Bioactive compounds from the scale insect fungus Conoideocrella tenuis BCC 44534

Article abstract of DOI:10.1016/j.tet.2018.01.004

Eleven new compounds, including two quinone derivatives of bioxanthracene, conoideocrellones A (1) and B (2), two bioxanthracenes 3 and 4, four isocoumarins and isocoumarin glycosides 7–10, two phenolic compounds 16 and 17, and a diterpenoid compound, conoideocin A (18), were isolated from culture of the scale-insect pathogenic fungus Conoideocrella tenuis BCC 44534. Seventeen known compounds, compounds 5 and 6, ES-242-2 and its atropisomer, isocoumarins and isocoumarin glycosides 11–15, 3,4,6-trihydroxymellein, cis-4,6-dihydroxymellein, metarhizins A (19) and B (20), BR-050 (21), 5α,8α-epidioxy-24(R)-methylcholesta-6,22-dien-3β-ol, zeorin, and conoideocrellide A, were also isolated from this fungus. Structures of these compounds were elucidated by NMR and MS data analyses. Compound 4 was active against Plasmodium falciparum K1 (IC₅₀ 6.6 μg/mL), while it did not show cytotoxicity. Conoideocrellone A (1) and compounds 3 and 7 exhibited cytotoxic activity, while conoideocin A (18) showed broad range of biological activities including antimalarial, antibacterial, and cytotoxic activities.

Full text of DOI:10.1016/j.tet.2018.01.004

Accepted Manuscript
Bioactive compounds from the scale insect fungus Conoideocrella tenuis BCC 44534
Siriporn Saepua, Jittra Kornsakulkarn, Wiwat Somyong, Pattiyaa Laksanacharoen,
Masahiko Isaka, Chawanee Thongpanchang
PII:
S0040-4020(18)30016-4
10.1016/j.tet.2018.01.004
TET 29219
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 3 November 2017
Revised Date: 12 December 2017
Accepted Date: 3 January 2018
Please cite this article as: Saepua S, Kornsakulkarn J, Somyong W, Laksanacharoen P, Isaka M,
Thongpanchang C, Bioactive compounds from the scale insect fungus Conoideocrella tenuis BCC
44534, Tetrahedron (2018), doi: 10.1016/j.tet.2018.01.004.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Bioactive compounds from the scale insect
Leave this area blank for abstract info.
fungus Conoideocrella tenuis BCC 44534
Siriporn Saepua, Jittra Kornsakulkarn, Wiwat Somyong, Pattiyaa Laksanacharoen, Masahiko Isaka, and
Chawanee Thongpanchang*
National Center for Genetic Engineering and Biotechnology, Thailand Science Park, Pathumthani, Thailand
1

ACCEPTED MANUSCRIPT
Tetrahedron
journal homepage: www.elsevier.com
Bioactive compounds from the scale insect fungus Conoideocrella tenuis BCC 44534
Siriporn Saepua, Jittra Kornsakulkarn, Wiwat Somyong, Pattiyaa Laksanacharoen, Masahiko Isaka, and
*
Chawanee Thongpanchang
National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road, Klong Luang, Pathumthani 12120,
Thailand
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Eleven new compounds, including two quinone derivatives of bioxanthracene,
conoideocrellones A (1) and B (2), two bioxanthracenes 3 and 4, four isocoumarins and
isocoumarin glycosides 7–10, two phenolic compounds 16 and 17, and a diterpenoid compound,
conoideocin A (18), were isolated from culture of the scale-insect pathogenic fungus
Conoideocrella tenuis BCC 44534. Seventeen known compounds, compounds 5 and 6, ES-
Available online
2
42-2 and its atropisomer, isocoumarins and isocoumarin glycosides 11–15, 3,4,6-
trihydroxymellein, cis-4,6-dihydroxymellein, metarhizins A (19) and B (20), BR-050 (21),
,8 -epidioxy-24(R)-methylcholesta-6,22-dien-3 -ol, zeorin, and conoideocrellide A, were
also isolated from this fungus. Structures of these compounds were elucidated by NMR and MS
data analyses. Compound 4 was active against Plasmodium falciparum K1 (IC50 6.6 µg/mL),
while it did not show cytotoxicity. Conoideocrellone A (1) and compounds 3 and 7 exhibited
cytotoxic activity, while conoideocin A (18) showed broad range of biological activities
including antimalarial, antibacterial, and cytotoxic activities.
5
α
α
β
Keywords:
Conoideocrella tenuis
Bioxanthracenes
Isocoumarins
Bioactive compounds
Insect fungi
2
009 Elsevier Ltd. All rights reserved.
1
. Introduction
quinone derivatives of bioxanthracene, conoideocrellones A (1)
and B (2), two new bioxanthracenes 3 and 4, four new
isocoumarins and isocoumarin glycosides 7–10, two new
phenolic compounds 16 and 17, and a new diterpenoid
compound, conoideocin A (18) (Fig. 1), together with seventeen
known compounds, including four bioxanthracenes, compounds
Insect pathogenic fungi are known to be a good source of
1
,2
various biologically active compounds.
Conoideocrella is a
small genus belonging to the Clavicipitaceae family, which
specifically attacks scale-insects. There are only two currently
described species, Conoideocrella luteorostrata and C. tenuis,
formerly named as Torrubiella luteorostrata and T. tenuis,
5,7
8,9
5
5
and 6, ES-242-2 and its atropisomer, five isocoumarins
10,6,11
and isocoumarin glycosides 11–15,
3
pyrone-diterpene hybrids, metarhizins A (19) and B (20), and
BR-050 (21),
methylcholesta-6,22-dien-3β-ol
conoideocrellide A.
two mellein derivatives,
13
3
respectively. Most of reported chemical studies on this genus
12
,4,6-trihydroxymellein and cis-4,6-dihydroxymellein, 14three
are those from our group, employing many strains isolated at
various locations in Thailand, and deposited and preserved in the
BIOTEC Culture Collection. Our studies have revealed that this
genus is a rich source of bioactive secondary metabolites with
diverse structures.
isocoumarin glycosides from T. tenuis BCC 12732,
paecilodepsipeptide A and naphthopyrone glycoside from T.
luteorostrata BCC 9617, hopane triterpenes and bioxanthracenes
from C. tenuis BCC 18627, and oxanthracenes from C.
luteorostrata BCC 31648. In continuation of our study, we
found an extract from C. tenuis BCC 44534 that showed a
15
two triterpenes, 5α,8α-epidioxy-24(R)-
16 17
and
zeorin,
and
5
Details of the isolation, structure
4-7
Examples of the isolated compounds are6
elucidation, and biological activities of these compounds are
presented herein.
