85
(286), ꢀ9.96 (310), þ1.05 (335), ꢀ2.47 (360); IR (CHCl3) nmax 3442,
2927, 2360, 1626, 1492, 1441, 1387, 1268, 1246, 1203, 1162,
1091 cmꢀ1 1H NMR (500 MHz, acetone-d6)
; d 1.40 (3H, s, 11-CH3),

J. Kornsakulkarn et al. / Tetrahedron 67 (2011) 7540e7547
7545
1.45 (1H, ddd, J¼2.0, 12.0, 13.2 Hz, Ha-4), 2.36 (1H, dd, J¼3.8,
1710, 1627, 1491, 1441, 1395, 1260, 1202, 1163, 1085, 1018 cmꢀ1
;
1H
1.07 (1H, ddd, J¼1.7, 13.2, 13.3 Hz, Ha-
12.0 Hz, Hb-4), 2.49 (1H, m, H-4a), 2.59 (1H, ddd, J¼5.1, 10.1, 13.1 Hz,
H-10a), 3.90 (3H, s, 12-OCH3), 3.96 (1H, dd, J¼10.1, 11.2 Hz, Ha-1),
4.02 (1H, dd, J¼5.1, 11.2 Hz, Hb-1), 4.63 (1H, d, J¼2.0 Hz, OH-3),
4.87 (1H, dd, J¼4.5, 9.8 Hz, H-5), 5.51 (1H, d, J¼4.5 Hz, OH-5),
6.45 (1H, s, ArH-8), 8.89 (1H, br s, OH-6), 12.48 (1H, s, OH-9); 13C
NMR (500 MHz, acetone-d6)
d
4), 1.20 (3H, s,11-CH3), 1.61 (1H, dd, J¼3.2,13.2 Hz, Hb-4), 2.80 (1H, t,
J¼3.8 Hz, H-10a), 3.10 (1H, m, H-4a), 3.37 (3H, s, 14-OCH3), 3.96 (3H,
s,12-OCH3), 4.02 (1H, dd, J¼3.8,11.2 Hz, Ha-1), 4.41 (1H, d, J¼11.2 Hz,
Hb-1), 4.63 (1H, s, OH-3), 4.64 (1H, s, H-5), 6.50 (1H, s, ArH-8), 7.64
(1H, br s, OH-6), 12.89 (1H, s, OH-9); 13C NMR (125 MHz, acetone-d6)
NMR (125 MHz, acetone-d6)
d 29.3 (C-11), 38.3 (C-4), 39.9 (C-4a),
45.0 (C-10a), 55.6 (C-12), 58.6 (C-1), 72.1 (C-5), 94.3 (C-3), 99.1 (C-
8), 107.9 (C-9a), 127.3 (C-5a), 137.8 (C-6), 155.9 (C-7), 158.8 (C-9),
201.3 (C-10); HRMS (ESITOF) m/z 333.0949 [MþNa]þ (calcd for:
C15H18O7Na, 333.0945).
d 29.3 (C-11), 33.6 (C-4a), 35.9 (C-4), 40.6 (C-10a), 55.7 (C-12), 56.0
(C-13), 58.1 (C-1), 72.6 (C-5), 94.0 (C-3), 99.2 (C-8), 108.3 (C-9a),
123.6 (C-5a),137.9 (C-6), 155.5 (C-7),159.1 (C-9), 202.2 (C-10); HRMS
(ESITOF) m/z 347.1108 [MþNa]þ (calcd for: C16H20O7Na, 347.1101).
