Bioactive compounds from the scale insect pathogenic fungus conoideocrella tenuis BCC 18627

Article abstract of DOI:10.1021/np100849x

A new cyclohexadepsipeptide, conoideocrellide A (1), its linear derivatives, conoideocrellides B-D (2-4), three new hopane triterpenoids (5-7), two new bioxanthracenes (9 and 10), and a new isocoumarin glycoside (13) were isolated from the scale insect pathogenic fungus Conoideocrella tenuis BCC 18627. Biological activities of the new compounds were evaluated.

Full text of DOI:10.1021/np100849x

ARTICLE
pubs.acs.org/jnp
Bioactive Compounds from the Scale Insect Pathogenic Fungus
Conoideocrella tenuis BCC 18627
Masahiko Isaka,* Somporn Palasarn, Sumalee Supothina, Somjit Komwijit, and J. Jennifer Luangsa-ard
National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road, Klong Luang,
Pathumthani 12120, Thailand
S Supporting Information
b
ABSTRACT: A new cyclohexadepsipeptide, conoideocrellide
A (1), its linear derivatives, conoideocrellides BꢀD (2ꢀ4),
three new hopane triterpenoids (5ꢀ7), two new bioxanthra-
cenes (9 and 10), and a new isocoumarin glycoside (13) were
isolated from the scale insect pathogenic fungus Conoideocrella
tenuis BCC 18627. Biological activities of the new compounds
were evaluated.
onoideocrella (family Clavicipitaceae, Hypocreales, Ascom-
ycota) is a recently proposed genus of insect pathogenic
the two tyrosine residues exhibited a phenolic proton signal at δH
9.21 (7-OH), while the other formed a prenyl ether. HMBC
correlations from the methylene protons of the prenyl group at
δH 4.45 to δC 158.0 of a quaternary carbon (C-7) and the
NOESY cross-peak between H2-10 and H-6/H-8 confirmed
the 7-O-prenyl group. The 3-phenyllactic acid (3-Ph-Lac) residue
was also elucidated on the basis of 2D NMR data. An oxymethine
at δC 74.4 (C-2) was attached to δC 37.4 of a methylene (C-3),
which was flanked by a phenyl group (HMBC). The sequence of
the six residues was also established by analysis of HMBC data
(Figure 1). Amide protons of Ala, Gly, Tyr, Ser, and O-prenyl-
Tyr were correlated respectively to carbonyl carbons of 3-Ph-Lac,
Ala, Gly, Tyr, and Ser. HMBC correlation from H-2 of 3-Ph-Lac
to C-1 of O-prenyl-Tyr indicated an ester linkage of O-prenyl-
Tyr-3-Ph-Lac to form a cyclohexadepsipeptide. This sequence
was further supported by NOESY correlations of H-2 of 3-Ph-
Lac to NH of Ala; H3-3 of Ala to NH of Gly; H2-2 of Gly to NH of
Tyr; H-3 of Tyr to NH of Ser; H2-3 of Ser to NH of O-prenyl-
Tyr; and H2-3 of O-prenyl-Tyr to H-2 of 3-Ph-Lac.
The absolute configurations of the amino acid residues in 1
were determined by Marfey’s method.9,10 The acid hydrolysate
of 1 was derivatized with NR-(2,4-dinitro-5-fluorophenyl)-L-
alaninamide (FDAA) and analyzed by HPLC using an ODS
column. The HPLC chromatogram (UV 340 nm) of the FDAA-
derivatized hydrolysate exhibited peaks of O-prenyl-D-Tyr, D-Ser, D-
Tyr, L-Ala, and Gly. The absolute configuration of the Ph-Lac
residue was addressed by isolation of this fragment from an acid
hydrolysate. The specific rotation of 3-phenyllactic acid ([R]26D ꢀ22,
c 0.18, EtOH), isolated from the hydrolysate, was consistent with that
of the commercial product (Sigma-Aldrich) (2S)-L-3-phenyllactic
acid. Conoideocrellide A (1) is structurally very similar to paecilo-
depsipeptide A (16), which was previously isolated in our laboratory
from T. luteorostrata BCC 9617 and its anamorph Paecilomyces
C
fungi that specifically attacks scale insects.1 There are only two
described species, C. luteorostrata and C. tenuis, which were
originally placed in Torrubiella as T. luteorostrata and T. tenuis,
respectively. There have been a few reports of compounds from
this genus: paecilodepsipeptide A and a naphthopyrone glyco-
side from T. luteorostrata BCC 9617,2 torrubiellutins AꢀC from
T. luteorostrata BCC 12904,3 and isocoumarin glycosides from
T. tenuis BCC 12732.4 In our research on the metabolites of insect
pathogenic fungi, we found an extract from C. tenuis strain BCC
1
18627 that exhibited a complex and unique H NMR spectro-
scopic profile suggesting the co-occurrence of several types of
secondary metabolites. Scale-up fermentation and chemical
studies led to the isolation of four new depsipeptides, conoideo-
crellides AꢀD (1ꢀ4), three new hopane-type triterpenes (5ꢀ7)
and the known zeorin (8), two new bioxanthracenes (9 and 10)
and the known ES-242-1 (11)5ꢀ7 and ES-242-2 (12),6ꢀ8 and a
new (13) and two known (14 and 15)4 isocoumarin glycosides.