4
2. Results and discussion
5
7
The molecular formula of conoideocrellone A (1), C H O ,
3
5
34 13
1
3
was determined by HRESIMS in combination with C NMR
1
1
prolific chemical profiles from HPLC and H NMR spectra and
spectroscopy. The H NMR spectrum showed similarity to those
8,9
exhibited antimalarial activity against Plasmodium falciparum
K1 (IC50 9.53 ꢀg/mL) and cytotoxic activity against human
small-cell lung cancer (NCI-H187, IC50 4.60 ꢀg/mL) cell lines.
of known co-metabolite ES-242-2. Notable differences were
the down field shift of one phenolic proton (δ 13.43) and the
H
absence of one methoxy group and one of the meta-coupled
13
—
The
—
c
—
hemical investigation led to the isolation of two new
aromatic proton. The C and 2D NMR spectroscopic data (in
*
Chawanee Thongpanchang. e-mail: chawanee@biotec.or.th
2

acetone-d ) revealed that conoideocrellone A was a non-
symmetrical C-10−C-10' dimer, wherein one of the monomeric
H-3, and the presence of broad singlet (narrow peak width) of H-
4 demonstrated the pseudoequatorial orientation of this proton.
With the same logic, the relative configuration at C-3' and C-4'
6
ACCEPTED MANUSCRIPT
unit was determined to be the same as that of ES-242-2. The
13
7-9
presence of two carbonyl groups in the C NMR spectrum and
the HMBC correlations from H-7' to C-5'/C-6'/C8'/C-8a'
established ring A of another monomeric unit as a quinone (Fig.
was determined in the same manner. The previous works
showed that the known bioxanthracenes (ES-242 derivatives)
isolated from invertebrate pathogenic fungi with a 4-acetoxy or
4-hydroxy group in a monomeric oxanthracene unit, including
ES-242-2 and its atropisomer (co-metabolites in the present
study), possess 3S,4S absolute configuration. Accordingly, it is
possible that 1 should also share the same absolute configuration,
3S,4S,3'S,4'S. The intense cross peak between H-4 and H-4' in
the NOESY spectrum suggested the same helicity of the chiral
axis as that of ES-242-2 (Fig. 2).
2
). The H-bonding between one of the quinone carbonyl and 9'-
OH resulting in the downfield chemical shifts of this quinone
carbonyl carbon (C-8', δC 192.7) and the phenolic hydroxyl
proton (9'-OH, δ 13.43). The cis-relationship between CH -11
H
3
and the acetoxy group, the same as known ES-242s, was deduced
on the basis of NOESY analysis and the coupling constants.
Thus, the correlation between H-3 and methylene proton H-1 in
the NOESY spectrum suggested the pseudoaxial orientation of
Fig. 1. Chemical structures of compounds 1 ‒ 21
Conoideocrellone B (2) possessed the same molecular formula
1
13
as conoideocrellone A (1). The UV, IR as well as H and
C
NMR spectra of both compounds were almost identical (Table 1).
Detailed analysis of 2D spectroscopic data, in particular HMBC
correlation, established the same structural feature to that of
conoideocrellone A. The relative configurations at C-3/C-4 and
C-3'/C-4' were also deduced to be identical to conoideocrellone A
(
1) and other ES-242 derivatives (cis relation between CH -11
3
and 4-OAc) on the same basis as previously described for 1.
Conoideocrellide (2) should share the same absolute
configuration with 1 and other known ES-242 derivatives,
S,4S,3'S,4'S. Therefore it should be the atropisomer of 1, which
B
3
differ in the configuration at C-10–C-10' junction. This
configurational assignment was confirmed by the intense
NOESY correlation between H-5 and H-4' (Fig. 1).
Fig. 2. Selected HMBC and NOESY correlations of
compounds 1 and 4
HRESIMS data of compounds 3 and 4 deduced the same
1
molecular formula as C H O . Their H NMR spectra showed
35
36 12
3

5
7
good similarity to those of known C-10−C-5' dimers 5 and 6,
242s. The difference between compounds 3 and 4 was
ACCEPTED MANUSCRIPT
respectively, except for the presence of an additional phenolic
determined by the analysis of NOESY correlations. The cross
peak between H-4 and H-10' in NOESY spectrum of compound 3
indicated the same helicity of the chiral axis as that of 5, while
the intense correlations from H-5 to H-10' and H-4 to OH-6' in
NOESY spectrum of compound 4 determined the opposite axial
stereochemistry to that of compound 3. The selected HMBC and
NOESY correlations of compound 4 are presented in Fig. 2.