4.3.5. Compound 5. Pale orange solid; ½a D26
ꢃ
ꢀ51.9 (c 0.06, acetone);
4.3.9. Compound 9. Red solid; ½a D24
ꢀ46.8 (c 0.16, acetone); UV
ꢃ
UV (MeOH) lmax (log
3
) 214 (4.01), 243 (4.07), 282 (3.97), 357 (3.86)
(MeOH) lmax (log ) 214 (3.91), 243 (3.93), 283 (3.85), 357 (3.74),
3
nm; CD (MeOH)
D
3
(nm) þ11.15 (219), ꢀ3.55 (245), þ4.30 (273),
423 (2.59) nm; CD (MeOH)
D
3
(nm) þ0.88 (215), ꢀ4.72 (238), þ4.05
þ1.12 (291), þ9.03 (315), ꢀ7.51 (350); IR (CHCl3) nmax 3443, 2925,
(271), þ1.18 (295), þ8.19 (315), ꢀ6.64 (351); IR (KBr) nmax 3378,
1627, 1494, 1442, 1387, 1293, 1204, 1164, 1105, 1032 cmꢀ1; 1H NMR
2925, 2853, 1707, 1627, 1490, 1441, 1396, 1364, 1258, 1163, 1091,
(500 MHz, acetone-d6)
d
1.08 (1H, dt, J¼1.7, 13.2 Hz, Ha-4), 1.21 (3H,
1034, 1019 cmꢀ1 1H NMR (500 MHz, acetone-d6)
; d 1.09 (1H, t,
s, 11-CH3), 1.63 (1H, dd, J¼3.2, 13.2 Hz, Hb-4), 2.94 (1H, m, H-10a),
2.98 (1H, m, H-4a), 3.94 (3H, s, 12-OCH3), 4.01 (1H, dd, J¼3.2,
11.2 Hz, Ha-1), 4.28 (1H, br s, OH-5), 4.47 (1H, dd, J¼1.4, 11.2 Hz, Hb-
1), 4.59 (1H, d, J¼1.7 Hz, OH-3), 4.99 (1H, s, H-5), 6.46 (1H, s, ArH-8),
7.52 (1H, br s, OH-6), 12.86 (1H, s, OH-9); 13C NMR (125 MHz, ac-
J¼13.2 Hz, Ha-4), 1.10 (3H, s, 11-CH3), 1.62 (1H, ddd, J¼1.0, 4.1,
13.2 Hz, Hb-4), 2.81 (1H, m, H-10a), 3.04 (1H, m, H-4a), 3.19 (3H, s,
13-OCH3), 3.36 (3H, s, 14-OCH3), 3.64 (1H, dd, J¼3.5, 11.2 Hz, Ha-1),
3.96 (3H, s, 12-OCH3), 4.44 (1H, d, J¼11.2 Hz, Hb-1), 4.63 (1H, d,
J¼3.0 Hz, H-5), 6.49 (1H, s, ArH-8), 7.66 (1H, s, OH-6), 12.86 (1H, s,
etone-d6)
d
29.3 (C-11), 36.2 (C-4a), 36.3 (C-4), 40.4 (C-10a), 55.7 (C-
OH-9); 13C NMR (125 MHz, acetone-d6)
d 22.8 (C-11), 33.2 (C-4a),
12), 58.1 (C-1), 63.92 (C-5), 94.0 (C-3), 98.9 (C-8), 108.2 (C-9a), 126.5
(C-5a), 137.2 (C-6), 155.6 (C-7), 158.7 (C-9), 202.3 (C-10); HRMS
(ESITOF) m/z 333.0946 [MþNa]þ (calcd for: C15H18O7Na, 333.0945).
35.9 (C-4), 40.3 (C-10a), 47.2 (C-13), 55.7 (C-12), 56.0 (C-14), 58.6
(C-1), 72.5 (C-5), 96.9 (C-3), 99.2 (C-8), 108.2 (C-9a), 123.5 (C-5a),
137.9 (C-6), 155.6 (C-7), 159.1 (C-9), 201.9 (C-10); HRMS (ESITOF) m/
z 361.1261 [MþNa]þ (calcd for: C17H22O7Na, 361.1258).
4.3.6. Compound 6. Orange solid; ½a D25
ꢀ64.0 (c 0.13, acetone); UV
ꢃ
(MeOH) lmax (log
3
) 214 (3.95), 243 (4.01), 282 (3.90), 357 (3.81)
4.3.10. Compound 10. Orange solid; ½a D25
ꢀ106.8 (c 0.23, acetone);
ꢃ
nm; CD (MeOH)
D
3
(nm) þ20.35 (211), ꢀ11.35 (229), þ6.25 (247),
UV (MeOH) lmax (log ) 214 (3.68), 243 (3.72), 282 (3.61), 357 (3.52)
3
ꢀ3.49 (270), þ1.75 (292), ꢀ3.16 (312), þ0.52 (341); IR (KBr) nmax
nm; CD (MeOH)
ꢀ1.86 (270), þ1.18 (292), ꢀ1.94 (312), þ0.56 (341); IR (KBr) nmax
3470, 2929,1745,1630,1554,1492,1441,1371,1226,1161,1054 cmꢀ1