We report here details of the isolation, structure elucidation, and
biological activities of the new compounds.
’ RESULTS AND DISCUSSION
Conoideocrellide A (1) was isolated as a colorless solid, and
the molecular formula was established as C40H47N5O10 by
HRESIMS. The IR spectrum exhibited intense broad absorption
bands at νmax 3500ꢀ3360 (amide NH and OH) and
1670ꢀ1630 cmꢀ1 (amide carbonyl) and a band of an ester
carbonyl at 1738 cmꢀ1. The 1H and 13C NMR data (DMSO-d6)
suggested that 1 was a hexadepsipeptide composed of a hydro-
xycarboxylic acid and five amino acid residues, showing six
carbonyl carbon signals at δC 172.5ꢀ168.2 and five amide NH
protons at δH 8.27ꢀ7.68. Five amino acid residues were assigned
on the basis of 2D NMR data (COSY, HMQC, and HMBC) as a
glycine (Gly), an alanine (Ala), a serine (Ser), a tyrosine (Tyr),
and an O-prenyltyrosine (O-prenyl-Tyr) (Table 1). Thus, one of
Received: November 22, 2010
Published: April 07, 2011
r
Copyright
2011 American Chemical Society and
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American Society of Pharmacognosy
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Table 1. NMR Spectroscopic Data (500 MHz, DMSO-d6) for Conoideocrellide A (1)
δH, mult.
(J in Hz)
δH, mult.
(J in Hz)
δH, mult.
position
δC, mult.
position
D-Ser
δC, mult.
position
δC, mult.
(J in Hz)
O-prenyl-D-Tyr
Gly
1
1
171.6, qC
1
170.5, qC
55.1, CH
61.9, CH2
169.5, qC
43.4, CH2
2
55.8, CH
35.5, CH2
128.3, qC
130.8, CH
115.0, CH
158.0, qC
64.7, CH2
120.5, CH
137.4, qC
25.8, CH3
18.4, CH3
4.38, m
2
4.26, m
2
3.63, m; 3.40, m
3
2.84ꢀ2.83, m
3
3.62ꢀ3.58, m
5.08, t (5.5)
NH
L-Ala
1
7.94, dd (5.8, 5.5)
4
3-OH
NH
D-Tyr
1
5,9
6,8
7
10
20
30
40
50
NH
6.94, d (8.6)
6.84, d (8.6)
7.68, d (8.0)
172.5, qC
48.9, CH
18.6, CH3
2
4.28, m
171.2, qC
56.2, CH
36.1, CH2
3
1.09, d (7.0)
7.70, d (6.9)
4.45, d (6.6)
5.35, m
2
4.19, m
NH
3
2.96, dd (14.0, 4.4)
2.72, m
L-3-Ph-Lac
168.2, qC
74.4, CH
37.4, CH2
1
2
3
1.67, s
4
128.5, qC
130.2, CH
115.5, CH
156.2, qC
5.05, dd (6.3, 4.5)
3.01, dd (14.0, 6.3)
2.74, dd (14.0, 4.5)
1.64, s
5,9
6,8
7
6.98, d (8.5)
6.63, d (8.5)
8.27, d (4.9)
4
136.2, qC
130.3, CH
128.4, CH
127.1, CH
7-OH
NH
9.21, s
5,9
6,8
7
6.89, d (7.9)
7.21, m
8.23, d (8.1)
7.19, m
cinnamomeus BCC 9616.2,11 Paecilodepsipeptide A is identical to
gliotide, which was isolated from Gliocladium sp.12,13 The absolute
configuration of 1 proved to be identical to gliotide/paecilodepsipep-
tide A (16). The only structural difference is that 1 possesses a D-Ser
residue instead of D-Ala in 16.