hydroxyl group replacing a methoxy group. By analysis of 2D
NMR spectroscopic data, the planar structures of compounds 3
and 4 were established to be the same. The HMBC correlations
from an additional hydroxyl proton to C-6' in both compounds
indicated the hydroxyl group at C-6'. The resonance as a broad
singlet of H-4/H-4' suggested the same relative configurations at
C-3/C-4 and C-3'/C-4' of both compounds to those of other ES-
Table 1. NMR spectroscopic data for compounds 1–4
a
a
b
a
Position
1 (in acetone-d
mult(J in Hz)
4.70 d (15.5)
.08 d (15.5)
6
)
2 (in acetone-d
6
)
3 (in acetone-d
6
)
4 (in acetone-d
6
)
δ
C
δ
H
δ
C
δ
H
mult(J in Hz)
δ
C
δ
H
mult(J in Hz)
δ
C
δ
H
mult(J in Hz)
1
65.3
66.0
4.72 d (15.6)
4.99 d (15.6)
64.5
4.72 d (15.6)
5.07 d (15.6)
65.5
4.64 d (15.5)
5.09 d (15.5)
5
3
4
73.5
3.81-3.90 m
73.7
3.60-3.64 m
72.9
3.72 dq (1.6, 6.5)
73.9
3.80 dq (0.7, 6.3)
67.7
5.30 brs
66.4
5.31 brs
66.9
5.19 d (1.6)
67.2
5.56 d (0.7)
4
5
6
7
8
8
9
9
1
1
1
1
a
a
129.1
98.7
-
131.1
96.8
-
133.9
98.0
-
134.5
99.2
-
6.11 s
6.07 s
6.13 s
6.10 s
158.4
98.5
-
158.9
98.6
-
157.9
98.0
-
158.5
98.5
-
6.61 s
6.57 s
6.64 s
6.64 s
158.5
111.5
150.3
116.0
125.6
128.9
17.0
-
158.5
111.6
150.7
116.9
125.2
128.2
16.7
-
157.6
110.8
149.9
115.5
121.9
136.3
16.4
-
158.5
111.3
150.3
116.6
123.6
136.8
17.2
-
-
-
-
-
-
-
-
-
a
-
-
-
-
0
0a
1
'
-
-
-
-
-
-
-
-
0.99 d (6.4)
4.76 d (17.5)
0.99 d (6.4)
0.96 d (6.5)
1.01 d (6.3)
64.8
65.1
4.77 d (17.6)
5.01 d (17.6)
63.8
4.62 d (15.4)
5.01 d (15.4)
64.9
4.60 d (15.4)
5.02 d (15.4)
5
.10 d (17.5)
3
4
4
5
6
7
8
8
9
9
1
1
1
4
4
6
8
9
4
4
6
8
9
'
73.8
65.5
140.4
177.8
162.9
108.9
192.7
114.9
158.2
135.2
132.9
136.1
17.2
169.7
20.3
55.5
57.0
-
3.81-3.90 m
73.6
64.8
141.0
179.4
162.8
109.1
192.7
114.9
158.7
135.3
133.0
134.5
16.8
169.9
20.3
55.5
56.9
-
3.71-3.75 m
71.9
68.4
133.4
109.7
153.4
98.5
157.8
110.3
149.6
114.0
115.4
135.6
15.8
168.7
19.7
54.5
56.2
-
3.86 dq (2.0, 6.0)
73.0
69.1
133.2
110.6
153.3
99.2
158.2
111.0
150.4
114.6
117.9
136.7
17.0
169.4
19.3
55.3
56.8
-
3.80 dq (0.7, 6.3)
'
5.35 brs
5.17 brs
5.52 d (2.0)
5.52 d (0.7)
a'
-
-
-
-
'
-
-
-
-
'
-
-
-
-
'
6.28 s
6.27 s
6.73 s
6.77 s
'
-
-
-
-
a'
-
-
-
-
'
-
-
-
-
a'
-
-
-
-
0'
-
-
6.37 s
-
6.34 s
-
0a'
-
-
1'
0.96 d (6.5)
-
0.88 d (6.4)
-
1.10 d (6.0)
-
1.10 d (6.3)
-
-OCOCH
3
-OCOCH
3
1.63 s
3.56 s
4.16 s
9.55 s
-
1.84 s
3.52 s
4.15 s
9.55 s
-
1.96 s
3.45 s
4.17 s
9.67 s
-
1.04 s
3.42 s
4.18 s
9.63 s
-
-OCH
-OCH
-OH
3
3
'-OCOCH
3
168.7
19.1
57.3
-
169.1
20.3
57.3
-
169.3
19.4
-
170.6
20.7
-
'-OCOCH
3
1.06 s
3.86 s
-
1.84 s
3.86 s
-
1.62 s
7.43 s
4.18 s
9.59 s
1.92 s
7.84 s
4.18 s
9.58 s
3
'- OCH /OH
'-OCH
3
56.2
-
57.0
-
'-OH
-
13.43 s
-
13.45 s
a
1
13
5
4
00 MHz for H and 125 MHz for 13C
b
1
00 MHz for H and 100 MHz for
C
4

ACCEPTED MANUSCRIPT
1
13
Table 2. NMR spectroscopic data for compounds 7–10 (500 MHz for H and 125 MHz for C)
Position
7 (in acetone-d
6
)
8 (in acetone-d
6
)
9 (in DMSO-d
6
)
6
10 (in DMSO-d )
δ
C
δ
H
mult(J in Hz)
δ
C
δ
H
mult(J in Hz)
δ
C
δ
H
mult(J in Hz)
δ
C
H
δ mult(J in Hz)
1
3
4
165.9
155.6
97.8
-
166.6
158.0
98.3
-
165.5
153.8
106.1
139.5
102.7
167.2
101.6
163.3
100.3
66.5
-
165.2
154.0
99.5
-
-
-
-
-
6.89 s
6.83 s
6.84 s
6.98 s
4
5
6
7
8
8
9
a
a
124.5
131.7
153.6
101.5
156.8
97.8
-
135.4
135.8
159.2
103.3
160.6
98.3
-
-
131.8
131.4
158.0
101.9
159.3
97.1
-
-
-
6.62 d (2.2)
-
-
-
-
-
6.48 s
6.49 s
6.57 d (2.2)
6.41 s
-
-
-
-
-
-
-
-
60.3
4.41 d (0.6)
61.2
4.43 s
4.42 d (13.9)
19.0
2.23 s
4
.58 d (13.9)
1
2
3
4
5
6
'
'
'
'
'
'
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
102.9
74.3
77.1
80.0
76.4
61.2
4.29 d (7.8)
3.01-3.07 m
3.26-3.32 m
2.95 t (9.3)
3.14-3.17 m
3.46-3.51 m
106.0
74.0
75.5
78.9
75.8
60.3
4.48 d (7.8)
3.31-3.35 m
3.38-3.42 m
3.07 t (9.3)
3.24 ddd (1.9, 4.8, 9.3)
3.49-3.53 m
3.59-3.62 m
3
-
.60-3.64 m
5
6
8
9
2
3
4
6
-OH/OCH
3
-
-
-
-
-
-
-
-
-
61.9
3.79 s
-
-
-
- OH/OCH
3
-
-
-
-
-
-
-
-
-
56.6
3.86 s
-
-
-OH
10.75 s
10.92 s
-
10.95 s
-
10.87 s
-OH
-
-
-
-
-
4.74 brs
-
-
-
-
'-OH
-
-
-
-
-
5.29 d (4.9)
5.19 d (5.2)
3.42 s
-
-
'-OH
-
-
5.43 d (5.8)
3.44 s
'- OCH
3
60.3
-
59.6
-
'-OH
4.71 brt (5.7)
4.75 brt (5.7)
Compound 7 was obtained as a light brown solid, possessing
the molecular formula C H O as determined by HRESIMS.