1H NMR (500 MHz, acetone-d6)
1.31 (3H, s, 11-CH3), 1.76 (1H, dd,
D
3
(nm) þ6.67 (211), ꢀ3.69 (229), þ3.65 (247),
3403, 2923, 2852, 1715, 1629, 1492, 1441, 1391, 1294, 1212, 1611,
1061, 1026 cmꢀ1; 1H NMR (500 MHz, acetone-d6)
d
1.40 (3H, s, 11-
;
CH3), 1.76 (1H, dd, J¼3.8, 12.8 Hz, Ha-4), 1.96 (1H, ddd, J¼2.0, 12.4,
12.8 Hz, Hb-4), 2.43 (1H, ddd, J¼3.1, 12.4, 12.0 Hz, H-4a), 3.00 (1H,
ddd, J¼5.1, 11.7, 12.0 Hz, H-10a), 3.92 (3H, s, 12-OCH3), 3.94 (1H, m,
Ha-1), 4.08 (1H, dd, J¼5.1, 11.2 Hz, Hb-1), 4.19 (1H, d, J¼4.5 Hz, OH-
5), 4.55 (1H, d, J¼2.0 Hz, OH-3), 4.98 (1H, m, H-5), 6.45 (1H, s, ArH-
8), 7.45 (1H, s, OH-6), 12.52 (1H, s, OH-9); 13C NMR (125 MHz, ac-
d
J¼3.5, 12.8 Hz, Ha-4), 1.98 (1H, dd, J¼12.3, 12.8 Hz, Hb-4), 2.37 (1H,
ddd, J¼3.5, 11.8, 12.3 Hz, H-4a), 3.01 (1H, ddd, J¼5.1, 11.4, 11.8 Hz, H-
10a), 3.17 (3H, s, 13-OCH3), 3.56 (1H, dd, J¼11.2, 11.4 Hz, Ha-1), 3.93
(3H, s,12-OCH3), 4.10 (1H, dd, J¼5.1,11.2 Hz, Hb-1), 4.21 (1H, br s, OH-
5), 4.96 (1H, s, H-5), 6.45 (1H, s, ArH-8), 7.55 (1H, br s, OH-6), 12.49
etone-d6)
d
29.3 (C-11), 37.3 (C-4a), 37.4 (C-4), 41.0 (C-10a), 55.7 (C-
(1H, s, OH-9); 13C NMR (125 MHz, acetone-d6)
d 22.9 (C-11), 37.0 (C-
12), 59.0 (C-1), 62.3 (C-5), 94.6 (C-3), 98.9 (C-8), 108.0 (C-9a), 128.7
(C-5a), 136.3 (C-6), 155.3 (C-7), 158.8 (C-9), 203.0 (C-10); HRMS
(ESITOF) m/z 333.0947 [MþNa]þ (calcd for: C15H18O7Na, 333.0945).
4a), 37.6 (C-4), 40.6 (C-10a), 47.0 (C-13), 55.7 (C-12), 59.5 (C-1), 62.1
(C-5), 97.5 (C-3), 98.9 (C-8), 107.9 (C-9a), 128.5 (C-5a), 136.3 (C-6),
155.4 (C-7), 158.8 (C-9), 202.7 (C-10); HRMS (ESITOF) m/z 347.1107
[MþNa]þ (calcd for: C16H20O7Na, 347.1101).
4.3.7. Compound 7. Red solid; ½a D26
ꢀ148.8 (c 0.09, acetone); UV
ꢃ
(MeOH) lmax (log
3
) 216 (4.09), 243 (4.18), 282 (4.09), 353 (3.97)
4.3.11. Compound 11. Red solid; ½a D24
ꢀ35.5 (c 0.25, acetone); UV
ꢃ
nm; CD (MeOH)
D
3
(nm) þ23.21 (243), ꢀ2.89 (264), þ18.0 (284),
(MeOH) lmax (log ) 214 (3.90), 243 (3.88), 282 (3.80), 357 (3.65),
3
ꢀ13.45 (310), þ1.14 (335), ꢀ3.98 (360); IR (CHCl3) nmax 3443, 2923,
424 (2.91) nm; CD (MeOH)
D
3
(nm) þ1771 (211), ꢀ5.26 (229), þ4.83
2360, 1626, 1493, 1441, 1388, 1289, 1245, 1203, 1161, 1045 cmꢀ1; 1H
(244), ꢀ2.09 (266), þ1.64 (290), ꢀ2.68 (312), þ0.95 (339); IR (KBr)
nmax 3416, 2922, 2851, 1715, 1628, 1490, 1460, 1441, 1390, 1291, 1261,
NMR (500 MHz, acetone-d6)
d 1.31 (3H, s, 11-CH3), 1.49 (1H, dd,
J¼11.7, 13.0 Hz, Ha-4), 2.35 (1H, dd, J¼3.9, 13.0 Hz, Hb-4), 2.44
(1H, m, H-4a), 2.61 (1H, ddd, J¼5.0, 10.7, 13.1 Hz, H-10a), 3.18 (3H, s,
13-OCH3), 3.59 (1H, dd, J¼10.7, 11.5 Hz, Ha-1), 3.90 (3H, s, 12-OCH3),
4.04 (1H, dd, J¼5.0, 11.5 Hz, Hb-1), 4.85 (1H, dd, J¼4.9, 10.1 Hz, H-5),