173.4. Therefore, conoideocrellide B (2) was identified as a linear
analogue of 1, wherein the ester moiety is hydrolyzed. This
conclusion was confirmed by alkaline hydrolysis of 1.
Conoideocrellide C (3) was assigned as the methyl ester
variant of 2. HMBC correlation from the methoxy protons (δH
3.56) to C-1 (δC 172.1) of O-prenyl-Tyr indicated that com-
pound 3 forms a methyl ester at the C-terminal position. The
structure designated as 3 was consistent with its molecular
formula of C41H51N5O11 (HRESIMS).
The molecular formula of conoideocrellide
B (2),
C40H49N5O11, was determined by HRESIMS. The 1H and 13
C
NMR data of 2 were similar to those of 1. The significant
difference was the upfield shift of H-2 of 3-Ph-Lac (δH 4.11,
dd, J = 8.5, 3.4 Hz) when compared with 1 (H-2, δH 5.05). In
addition, C-1 of O-prenyl-Tyr appeared as a broad signal at δC
Conoideocrellide D (4) was structurally related to 3, with the
only difference being that the prenyl group was oxygenated in 4.
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Thus, CH3-40 of O-prenyl-Tyr was replaced by a hydroxymethyl
group resonating at δC 66.0/δH 3.82 and 4.87. The E-geometry
of the trisubstituted olefin was assigned on the basis of the
NOESY correlations for H2-10/H3-50 and H-20/H2-40.
Similar to 16, a unique structural feature of conoideocrellide A
(1) is that it possesses three D-amino acid residues, including an
unusual O-prenyl-D-Tyr, whereas it contains only one L-amino acid
(L-Ala). Linear analogues 2 and 3 are considered respectively to be
hydrolysis and methanolysis products of 1. Probably these minor
constituents are artifacts, formed during a methanolic mycelia
extraction process and/or extensive column chromatography. We
did not isolate the cyclic analogue of 4, the plausible precursor.
Compound 5 was isolated as a colorless solid, and the molecular
formula was established as C30H50O4 by HRESIMS. The IR
spectrum exhibited broad absorption bands of hydroxy groups at
νmax 3531 and 3484 cmꢀ1 and a carbonyl absorption at 1697 cmꢀ1
.
Figure 1. Selected HMBC and NOESY correlations for 1, indicating
the sequence of five amino acids and 3-Ph-Lac.
1
The H and 13C NMR data in DMSO-d6 suggested that 5 is a
Table 2. NMR Spectroscopic Data (500 MHz, DMSO-d6) for Triterpenes 5ꢀ7
5
6
7
position
δC, mult.
δH, mult. (J in Hz)
δC, mult.
δH, mult. (J in Hz)
δC, mult.