C H O as established by HRESIMS suggested that 9 has an
18 22 10
additional methyl group as compared to 12. The additional
10
8
6
1
1
The H NMR spectrum showed one D O exchangeable phenolic
methoxy signal at δ 3.86 in H NMR spectrum was indicated to
2
H
proton (when a few drop of D O was added), two aromatic
present at C-6 instead of the hydroxyl group in 12 on the basis of
HMBC correlation analysis. The identical sugar moiety to that of
12 was assigned as 4'-O-methyl-β-glucopyranose. It was
suggested to attached at C-9 by the HMBC correlation from an
anomeric proton (H-1', δH 4.29) to C-9. The large coupling
constants of J1',2' = 7.8 Hz, J3',4' = J4',5' = 9.3 Hz confirmed the
axial orientation of all methine protons (H-1' to H-5') in the sugar
2
protons, and two oxygenated methylene protons (Table 2). The
presence of a conjugated lactone functional group was indicated
13
by the signal at δC 165.9 in the C NMR spectrum,
-
1
corresponding to the absorption band at 1681 cm in IR
5,6
spectrum.
The HMBC correlations from H-4 to C-3/C-4a/C-
5
/C-8a/C-9, H-7 to C-5/C-6/C-8/C8a, and H-9 to C-3/C-4
revealed the 3-methylisocoumarin unit. The hydroxyl group at
C-5, C-6, C-8 and C-9 were established on the basis of the
molecular formula and their chemical shifts. Thus, compound 7
unit. The correlation from methoxy protons (4'- OCH
3
, δ 3.42) to
H
C-4' indicated the methoxy group at C-4'. The configuration of
D
4'-O-methyl-β-glucopyranose was established by comparison of
was
isocoumarin.
determined
as
3-hydroxymethyl-5,6,8-trihydroxy
the specific rotations of the aqueous layer of its acid hydrolysate
25
([α]
+55.4, c 0.08, MeOH) with that of 4-O-methyl-D-
D
25 18 1
1
glucopyranose ([α] +80, c 1.30, MeOH). The H NMR
D
The H NMR spectrum of compound 8 showed a resemblance
to compound 7 except for the presence of an additional methoxy
signal at δ 3.79. The molecular formula C H O as deduced by
HRESIMS also indicated an atomic mass 14 units higher than
that of 7. The correlation form the methoxy protons to C-5 in
HMBC spectrum suggested the 5-O-methyl derivative of 7.
Compound 8 was, therefore, determined to be 6,8-dihydroxy-3-
hydroxymethyl-5-methoxyisocoumarin.
spectrum of the aglycone unit, obtained by acid hydrolysis of 9,
19
was identical to that of cytogenin. Therefore compound 9 was
H
11 10
6
determined as 9-O-(4'-O-methyl-β-
D-glucopyranosyl)cytogenin.
The molecular formula of compound 10 was deduced to be
1
3
C H O by HRESIMS in combination with C NMR
17
20 10
1
13
spectroscopy. The H and C NMR spectra revealed the closely
related structure of 10 and the known co-metabolite 14 except for
the presence of the sugar unit and the missing of one of the meta-
coupled aromatic proton. The HMBC correlation from an
anomeric proton (H-1') to C-5 suggested the linkage of the sugar
1
13
The H and C NMR spectra of compound 9 were similar to
those of the known co-metabolite 12. The molecular formula
6
5

moiety to C-5. The 2D NMR spectroscopic data of the sugar unit
were in good agreement with those of the sugar moiety in 9.
H -21 to C-1/C-5/C-9/C-10, and H -18 to C-7/C-8/C-9 established a
3 2
substituted decalin skeleton. The existence of the prenyl unit at C-
11 was deduced by the correlations between H-11, H-12, and H-13
ACCEPTED MANUSCRIPT
Comparison of the specific rotation of the aqueous layer of
2
3
hydrolysate ([α]
+58.7, c 0.08, MeOH) with that of 4-O-
in COSY spectrum and the correlations from H-5 and H
3
-17 to C-
D
25
18
methyl-
D
-glucopyranose ([α] +80, c 1.30, MeOH) indicated
11, H -15 and H -16 to C-13/C-14 in HMBC spectrum. The 2-
3
3
D
its configuration.
D
hydroxy-3-methylbutanoyloxy moiety at C-3 was indicated by the
HMBC correlations from H-3 and H-2' to C-1' and H -4' and H -5'
3
3
to C-2'/C-3'. The correlations from H-9 to C-20 and H -19 to C-
Table 3. NMR spectroscopic data for compounds 16 and 17 (500
MHz for H and 125 MHz for C)
2
1
13
8
/C-9/C-10/C-20 suggested an acetyl group at C-9. The observed
NOESY correlations, presented in Fig. 4, showed the resemblance
relative configurations of decalin system to those of the known co-
metabolites 19–21. The absolute configurations at C-3, C-4, C-5,
C-9, and C-10 were determined by converting conoideocin A (18)
into 22 by metholysis with NaOMe. The similar specific rotation of
Position
16 (in DMSO-d
6
)
6
17 (in DMSO-d )
δ
C
δ
H
mult(J in Hz)
δ
C
H
δ mult(J in Hz)
1
2
3
79.2
208.4
24.8
141.2
104.6
158.4
101.9
-
4.81 d (3.6)
201.5
68.7
20.8
136.6
106.4
158.5
107.2
-
-
24
24
-
4.90, dq (6.7)
2
2 ([α]
−44.9, c 0.03, MeOH) to that of BR-050 (21, [α]
D D
14
2.00 s
1.24 d (6.7)
−32.1, c 0.03, MeOH) indicated the same absolute configurations
of these two compounds. Many attempts to converted conoideocin
A (18) to (R)- and (S)-MTPA esters failed, no desired products were
found in the reaction. Thus, the absolute configuration at C-2' could
not be determined.
1
2
3
4
1
2
3
'
-
-
', 6'
6.23 d (1.6)
6.81 d (2.1)
', 5'
-
-
'
6.11 d (1.6)
5.83 d (3.6)
-
6.44 d (2.1)
-
O
-OH
-OH
', 5'-OH
H
20
-
-
5.19 d (6.7)
9.61 s
H
19
OH
21
H C
H
18
3
-
9.26 s
-
H
1
9
H
CH3
O
10
H
5
1'
The HRMS data of compounds 16 and 17 indicated the same
3
7
3'
O
1
13
H C
3
H
molecular formula as C H O . The H and C NMR spectra
9
10
4
H C 11
3
HO
H
were also similar (Table 3), which showed the presence of three
hydroxyl groups, 1,3,5-trisubstitued benzene ring, one
oxygenated methine proton, one methyl and one carbonyl group.
Detailed analysis of 2D spectroscopic data established 1-(3,5-
17
13
COSY
HMBC
CH3
H C
3
dihydroxyphenyl)-1-hydroxypropane-2-one
and
1-(3,5-
dihydroxyphenyl)-2-hydroxypropane-1-one for 16 and 17,
respectively. The assignment of these two compounds were
assured by the HMBC correlations, depicted as shown in Fig. 3.