5.49 (1H, d, J¼4.9 Hz, OH-5), 6.45 (1H, s, ArH-8), 8.86 (1H, br s, OH-
1243, 1203, 1161, 1093 cmꢀ1; 1H NMR (500 MHz, acetone-d6)
d 1.29
(3H, s, 11-CH3), 1.81 (1H, dd, J¼3.9, 13.0 Hz, Hb-4), 1.92 (1H, dd,
J¼12.6, 13.0 Hz, Ha-4), 2.41 (1H, m, H-4a), 2.90 (1H, m, H-10a), 3.15
(3H, s, 13-OCH3), 3.36 (3H, s, 14-OCH3), 3.50 (1H, t, J¼11.1 Hz, Ha-1),
3.95 (3H, s,12-OCH3), 4.09 (1H, dd, J¼5.0,11.1 Hz, Hb-1), 4.63 (1H, d,
J¼2.0 Hz, H-5), 6.48 (1H, s, ArH-8), 7.75 (1H, s, OH-6), 12.63 (1H, s,
6), 12.44 (1H, s, OH-9); 13C NMR (125 MHz, acetone-d6)
d 22.8 (C-
11), 38.5 (C-4), 39.5 (C-4a), 44.6 (C-10a), 47.0 (C-13), 55.6 (C-12),
59.1 (C-1), 71.9 (C-5), 97.3 (C-3), 99.1 (C-8), 107.9 (C-9a), 127.2 (C-
5a), 137.8 (C-6), 160.0 (C-7), 158.8 (C-9), 201.1 (C-10); HRMS (ESI-
TOF) m/z 347.1103 [MþNa]þ (calcd for: C16H20O7Na, 347.1101).
OH-9); 13C NMR (125 MHz, acetone-d6)
d 22.8 (C-11), 37.5 (C-4a),
37.7 (C-4), 41.4 (C-10a), 47.0 (C-13), 55.8 (C-12), 56.9 (C-14), 59.7
(C-1), 70.9 (C-5), 97.5 (C-3), 99.3 (C-8), 107.9 (C-9a), 125.5 (C-5a),
136.9 (C-6), 155.3 (C-7), 159.6 (C-9), 202.4 (C-10); HRMS (ESITOF)
m/z 361.1264 [MþNa]þ (calcd for: C17H22O7Na, 361.1258).
4.3.8. Compound 8. Yellow solid; ½a D27
ꢀ84.0 (c 0.06, acetone); UV
ꢃ
(MeOH) lmax (log
3
) 216 (4.06), 243 (4.10), 282 (4.01), 357 (3.92) nm;
4.3.12. Compound 12. Orange solid; ½a D24
ꢀ172.5 (c 0.03, acetone);
ꢃ
CD (MeOH)
D
3
(nm) þ5.06 (217), ꢀ4.62 (240), þ5.03 (271), þ1.03
UV (MeOH) lmax (log ) 217 (3.94), 243 (4.06), 282 (3.97), 354 (3.87)
3
(290), þ11.29 (315), ꢀ9.34 (351); IR (KBr) nmax 3415, 2928, 2854,
nm; CD (MeOH)
D
3 (nm) ꢀ68.04 (233), þ31.25 (254), ꢀ17.35 (274),

7546
J. Kornsakulkarn et al. / Tetrahedron 67 (2011) 7540e7547
þ10.31 (293), ꢀ8.90 (318), þ4.54 (338), þ8.78 (362); IR (KBr) nmax
Crystal data for compound 14: C29H22Br2O8, MW¼658.31, tri-
clinic, dimensions: 0.15ꢂ0.10ꢂ0.10 mm3, D¼1.441 g/cm3, space
3274, 2935, 2852, 1624, 1492, 1459, 1442, 1410, 1382, 1369, 1258,
1224, 1204, 1155, 1125, 1097 cmꢀ1; 1H NMR (500 MHz, CDCl3)
d
1.58
group P1, Z¼2, a¼9.3789 (8ꢁ), b¼12.2707 (ꢁ11), c¼14.6566 (13) A,
ꢀ
3
ꢁ
ꢀ
(3H, s, 11-CH3), 1.99 (1H, dd, J¼4.0, 11.6 Hz, Ha-4), 2.03 (1H, d,
J¼11.6 Hz, Hb-4), 2.94 (1H, ddd, J¼3.4, 3.8, 4.0 Hz, H-4a), 3.00 (1H,
ddd, J¼3.8, 8.7, 9.5 Hz, H-10a), 3.80 (1H, dd, J¼9.5, 11.3 Hz, Ha-1),
3.93 (1H, s, OH-5), 3.94 (3H, s, 12-OCH3), 4.00 (1H, dd, J¼8.7,
11.3 Hz, Hb-1), 5.34 (1H, d, J¼3.4 Hz, H-5), 5.65 (1H, br s, OH-6), 6.48
a¼100.09 (1) , b¼102.66(1) , g¼107.43(1) , V¼1516.8(2) A , re-
flections collected/unique: 14,278/8742 (Rint¼0.0433), number of
observation [>2
wR2¼0.2267. Flack parameter¼0.11 (2).