δH, mult. (J in Hz)
1
44.5, CH2
19.1, CH2
44.23, CH2
34.7, qC
R 0.81, dt (3.2, 13.5); β 2.73, m
R 1.24, m; β 1.59, m
45.0, CH2
19.2, CH2
44.5, CH2
34.8, qC
R 0.82, m; β 2.68, br d (13.3)
R 1.23, m; β 1.61, m
42.8, CH2
18.9, CH2
44.0, CH2
34.2, qC
R 0.70, m; β 1.57, m
R 1.31, m; β 1.59, m
R 1.10, m; β 1.26, m
2
3
R 1.06, m; β 1.19, m
R 1.11, dt (4.0, 13.4); β 1.21, m
4
5
56.6, CH
65.9, CH
42.6, CH2
43.9, qC
0.55, br s
56.8, CH
66.4, CH
42.1, CH2
43.6, qC
0.70, br s
55.4, CH
72.11,a CH
72.13,a CH
46.8, qC
0.68, br s
4.04, m
3.44, m
6
4.18, m
4.27, br s
7
R 1.08, m; β 1.47, m
R 1.76, dd (12.2, 4.5); β 1.37, m
8
9
57.7, CH
39.9, qC
1.42, m
56.6, CH
39.2, qC
1.21, m
50.9,b CH
1.13, m
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
37.2, qC
69.4, CH
35.6, CH2
49.2, CH
55.9, qC
4.13, m
69.2, CH
36.7, CH2
48.4, CH
45.1, qC
3.83, m
20.9, CH2
24.6, CH2
49.5, CH
43.5, qC
R 1.54, m; β 1.36, m
R 1.38, m; β 1.30, m
1.33, m
R 2.12, m; β 1.80, m
1.78, m
R 1.36, m; β 1.51, m
1.44, m
27.4, CH2
22.1, CH2
54.0, CH
44.21, qC
39.7, CH2
26.6, CH2
50.5, CH
71.7, qC
R 1.98, m; β 0.90, m
R 1.25, m; β 1.81, m
1.38, m
28.7, CH2
23.5, CH2
54.6, CH
44.2, qC
R 1.70, m; β 0.95, m
R 1.21, m; β 1.79, m
1.32, m
38.4, CH2
22.3, CH2
54.1, CH
44.3, qC
R 1.65, m; β 1.47, m
R 1.51, m; β 1.84, m
1.30, m
R 0.99, m; β 1.44, m
R 1.45, m; β 1.62, m
2.06, m
41.3, CH2
26.6, CH2
51.1, CH
72.1, qC
R 0.90, m; β 1.45, m
R 1.46, m; β 1.64, m
2.11, m
41.9, CH2
26.4, CH2
51.0,b CH
72.0, qC
R 0.88, m; β 1.45, m
R 1.45, m; β 1.60, m
2.09, m
34.0, CH3
24.1, CH3
18.3, CH3
19.3, CH3
210.7, CH
0.85, s
1.14, s
1.30, s
1.29, s
10.32, s
34.2, CH3
24.3, CH3
18.2, CH3
19.4, CH3
60.4, CH2
0.89, s
33.3, CH3
24.3, CH3
17.4, CH3
11.6, CH3
17.7, CH3
0.89, s
1.14, s
1.09, s
1.12, s
0.93, s
1.16, s
1.28, s
1.22, s
3.70, dd (12.2, 5.3)
3.62, dd (12.2, 4.5)
0.81, s
28
14.8, CH3
31.3, CH3
29.7, CH3
0.58, s
0.98, s
1.04, s
4.14, m
15.3, CH3
31.1, CH3
29.6, CH3
16.7, CH3
31.2, CH3
29.6, CH3
0.71, s
29
1.04, s
1.06, s
30
1.03, s
1.02, s
6-OH
7-OH
11-OH
22-OH
27-OH
3.99, d (3.4)
4.03, m
3.86, br d (7.5)
4.17, d (6.0)
3.88, s
3.83, m
3.80, s
3.81, s
4.19, dd (5.3, 4.5)
a,b Carbon chemical shifts may be interchanged.
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Figure 3. Selected HMBC and NOESY correlations for 9.
Figure 2. Key NOESY correlations for 5.
Table 3. NMR Spectroscopic Data (500 MHz, CDCl3) for 9
position
δC, mult.
δH, mult. (J in Hz)
1, 10
3, 30
4, 40
4a, 4a0
5, 50
6, 60
7, 70
8, 80
8a, 8a0
9, 90
65.5, CH2
73.4, CH
65.6, CH
131.8, qC
96.8, CH
157.8, qC
98.2, CH
157.3, qC
110.9, qC
149.8, qC
115.6, qC
125.1, qC
134.9, qC
16.6, CH3
169.3, qC
20.4, CH3
55.3, CH3
56.2, CH3
5.19, d (15.7); 4.84, d (15.7)
3.65, dq (1.8, 6.4)
5.30, d (1.8)
6.09, d (2.2)
6.51, d (2.2)
Figure 4. Selected HMBC and NOESY correlations for 10.
(H-11) to H3-25, H3-26, and H-13 and from 11-OH to Hβ-1 and
HR-12. The unusual downfield shift of Hβ-1 (δH 2.73) can be
explained by the deshielding of this proton by the 11R-OH group.