The absolute configuration at C-1 of 16 was determined as R by
H
H
CH3
10
CH
H
O
3
1
9
H
OH
H
3
8
H C
3
O
5
7
23
OH
H
comparing the optical rotation ([α] −230.0, c 0.002, MeOH) to
CH3
O
D
H
H
H
those of phenylhydroxypropanone analogues, e.g. −181 for (R)-(–
)
-1-hydroxy-1-phenylpropane-2-one and −225 for (R)-(–)-1-(4-
20
hydroxyphenyl)-1-hydroxypropane -2-one. On the same basis,
comparison of the optical rotation of 17 ([α]
NOESY
25
−81.1, c 0.01,
D
MeOH) to those of similar derivatives, e.g. −83 for (S)-(–)-2-
hydroxy-1-phenylpropane-1-one and −79 for (S)-(–)-2-hydroxy-
Fig. 4. Selected COSY, HMBC and NOESY correlations of
compounds 18
21
1
-(4-methoxyphenyl)propane -1-one, deduced S configuration
at C-2.
The structures of seventeen known compounds, compounds 5–
5,7
8,9
5
6
,
ES-242-2, atropisomer of ES-242-2, isocoumarins and
1
0,6,11
12
isocoumarin glycosides 11–15,
3,4,6-trihydroxymellein cis-
13
14
4
(
,6-dihydroxymellein, metarhizins A (19) and B (20), BR-050
15
16
21),
5α,8α-epidioxy-24(R)-methylcholesta-6,22-dien-3β-ol,
17
5
zeorin, and conoideocrellide A, were elucidated by analysis of
their spectroscopic and physical property data which were in good
agreement with those appeared in the literature data.
All new compounds apart from 2 and 8, due to the sample
shortage, were tested for antibacterial (Bacillus cereus),
antimalarial (P. falciparum K1), and cytotoxicity against KB, MCF-
Fig. 3. Selected HMBC correlations of compounds 16 and 17
Conoideocin A (18) with the molecular formula C H O by 7, NCI-H187, and Vero cells (Table 4). Cyclohexadepsipeptides
26
42
5
HRESIMS was obtained as a pale yellow solid. The IR spectrum and most of isocoumarins and isocoumarin glycosides, including
-
1
displayed major absorption bands at 3432 and 1723 cm new compounds 9 and 10, were inactive in our biological assays at
5,6
corresponding to a hydroxyl and a carbonyl group, respectively. the maximum tested concentrations, however, compound 7 was
1
13
The H and C NMR spectra showed the presence of two carbonyl, moderately active to NCI-H187 and Vero cells (IC50 27.7 and 9.6
six methyl, eight methylene, and six methine groups. The COSY µg/mL, respectively). Bioxanthracenes are known to exhibit
2
2,23
correlations observed for H-1–H-2–H-3 and H-5–H-6–H-7 together antimalarial activity
and a new analogue 4 also showed activity
with the HMBC correlations from H -11 and H -17 to C-3/C-4/C-5, against P. falciparum K1 (IC50 6.6 µg/mL) without cytotoxicity,
2
3
6

while conoideocrellone A (1) and compound 3 had no antimalarial and 47.4 µg/mL, respectively). Pyrone diterpene compounds 19-21
ACCEPTED MANUSCRIPT
activity but exhibited cytotoxic activity. Conoideocin A (18), was were reported to exhibit antimalarial and antiproliferative activity
14,24
active against P. falciparum (IC50 6.6 µg/mL), B. cereus (MIC 25 on both insect and human cancer cell lines.
µg/mL), and human cancer cell lines KB and NCI-H187 (IC50 25.8 17 were not active in our biological assays.
Compounds 16 and
Table 4. Biological activities of compounds 1, 3, 4, 7, 9, 10 and 16–18
compound
P. falciparum, IC50
µg/mL)
B. cereus, MIC
(µg/mL)
cytotoxicity, IC50 (µg/mL)
(
KB
29.1
31.4
>50
>50
>50
>50
>50
>50
25.8
-
MCF-7
11.5
43.0
>50
>50
>50
>50
>50
>50
>50
-
NCI-H187
7.3
Vero
23.6
>50
>50
9.6
>50
>50
>50
>50
>50
-
Conoideocrellone A (1)
Compound 3
>10
>10
6.6
>50
>50
>50
>50
>50
>50
>50
>50
25.0
-
28.1
>50
27.7
>50
>50
>50
>50
47.4
-
Compound 4
Compound 7
>10
>10
>10
>10
>10
6.6
Compound 9
Compound 10
Compound 16
Compound 17
Conoideocin A (18)
a
Dihydroartemisinin
0.0010
0.020
-
a
Mefloquine hydrochloride
-
-
-
-
-
b
Vancomycin hydrochloride
2.0
-
-
-
-
-
c
Doxorubicin hydrochloride
-
0.5
6.9
-
0.1
-
c
Ellipticine
-
-
2.8
4.0
1.9
a
b
c
Positive control for the antimalarial assay. Positive control for the antibacterial assay. Positive control for the cytotoxicity assay.
BIOTEC Culture Collection as BCC 44534 on November 12,
010. This fungus was identified as Conoideocrella tenuis of the
3
. Conclusion
2
In conclusion, the present study further demonstrated that the
order Hypocreales, family Clavicitaceae, on the basis of
morphology, by Ms. Kanoksri Tasanathai, BIOTEC, and the
partial nuclear large subunits rDNA (nc28S) (GenBank accession
number MG198773), the internal transcribed spacer (ITS1-5.8S-
ITS2) rDNA (GenBank accession number MG230251), and the
partial translation elongation factor 1-alpha region (EF-1alpha)
(GenBank accession number MG230541), analysed by Dr.