s
(I)] 7,308, final R indices [I>2
s
(I)]: R1¼0.0751,
Crystallographic data (excluding structure factors) for the
structures 13 and 14 in this paper have been deposited with the
Cambridge Crystallographic Data Centre as supplementary publi-
cation no. CCDC 818559 and 818560, respectively. Copies of the
data can be obtained free of charge, on application to CCDC, 12
Union Road, Cambridge CB2 1EZ, UK, (fax: þ44 (0) 1223 336033 or
e-mail: deposit@cdcc.cam.ac.uk).
(1H, s, ArH-8),12.00 (1H, s, OH-9); 13C NMR (125 MHz, CDCl3)
d 23.9
(C-11), 38.9 (C-4a), 40.1 (C-4), 45.0 (C-10a), 56.4 (C-12), 62.8 (C-1),
72.0 (C-5), 100.1 (C-8), 106.2 (C-3), 107.2 (C-9a), 121.2 (C-5a), 137.8
(C-6), 154.5 (C-7), 158.7 (C-9), 203.1 (C-10); HRMS (ESITOF) m/z
309.0961 [MꢀH]ꢀ (calcd for: C15H17O7, 309.0969).
4.4. Preparation of bis-O-(bromobenzoyl) derivatives 13 and 14
4.6. Preparation of the THENA esters 15a and 15b
To the suspension of 2 (10 mg) and NaH (2.9 mg) inTHF (1 mL) was
added the solution of 3-bromobenzoyl chloride (10.0
mL) in THF
Compound 4 (3.8 mg) was methylated with MeI (2.5 equiv) in
K2CO3 (2.5 equiv) and acetone (0.5 mL) at room temperature for
18 h. The reaction mixture was evaporated then diluted with H2O
and extracted with EtOAc. The organic layer was concentrated
under reduced pressure to leave dark brown solid, which was pu-
rified by column chromatography on silica gel (using 40% EtOAc/n-
hexane as eluent) to furnish the dimethylated product (4.0 mg) as
a pale orange solid. The replacement of hydroxyl groups at C-3
and C-6 with methoxy groups (dH 3.17 and 3.78) was revealed in the
3,6-O-dimethyl ether derivative.
(0.5 mL), at 0 ꢁC. The mixturewas left stirring at room temperature for
1 h, diluted with satd NaHCO3 (2 mL) and then extracted with EtOAc
(2 mL). The organic layer was concentrated under reduced pressure,
then purified by preparative HPLC (step gradient elution with
50e100% MeCN/H2O) to yield a white solid of 13 (10.1 mg). Com-
pound 14 was prepared from 3 (10 mg) and 4-bromobenzoylchloride
(2 equiv) and was obtained in 11.1 mg as a white solid after purifi-
cation by preparative HPLC. Recrystallization of both 13 (10.1 mg) and
14 (11.1 mg) in 1:1 CH2Cl2/MeOH gave colorless needles.
The dimethylated product (1.8 mg) was treated with (S)-THE-
4.4.1. Bis-O-(3-bromobenzoyl) derivative 13. Colorless needles; mp
NACl (20
mL) in CH2Cl2 (0.2 mL) and pyridine (0.2 mL) at room
171.6e173.5 ꢁC; 1H NMR (400 MHz, CDCl3)
d 2.08 (3H, s, 11-CH3),
temperature for 16 h. The mixture was diluted with EtOAc (2 mL)
and washed with H2O (2 mL), and the organic layer was evaporated
under reduced pressure. The residue was purified by preparative
HPLC (step gradient elution with 50e100% MeCN/H2O) to obtained
the (S)-THENA ester 15a (1.4 mg). Similarly, (R)-THENA ester 15b
was prepared from the dimethylated product (1.8 mg) and (R)-
THENACl (20 mL) and was obtained in 1.5 mg after purification by
preparative HPLC. The assignment of protons for 15a and 15b was
achieved by analysis of COSY and NOESY data.