On the basis of these data, 5 was assigned as a new hopane-type
triterpene, hopan-27-al-6β,11R,22-triol.
9a, 9a0
10, 100
10a, 10a0
11, 110
4-OCOCH3, 40-OCOCH3
4-OCOCH3, 40-OCOCH3
6-OCH3, 60-OCH3
Compound 6 possessed the molecular formula C30H52O4
(HRESIMS), containing two more hydrogen atoms than 5. The 1H
and 13C NMR spectroscopic data were similar to those of 5. Significant
differences were the absence of the 1H and 13C resonances of a formyl
group (CH-27 of 5) instead of the presence of a hydroxymethyl group
(δC 60.4, δH 3.70 and 3.62; 27-OH δH 4.19, dd, J = 5.3, 4.5 Hz). The
location of the hydroxymethyl group was confirmed by the HMBC
correlations from H2-27 to C-8 and C-13 and from 27-OH to C-14.
The molecular formula of compound 7 was determined by
HRESIMS as C30H52O3. The 1H and 13C NMR spectroscopic data
suggested that 7 is structurally related to 5 and 6; however, the
11-OH functionality was absent and CH3-27 was not oxygenated.
Instead, 7 possessed a 7-OH group, as confirmed by a weak COSY
correlation between H-7 (δH 3.44) and H-6 (δH 4.04) and an
HMBC correlation from 7-OH to C-8. Similar to 5 and 6, H-5
resonated as a broad singlet with a narrow peak width and exhibited
an intense NOESY cross-peak to H-6, which confirmed an axial
orientation of 6-OH. NOESY correlations from H-7 to H-5, H-9,
and H3-27 indicated that H-7 occupied an axial position. Therefore,
compound 7 was assigned as hopane-6β,7β,22-triol.
The molecular formula of 9 (C36H38O12), determined by
HRESIMS, was the same as the known co-metabolite ES-242-2
(12).6ꢀ8 The 1H and 13C NMR spectroscopic data of compound
9 were similar to those of 12 and also revealed that 9 is a
symmetrical dimer (Table 3). The small coupling constant (J =
1.8 Hz) of H-3/H-4 indicated the cis-relation of CH3-11 and the
acetoxy group. Analysis of HMBC correlations (Figure 3) re-
vealed that 9 is a C-10ꢀC-100 dimer. The intense NOESY
correlation of H-4 (H-40) and H-50 (H-5) indicated that the
helicity of the chiral axis is opposite that of 12. Therefore,
compound 9 was assigned as the atropisomer of 12.
1.05, d (6.4)
1.88, s
3.51, s
4.08, s
9.55, s
8-OCH3, 80-OCH3
9-OH, 90-OH
hopane-type triterpene related to the known co-metabolite, zeorin
1
(8). The H and 13C NMR, DEPT135, and HMQC data for 5
supported the presence of an aldehyde (δC 210.7/δH 10.32), an
oxygenated quaternary carbon (δC 71.7), five quaternary carbons,
two oxymethines (δC 65.9/δH 4.18 and δC 69.4/δH 4.13), five
methines, nine methylenes, and seven methyl groups (Table 2). The
planar structure of 5 was deduced by analyses of COSY and HMBC
data. The location of the aldehyde functionality was assigned by the
HMBC correlations from the formyl proton (δH 10.32) to C-14
and C-15. The key HMBC correlations were those from seven
methyl groups (H3-23, H3-24, H3-25, H3-26, H3-28, H3-29, and H3-
30) attached to quaternary sp3 carbons C-4, C-4, C-10, C-8, C-18,
C-22, and C-22, respectively. The relative configuration of 5 was
addressed on the basis of J-values and NOESY correlations
(Figure 2). Key NOESY correlations were those for protons and
methyl protons at axial positions. NOESY cross-peaks at the β-face
were observed from H3-25 to Hβ-2 and H-11 and from H3-26 to
H-11, H-13, and Hβ-15. Important NOESY correlations on the R-
face were those from H-5 to HR-1, HR-3, H-6, and H-9 and from
H-27 to HR-7, H-9, HR-12, and H3-28. H-5 resonated as a broad
singlet with a narrow signal width and exhibited a weak COSY cross-
peak and an intense NOESY cross-peak with H-6. These data
indicated an equatorial orientation of H-6. An axial orientation of
H-11 was evident from the NOESY correlations from this proton
Compound 10 possessed the same molecular formula
(HRESIMS) as 9 and 12. Analysis of the 2D NMR data, in
particular HMBC correlations, demonstrated that it is a C-10ꢀC-
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Table 4. NMR Spectroscopic Data (500 MHz, CDCl3) for 10
position
δC, mult.