Nattawut Boonyuen, BIOTEC.
genus Conoideocrella is a potent source of the diverse structures
of bioactive secondary metabolites. Various types of
compounds, including isocoumarins and isocoumarin glycosides,
bioxanthracenes, cyclohexadepsipeptides, diterpenes, and
triterpenes, were reported from the different strains of the fungi
in this genus. Interestingly, all these type of compounds could be
isolated from the strain in this study, BCC 44534, and the
quinone derivatives of bioxanthracene, conoideocrellone A (1)
and B (2), were reported here for the first time. Compound 4 was
active against P. falciparum, conoideocrellone A (1) and
4
.3. Fermentation and isolation
The fungus BCC 44534 was maintained on potato dextrose
compounds
conoideocin
3
and
7
exhibited cytotoxic activity while
o
agar at 25 C. The agar was cut into pieces (1ꢀ1 cm) and
inoculated into 8ꢀ250-mL Erlenmeyer flasks containing 25 mL
A
(18) showed various activities including
antimalarial, antibacterial, and cytotoxic activities.
of potato dextrose broth (PDB, potato starch 4.0 g/L, dextrose
o
2
0.0 g/L). After incubation at 25 C for 7 days on a rotary shaker
4
4
. Experimental
(
200 rpm), each primary culture was transferred into 1 L
Erlenmeyer flask containing 250 mL of the same liquid medium
PDB) and incubated under the same conditions for 5 days. Each
.1. General procedures
(
Melting points were measured using a Metler MP90 melting
2
1
5 mL portion of the secondary culture was transferred into 40 ꢀ
L Erlenmeyer flasks containing 250 mL of M102 medium [40
point apparatus. Optical rotation measurements were conducted
by using a JASCO P-1030 digital polarimeter. UV and FT-IR
spectra were recorded on an Analytic Jena Spekol 1200
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. Column chromatography was
performed on silica gel 60 (70-230 Mesh ASTM, Merck). HPLC
were performed using Dionex-Ultimate 3000 series equipped
with a binary pump, an autosampler, and a diode array detector.
ꢀ
250 mL, composition: sucrose (30.0 g/L), malt extract (20.0
g/L), bacto-peptone (2.0 g/L), yeast extract (1.0 g/L), KCl (0.5
g/L), MgSO .7H O (0.5 g/L), and KH PO (0.5 g/L)] and the
4
2
2
4
fermentation was carried out under static conditions, 25 °C for 19
days.
After filtration of the mycelium, the cells were macerated in
MeOH (2 L) for 3 days, followed by CH Cl (2 L) for 3 days.
2
2
The MeOH and CH Cl extracts were combined and evaporated
2
2
under reduced pressure. Water (500 mL) was added, and the
mixture was extracted with hexanes (3ꢀ500 mL), followed by
EtOAc (3ꢀ500 mL). The brown solid (6.46 g), obtained from
EtOAc extract, was fractionated using Sephadex LH-20, eluted
with 100% MeOH, to give seven fractions (A1–A7). Compound
4
.2. Fungal material
The fungus was isolated from a scale-insect (Hemiptera),
collected in Khao Yai National Park, Nakhon Ratchasima
Province, Thailand. The living culture was deposited in the
1
1 (44.8 mg) was obtained from fraction A7. Further purification
7

of fraction A2 (815.4 mg) by preparative HPLC using reverse
phase column (SunFire C18 OBD, 10 ꢀm, 19ꢀ250 mm, step
(190.2 mg), 13 (20.9 mg), 14 (23.1 mg), 15 (217.0 mg), 16 (97.4
mg), 17 (42.1 mg), ES-242-2 (40.2 mg), atropisomer of ES-242-2
(7.3 mg), 3,4,6-trihydroxymellein (441.7 mg), cis-4,6-
dihydroxymellein (15.3 mg), and conoideocrellide A (133.2 mg).
Further purification of fraction B3 (382.2 mg) yielded
compounds 7 (51.5 mg), 11 (202.5 mg), and 14 (13.3 mg).
ACCEPTED MANUSCRIPT
gradient elution with 40−95% MeCN/H O, flow rate 15.0
2
mL/min) afford compound 18 (8.5 mg). Fraction A3 (4.25 g)
was subjected to silica gel column chromatography, using 50%
EtOAc/hexanes as an eluent, to provide thirteen fractions (A3-1–
A3-13). Zeorin (265.7 mg) and conoideocrellide A (2.2 g) were
obtained, respectively, from fractions A3-4 and A3-13. Fraction
A3-5 and A3-12 were purified by preparative HPLC, step
23
4
.3.1 Conoideocrellone A (1). Yellow solid; [α] −47.6 (c 0.13,
D
MeOH); UV (MeOH) λmax (log ε) 239 (4.29), 293 (3.86), 339
(
2
3.47), 355 (3.47), 423 (3.30), nm; IR (ATR) ν
854, 1737, 1628, 1364, 1214, 1157, 1098 cm ; H NMR and C
3388, 2926,
max
13
gradient elution with 40−95% MeCN/H O, to furnish 5α,8α-
-1
1
2
epidioxy-24(R)-methylcholesta-6,22-dien-3β-ol (5.2 mg) and
compound 5 (7.1 mg), respectively. Consecutive purification of
fraction A3-6 by preparative HPLC, step gradient elution with
+
NMR data, see Table 1; HRESIMS m/z 685.1890 [M + Na]
calcd. for: C H O Na, 685.1892).
(
35
34 13
23
4
0−95% MeCN/H O, yielded compounds 19 (2.1 mg) and 20
4.3.2 Conoideocrellone B (2). Yellow solid; [α]
−129.9 (c
D
2
(320.4 mg). Compounds 18 (17.3 mg), 20 (4.7 mg), 21 (3.3 mg),
0.06, MeOH); UV (MeOH) λmax (log ε) 239 (4.41), 295 (4.01),
339 (3.72), 354 (3.75), 420 (3.60), nm; IR (ATR) ν 3386,
and atropisomer of ES-242-2 (6.4 mg) were obtained from
fraction A3-7 after purification by preparative HPLC, step
max
-
1 1
2926, 2854, 1732, 1627, 1366, 1216, 1158, 1099 cm ; H NMR
13
gradient elution with 60−95% MeCN/H O. Fraction A3-8 was
and+ C NMR data, see Table 1; HRESIMS m/z 685.1896 [M +
2
subjected to preparative HPLC, step gradient elution with
Na] (calcd. for: C H O Na, 685.1892).
35
34 13
6
0−80% MeCN/H O, to give compounds 18 (1.3 mg), 21 (3.5
2
4
.3.3 Compound 3. Light brown solid; dec. 241.6−243.2 °C;
25
mg), ES-242-2 (1.1 mg), and atropisomer of ES-242-2 (4.6 mg).