2.47 (1H, dd, J¼7.9, 18.9 Hz, Ha-9), 2.58 (1H, dd, J¼8.0, 18.9 Hz,
Hb-9), 2.98 (1H, m, H-3), 3.18 (1H, t, J¼4.8, H-2), 3.80 (1H, d,
J¼9.0 Hz, Ha-13), 3.88 (3H, s, 12-OCH3), 4.19 (1H, dd, J¼4.8, 9.0 Hz,
Hb-13), 5.30 (1H, m, H-4), 6.81 (1H, s, ArH-7), 7.42 (2H, dt, J¼2.0,
7.8 Hz, 3-bromobenzoyl), 7.79 (2H, t, J¼8.8 Hz, 3-bromobenzoyl),
8.15 (2H, d, J¼7.4 Hz, 3-bromobenzoyl), 8.36 (2H, d, J¼2.0 Hz,
3-bromobenzoyl); HRMS (ESITOF) m/z 656.9824 and 660.9752
[MþH]þ (calcd for: C29H23O879Br2, 656.9754; calcd for:
C29H23O881Br2, 660.9723).
4.6.1. (S)-THENA ester 15a. White solid; 1H NMR (500 MHz, ace-
4.4.2. Bis-O-(4-bromobenzoyl) derivative 14. Colorless needles; mp
tone-d6)
d
1.17 (3H, s, 11-CH3), 1.24 (1H, t, J¼13.3 Hz, Ha-4), 1.37 and
110.0e112.0 ꢁC; 1H NMR (400 MHz, acetone-d6)
d 2.08 (3H, s,11-CH3),
1.63 (2H, m, CH2 of THENA), 1.87 (1H, dd, J¼3.5, 13.3 Hz, Hb-4), 2.16
(2H, m, CH2 of THENA), 2.97 (1H, m, H-4a), 3.19 (3H, s, 13-OCH3),
3.23 (1H, m, H-10a), 3.70 (1H, dd, J¼3.2, 11.3 Hz, Ha-1), 3.71 (3H, s,
15-OCH3), 3.96 (3H, s, 12-OCH3), 4.49 (1H, d, J¼11.3 Hz, Hb-1), 5.41
(1H, d, J¼4.6 Hz, CH of THENA), 6.31 (1H, d, J¼2.4 Hz, H-5), 6.62 (1H,
s, ArH-8), 7.20 (2H, m, ArH of THENA), 7.30 (1H, dd, J¼2.1, 6.8 Hz,
ArH of THENA), 7.46 (1H, dd, J¼2.1, 6.9 Hz, ArH of THENA); HRMS
(ESITOF) m/z 533.1785 [MþNa]þ (calcd for: C28H30O9Na, 533.1782).
2.61 (1H, dd, J¼6.6,18.2 Hz, Ha-9), 2.71 (1H, dd, J¼6.8,18.2 Hz, Hb-9),
2.82(1H, m, H-3), 3.09 (1H, d, J¼5.1 Hz, H-2), 3.61 (1H, d, J¼9.1 Hz, Ha-
13), 3.94 (3H, s,12-OCH3), 4.16 (1H, dd, J¼5.5, 9.1 Hz, Hb-13), 5.20 (1H,
m, H-4), 7.14 (1H, s, ArH-7), 7.79 (2H, d, J¼8.3 Hz, 3-bromobenzoyl),
7.81 (2H, t, J¼8.4 Hz, 3-bromobenzoyl), 8.09 (2H, d, J¼8.3 Hz, 3-
bromobenzoyl), 8.14 (2H, d, J¼8.4 Hz, 3-bromobenzoyl); HRMS
(ESITOF) m/z 656.9843 and 660.9760 [MþH]þ (calcd for:
C29H23O879Br2, 656.9754; calcd for: C29H23O881Br2, 660.9723).
4.6.2. (R)-THENA ester 15b. White solid; 1H NMR (500 MHz, ace-
4.5. X-ray crystallographic analysis of 13 and 14
tone-d6)
d
1.16 (3H, s, 11-CH3), 1.23 (1H, t, J¼13.2 Hz, Ha-4), 1.36 and
1.64 (2H, m, CH2 of THENA), 1.86 (1H, dd, J¼3.8, 13.2 Hz, Hb-4), 2.14
(2H, m, CH2 of THENA), 2.86 (1H, m, H-4a), 3.18 (3H, s, 13-OCH3),
3.19 (1H, m, H-10a), 3.66 (1H, dd, J¼3.2, 11.4 Hz, Ha-1), 3.78 (3H, s,
15-OCH3), 4.00 (3H, s, 12-OCH3), 4.45 (1H, d, J¼11.4 Hz, Hb-1), 5.42
(1H, d, J¼4.6 Hz, CH of THENA), 6.34 (1H, d, J¼2.5 Hz, H-5), 6.66 (1H,
s, ArH-8), 7.12e7.21 (2H, m, ArH of THENA), 7.31 (2H, d, J¼7.5 Hz,
ArH of THENA); HRMS (ESITOF) m/z 533.1787 [MþNa]þ (calcd for:
C28H30O9Na, 533.1782).
X-ray diffraction data of 13 and 14 were measured on a
BrukereNonius kappaCCD diffractometer with graphite mono-
ꢀ
chromated Mo K
a
radiation (
l
¼0.71073 A) at 298 (2) K for 13 and at
150 (2) K for 14. The structures were solved by direct methods by
SIR97,17 and refined with full-matrix least-squares calculations on
F2 using SHELXL-97.18
Crystal data for compound 13: 2(C29H22Br2O8)$CH2Cl2, MW¼
1401.523, monoclinic, dimensions: 0.15ꢂ0.15ꢂ0.10 mm3,
D¼1.663 g/cm3, space group C2, Z¼ꢁ2, a¼13.8367 (3), b¼9.64360
3
ꢀ
ꢀ
(10), c¼20.9872 (4) A,
b
¼91.496 (1) , V¼2799.5 (1) A , reflections
4.7. Preparation of the bis-MTPA esters 16a and 16b
collected/unique: 24,140/5404 (Rint¼0.0239), number of observa-
tion [>2 (I)] 4,978, final indices [I>2
(I)]: R1¼0.0391,
wR2¼0.0926. Flack parameter¼0.048 (7).
s
R
s
The bis-MTPA esters, 16a and 16b, were prepared from com-
pound 6 with (ꢀ)-(R)-MTPACl and (þ)-(S)-MTPACl, respectively, by

J. Kornsakulkarn et al. / Tetrahedron 67 (2011) 7540e7547
7547
the same method as that used for 15a and 15b and were obtained as
a white solid in 1.5 mg and 1.7 mg, respectively.
National Research Universities Initiative and Center of Excellence
for Innovation in Chemistry (PERCH-CIC) for support and the X-ray
facility.
4.7.1. Bis-(S)-MTPA ester 16a. White solid; 1H NMR (400 MHz,
acetone-d6)
d
1.19 (3H, s, 11-CH3), 1.23 (1H, t, J¼12.7 Hz, Ha-4), 1.85
Supplementary data
(1H, dd, J¼3.5, 12.7 Hz, Hb-4), 2.50 (1H, m, H-4a), 2.77 (1H, m,
H-10a), 3.14 (3H, s, 13-OCH3), 3.46 (1H, m, Ha-1), 3.47 (3H, s, OCH3
of MTPA), 3.76 (3H, s, OCH3 of MTPA), 3.88 (1H, dd, J¼5.1, 11.6 Hz,
Hb-1), 3.96 (3H, s, 15-OCH3), 4.01 (3H, s, 12-OCH3), 6.69 (1H, d,
J¼2.3 Hz, H-5), 6.80 (1H, s, ArH-8), 7.44 (3H, m, ArH of MTPA), 7.53
(3H, m, ArH of MTPA), 7.60 (2H, m, ArH of MTPA), 7.75 (2H, m, ArH
of MTPA); HRMS (ESITOF) m/z 793.2059 [MþNa]þ (calcd for:
C37H36O11F6Na, 793.2054).
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.tet.2011.07.078. These data
include NMR spectra of the most important compounds described
in this article.
References and notes
1. McLean, M. Mycopathologia 1996, 133, 163e179.
4.7.2. Bis-(R)-MTPA ester 16b. White solid; 1H NMR (400 MHz,
2. Medentsev, A. G.; Akimenko, V. K. Phytochemistry 1998, 47, 935e959.
3. Thrane, U.; Adler, A.; Clasen, P.; Galvano, F.; Langseth, W.; Lew, H.; Logrieco, A.;
Nielsen, K. F.; Ritieni, A. Int. J. Food Microbiol. 2004, 95, 257e266.