Further purification of fraction A3-9 by preparative HPLC, step
[
(
α]
+122.0 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 239
D
4.56), 296 (4.11), 310 (4.07), 341 (3.98), 355 (3.98), 425 (3.75),
gradient elution with 50−90% MeCN/H O, afforded compound 3
2
nm; IR (ATR) ν 3407, 2927, 2856, 1733, 1625, 1369, 1228,
max
(
(
4.6 mg), ES-242-2 (48.6 mg), and atropisomer of ES-242-2
20.5 mg). Fraction A3-10 was purified by preparative HPLC,
-
1
1
13
1
159, 1097 cm ; H NMR and C NMR data, see Table 1;
HRESIMS m/z 671.2090 [M + Na] (calcd. for: C H O Na,
+
35
36 12
step gradient elution with 50−70% MeCN/H O, followed by
2
6
71.2099).
preparative
EtOAc/hexanes as an eluent, to provide compounds 3 (1.5 mg), 4
0.9 mg), and ES-242-2 (16.3 mg). Compound 6 (4.5 mg) was
obtained after trituration of fraction A3-11 with MeOH followed
by filtration. Further purification of the filtrate yielded
thin
layer
chromatography,
using
40%
25
4
.3.4 Compound 4. Yellow solid; [α] −38.9 (c 0.11, MeOH);
D
(
UV (MeOH) λmax (log ε) 238 (4.44), 295 (4.00), 307 (4.00), 340
(
2
3.92), 355 (3.96), 426 (3.66), nm; IR (ATR) νmax 3- 387, 2925,
1
1
854, 1735, 1625, 1459, 1366, 1232, 1157, 1096 cm ; H NMR
13
compound 5 (3.2 mg). Fraction A4 (141.5 mg) was subjected to
preparative HPLC, step gradient elution with 5−100%
and+ C NMR data, see Table 1; HRESIMS m/z 671.2097 [M +
Na] (calcd. for: C H O Na, 671.2099).
35
36 12
MeCN/H O, to furnish compounds 1 (1.8 mg), 3 (3.5 mg), 4 (4.2
2
4
.3.5 Compound 7. Light brown solid; mp 222.8−224.0 °C; UV
mg), 5 (3.2 mg), 12 (5.5mg), 15 (45.4 mg), conoideocrellide A
(MeOH) λmax (log ε) 238 (4.35), 261 (4.10), 350 (4.04), 425
(4.2 mg), ES-242-2 (4.0 mg), and atropisomer of ES-242-2 (7.1
(
1
3.55) nm; IR (ATR) ν 3430, 1681, 1638, 1630, 1479, 1295,
mg). More of compound 12 (40.7 mg) was obtained after
trituration of fraction A5 (213.7 mg) with MeOH followed by
filtration. Consecutive purification of the filtrate with preparative
max
-
1
1
13
176, 1080 cm ; H NMR and C NMR data, see Table 2;
+
HRESIMS m/z 247.0204 [M + Na] (calcd. for: C H O Na,
10
8
6
2
47.0213).
HPLC, step gradient elution with 5−70% MeCN/H O, give
2
compounds 10 (3.8 mg), 12 (32.4 mg), 13 (9.7 mg), 14 (5.5 mg),
4
2
3
.3.6 Compound 8. Light brown solid; UV (MeOH) λmax (log ε)
40 (4.25), 258 (4.07), 335 (3.93), 422 (3.79) nm; IR (ATR) νmax
287, 2925, 2854, 1682, 1639, 1618, 1482, 1352, 1301, 1169,
1
5 (21.1 mg), and 3,4,6-trihydroxymellein (13.6 mg). Fraction
F6 (32.9 mg) was further purified by preparative HPLC, step
-
1
1
13
gradient elution with 10−70% MeCN/H O, to afford compounds
2
1074 cm ; H NMR and C NMR data, see Table 2; HRESIMS
m/z 261.0364 [M + Na] (calcd. for: C H O Na, 261.0370).
+
1
1 (11.5 mg) and 14 (5.7 mg). Purification of the crude hexanes
extract (1.51 g) by silica gel column chromatography, using
00% CH Cl as an eluent, followed by preparative HPLC, step
11
10
6
25
4
.3.7 Compound 9. Light brown solid; [α]
−17.6 (c 0.05,
D
1
2
2
MeOH); UV (MeOH) λmax (log ε) 239 (4.17), 276 (3.64), 321
gradient elution with 30−80% MeCN/H O, provided compounds
2
(3.55) nm; IR (ATR) νmax 3340, 2929, 2854, 1686, 1621, 1573,
1
(
2
(2.6 mg), 2 (1.1 mg), 3 (7.8 mg), 5 (17.6 mg), 11 (3.3 mg), 15
5.6 mg), 18 (17.6 mg), ES-242-2 (58.2 mg), atropisomer of ES-
42-2 (50.5 mg), zeorin (24.1 mg), 5α,8α-epidioxy-24(R)-
methylcholesta-6,22-dien-3β-ol (4.6 mg), and conoideocrellide A
-
1
1
13
1
506, 1455, 1382, 1238, 1199, 1166, 1080 cm ; H NMR and C
+
NMR data, see Table 2; HRESIMS m/z 421.1116 [M + Na]
calcd. for: C H O Na, 421.1105).
(
18 22 10
(
24.7 mg).
4
.3.8 Compound 10. . Light brown solid; mp 206.7−208.1 °C;
24
[
(
α]
+23.4 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 239
D
The culture broth was then extracted with EtOAc (3ꢀ20 L)
4.25), 280 (3.81), 332 (3.71), 427 (3.19) nm; IR (ATR) νmax
and evaporated to dryness, leaving a dark brown solid (5.56 g).
The crude extract was pass through Sephadex LH-20 with MeOH
as an eluent to give six fractions (B1–B6). Conoideocrellide A
3
1
350, 2970, 2928, 1738, 1685, 1617, 1481, 1445, 1366, 1229,
-1 1 13
217, 1073 cm ; H NMR and C NMR data, see Table 2;
+
HRESIMS m/z 407.0950 [M + Na] (calcd. for: C H O Na,
4
(6.4 mg) was obtained from fractions B1 after purification by
17 20 10
07.0949).
preparative HPLC, step gradient elution with 40−65%
MeCN/H O. Trituration of fraction B2 (4.3 g) with MeOH
23
2
4
.3.9 Compound 16. Light brown oil; [α]
−230.0 (c 0.002,
D
followed by filtration provided compound 12 (405.7 mg). The
filtrate of fraction B2 was subjected to Sephadex LH-20, eluted
with 100% MeOH, and then further purified by consecutive
preparative HPLC to furnish compounds 3 (11.5 mg), 5 (33.7
mg), 8 (2.1 mg), 9 (19.6 mg), 10 (37.9 mg), 11 (16.4 mg), 12
MeOH); UV (MeOH) λmax (log ε) 230 (3.65), 285 (3.51), 423
3.20) nm; IR (ATR) ν 3320, 2925, 2854, 1713, 1603, 1456,
341, 1306, 1161 cm ; H NMR and C NMR data, see Table 3;
HRESIMS m/z 181.0505 [M − H] (calcd. for: C H O ,
(
1
max
-1
1
13
−
9
9
4
1
81.0506).