4. Arnstein, H. R. V.; Cook, A. H. J. Chem. Soc. 1947, 1021e1028.
5. Baker, R. A.; Tatum, J. H.; Nemee, S. J. Mycopathologia 1990, 111, 9e15.
6. McCulloch, A. W.; McInnes, A. G.; Smith, D. G. Can. J. Chem. 1982, 60,
2943e2949.
7. Tatum, J. H.; Baker, R. A.; Berry, R. E. Phytochemistry 1987, 26, 2499e2500.
8. Claydon, N.; Grove, J. F.; Pople, M. J. Invertebr. Pathol. 1977, 30, 216e223.
9. Kurobane, I.; Zaita, N.; Fukuda, A. J. Antibiot. 1986, 39, 205e214.
10. Itsuo, K.; Shunji, S.; Akihiko, M.; Akio, F. EP 0234431 A2, 1987.
11. Arsenault, G. P. Tetrahedron Lett. 1965, 45, 4033e4037.
acetone-d6)
d
1.27 (3H, s, 11-CH3), 1.49 (1H, t, J¼12.7 Hz, Ha-4), 1.93
(1H, dd, J¼3.4, 12.7 Hz, Hb-4), 2.77 (1H, m, H-4a), 2.78 (1H, m,
H-10a), 3.18 (3H, s, 13-OCH3), 3.57 (1H, t, J¼11.0 Hz, Ha-1), 3.46 (3H,
s, OCH3 of MTPA), 3.75 (3H, s, OCH3 of MTPA), 3.94 (3H, s, 14-OCH3),
3.99 (3H, s,12-OCH3), 4.01 (1H, dd, J¼4.0,11.0 Hz, Hb-1), 6.70 (1H, d,
J¼2.1 Hz, H-5), 6.79 (1H, s, ArH-8), 7.47 (3H, m, ArH of MTPA), 7.53
(3H, m, ArH of MTPA), 7.57 (2H, m, ArH of MTPA), 7.77 (2H, m, ArH
of MTPA); HRMS (ESITOF) m/z 793.2053 [MþNa]þ (calcd for:
C37H36O11F6Na, 793.2054).
12. Ruelius, H. W.; Gauhe, A. Ann. Chem. 1950, 569, 38e59.
13. Intrinsically, the crystal of compound 14 did not diffract well at ambient
temperature and the crystal structure could not be obtained. Diffraction
4.8. Biological assays
quality was improved by data collection at 150
K and structure de-
termination was then possible. Extensive re-crystallization efforts failed to
yield better quality diffraction data. From the “checkcif” results, analysis of
crystal packing was carried out. There was a void space in the crystal and
that might contribute to the poor crystal packing and hence the relatively
high wR2 value. It should be noted that no solvent molecules were observed
in the void cavity. The relatively poor refinement numbers are due to the
intrinsic diffraction quality of the crystal. However, determination of the
crystal structures is satisfactory.
Growth inhibitory activity against M. tuberculosis H37Ra and
cytotoxicity to Vero cells (African green monkey kidney fibroblasts)
were performed using the green fluorescent protein (GFP)-based
method.19 Antifungal activity against C. albicans and anticancer
activities against KB cells (oral human epidermoid carcinoma),
MCF-7 cells (human breast cancer), and NCI-H187 cells (human
small-cell lung cancer) were evaluated using the resazurin micro-
plate assay.20
14. Sungsuwan, S.; Ruangsupapichart, N.; Prabpai, S.; Kongsaeree, P.; Thongpan-
chang, T. Tetrahedron Lett. 2010, 51, 4965e4967.
15. The modified Mosher method16 was also applied with compound 4 and gave
the same result.
16. Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1973, 95, 512e519.
17. Altomare, A.; Burla, M. C.; Camalli, M.; Cascarano, G. L.; Giacovazzo, C.; Gua-
gliardi, A.; Moliterni, A. G. G.; Polidori, G.; Spagna, R. J. Appl. Crystallogr. 1999, 32,
115e119.
18. Sheldrick, G. M. Acta Crystallogr. 2008, A64, 112e122.
19. Changsen, C.; Franzblau, S. G.; Palittapongarnpim, P. Antimicrob. Agents Che-
mother. 2003, 47, 3682e3687.
Acknowledgements
Financial support from the Bioresources Research Network,
National Center for Genetic Engineering and Biotechnology (BIO-
TEC), is gratefully acknowledged. P.K. thanks the Office of the
Higher Education Commission and Mahidol University under the
20. O’Brien, J.; Wilson, I.; Orton, T.; Pongnan, F. Eur. J. Biochem. 2000, 267,
5421e5426.