8

2
5
4
.3.10 Compound 17. Light brown oil; [α]
−81.1 (c 0.01,
3.9, 10.4 Hz, H-3), 3.45 (1H, brs, OH-3), 2.63 (1H, dd, J = 9.5,
ACCEPTED MANUSCRIPT
D
MeOH); UV (MeOH) λmax (log ε) 231 (3.76), 259 (3.73), 417
18.6 Hz, Ha-19), 2.30 (1H, dd, J = 9.5, 18.6 Hz, Hb-19), 2.20
(3H, m, H-7, H-9), 1.95 (2H, m, H-12), 1.70 (2H, m, H-2), 1.66
(3H, s, H-16), 1.62 (3H, s, H-15), 1.55 (1H, m, H-5), 1.37 (2H,
m, H-6), 1.33 (2H, m, H-1), 1.22 (2H, m, H-11), 0.99 (3H, s, H-
(
1
3.34) nm; IR (ATR) ν 3248, 2925, 2855, 1686, 1601, 1454,
341, 1305, 1162 cm ; H NMR and C NMR data, see Table 3;
HRESIMS m/z 181.0507 [M − H] (calcd. for: C H O ,
max
-
1
1
13
−
9
9
4
+
1
81.0506).
21), 0.78 (3H, s, H-17); HRESIMS m/z 357.2415 [M + Na]
2
4
(calcd. for: C21H O Na, 357.2400).
34 3
4
0
4
1
.3.11 Conoideocin A (18). Pale yellow solid; [α]
−4.1 (c
D
.51, MeOH); UV (MeOH) λmax (log ε) 237 (3.18), 266 (3.11),
23 (2.34), nm; IR (ATR) νmax 3432, 2938, 2876, 1723, 1454,
387, 1257, 1216, 1177, 1137 cm ; H NMR (400 MHz, CDCl )
δ 5.01 (1H, brt, J = 6.9 Hz, H-13), 4.85 (1H, dd, J = 5.7, 10.2 Hz,
H-3), 4.73 (1H, brs, Ha-18), 4.68 (1H, brs, Hb-18), 4.01 (1H, d, J
3.4 Hz, H-2'), 2.63 (1H, dd, J = 4.1, 13.4 Hz, Ha-19), 2.37 (1H,
dd, J = 9.7, 13.4 Hz, Hb-19), 2.30 (1H, dd, J = 4.1, 9.7 Hz, H-9),
.23 (1H, m, Ha-7), 2.16 (1H, m, Hb-7), 2.04 (1H, m, H-3'), 1.93
4.7. Biological assays
-
1
1
Antimalarial activity against P. falciparum K1 was performed
by using microculture radioisotope technique. Cytotoxicity to
Vero cells (African green monkey kidney fibroblasts) was carried
3
25
out by using the green fluorescent protein (GFP) based
=
26,27
method.
The activities for antibacterial to B. cereus and
anticancer 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
2
(
(
1H, m, Ha-12), 1.82 (1H, m, Hb-12), 1.77 (2H, m, H-2), 1.66
3H, s, H-16), 1.58 (3H, s, H-15), 1.51 (1H, m, H-5), 1.48 (1H,
m, Ha-6), 1.39 (1H, m, Hb-6), 1.29 (1H, m, Ha-11), 1.28 (2H, m,
28
microplate assay.
H-1), 1.15 (1H, m, Hb-11), 1.02 (3H, s, H-21), 1.01 (3H, d, J =
Acknowledgments
7
.1 Hz, H-5'), 0.87 (3H, s, H-17), 0.86 (3H, d, J = 6.6 Hz, H-4');
This work was supported by Thailand Research Fund (Grant
No. DBG5980002) and National Center for Genetic Engineering
and Biotechnology (BIOTEC), National Science and Technology
Development Agency (NSTDA).
1
3
C NMR (100 MHz, CDCl ) δ 178.4 (C, C-20), 174.6 (C, C-1'),
3
1
46.8 (C, C-8), 131.7 (C, C-14), 124.0 (CH, C-13), 111.3 (CH2,
C-18), 77.6 (CH, C-3), 74.8 (CH, C-2'), 53.6 (CH, C-9), 40.0 (C,
C-4), 39.5 (CH, C-5), 37.6 (CH , H-11), 37.1 (C, C-10), 34.2
2
(
2
CH , C-1), 33.9 (CH , C-19), 32.2 (CH, C-3'), 30.6 (CH , C-7),
5.7 (CH , C-16), 23.9 (CH , C-2), 22.5 (CH , C-21), 22.5 (CH ,
3 2 3 2
2
2
2
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2
3
3
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26
42
5
1
4
.4. Hydrolysis of compound 9
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4
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25
-O-methyl-D-glucopyranose (0.78 mg, 80% yield, [α] +55.4,
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1
method described for compound 9. The aqueous layer was
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1
D
1
23
(
D
layer was evaporated to dryness under reduced pressure to obtain
1
aglycone unit (0.83 mg, 80% yield); H NMR (400 MHz,
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6
1
4. Kikuchi, H.; Hoshi, T.; Kitayama, M.; Sekiya, M.; Katou, Y.; Ueda, K.;
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−
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10
7
5
2
07.0299).
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4
.6. Methanolysis of compound 18
1
6. Sheffer, M.; Fried, A.; Gottlieb, H. E.; Tietz, A.; Avron, M. Biochim.
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1
1
1
aqueous HCl, diluted with H O (4.0 mL), and extracted with
2
EtOAc (4.0 mL). The organic layer was evaporated and the
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1
507.
ꢀ
2
2
2
0. Engel, S.; Vyazmensky, M.; Berkovich, D.; Barak, Z. e.; Chipman, D. M.
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2
24
1
mg, 0.001 mmol, 7% yield, [α]
−44.9, c 0.03, MeOH); H
D
NMR (400 MHz, acetone-d ) δ 5.12 (1H, brt, J = 7.2 Hz, H-13),
6
4
.67 (1H, brs, Ha-18), 4.61 (1H, brs, Hb-18), 3.52 (1H, dd, J =
9

2
3. Jaturapat, A.; Isaka, M.; Hywel-Jones, N. L.; Lertwerawat, Y.;
ACCEPTED MANUSCRIPT
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Supplementary data
2
2
2
2
2
Supplementary data associated with this article can be found
in the online version, at doi:XXX. These data include NMR
spectra of most important compounds described in this article.
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2
1
0