δH, mult. (J in Hz)
position
δC, mult.
δH, mult. (J in Hz)
1
65.0, CH2
5.28, d (15.6)
4.90, d (15.6)
3.79, br q (6.4)
5.26, br s
10
64.7, CH2
5.21, d (15.6)
4.76, d (15.6)
3.91, dq (1.6, 6.4)
5.63, d (1.6)
3
4
72.8, CH
67.3, CH
30
40
72.4, CH
68.8, CH
4a
132.0, qC
98.3, CH
4a0
50
60
70
133.0, qC
113.8, qC
155.5, qC
94.5, CH
5
6.00, d (2.1)
6.45, d (2.1)
6
157.1, qC
97.9, CH
7
6.67, s
8
157.3, qC
110.89,a qC
149.64,b qC
115.1, qC
123.8, qC
135.6,c qC
17.0, CH3
169.5, qC
20.8,d CH3
55.1, CH3
56.4,e CH3
80
8a0
90
9a0
100
10a0
110
40-OCOCH3
40-OCOCH3
60-OCH3
80-OCH3
90-OH
157.7, qC
110.86,a qC
149.59,b qC
114.6, qC
116.3, CH
135.5,c qC
16.8, CH3
170.4, qC
20.7,d CH3
56.8, CH3
56.3,e CH3
8a
9
9a
10
6.39, s
10a
11
1.08, d (6.4)
1.23, d (6.4)
4-OCOCH3
4-OCOCH3
6-OCH3
8-OCH3
9-OH
1.98, s
3.45, s
4.07, s
9.53, s
1.77, s
3.69, s
4.18, s
9.51, s
aꢀe The assignments of carbons can be interchanged.
Table 5. NMR Spectroscopic Data (500 MHz, acetone-d6)
for 13
position
δC, mult.
δH, mult. (J in Hz)
1
166.0, qC
154.3, qC
98.7, CH
131.1, qC
135.3, qC
157.8, qC
101.8, CH
159.2, qC
99.2, qC
3
4
6.65, br s
4a
Figure 5. Selected HMBC and NOESY correlations for 13.
5
6
50 dimer (Figure 4, Table 4). H-4 (δH 5.26) resonated as a broad
singlet, whereas H-40 (δH 5.63) was a doublet with a small coupling
constant (J = 1.6 Hz). These data indicated that the relative
configurations of C-3/C-4 and C-30/C-40 are the same as other
ES-242 derivatives. The intense NOESY correlation observed
between H-4 and H-100 (δH 6.39, s) revealed the helicity of the
chiral axis as shown in the structural formula. The deacetyl analogue
of 10 and its atropisomer were previously reported.6,7
The molecular formula of 13 was established as C18H22O10 by
HRESIMS. The 1H and 13C NMR spectroscopic data showed a
resemblance to the known co-metabolite 14.4 Notable differ-
ences were the presence of a methoxy group (δC 60.9; δH 3.81,
3H, s) and the absence of one of the meta-coupled aromatic
protons (H-5). Analysis of the 2D NMR data (COSY, HMQC,
and HMBC) revealed that 13 was the 5-methoxy derivative of 14
(Figure 5, Table 5). The sugar moiety was assigned as 40-O-
methyl-β-glucopyranose, which was identical to 14 and 15. Thus,
7
6.74, s
8
8a
9
18.6, CH3
60.9, CH3
10.92, br s
100.2, CH
74.0, CH
77.1, CH
79.1, CH
76.3, CH
61.1, CH2
59.6, CH3
2.28, d (0.6)
3.81, s
5-OCH3
8-OH
10
20
30
40
50
60
5.17, d (7.7)
3.57, t (8.4)
3.67, t (9.0)
3.24, t (9.3)
3.61, m
3.85, br d (11.9); 3.71, m
3.56, s
40-OCH3
from the HMBC correlation from the anomeric proton (H-10, δH
5.17) to C-6. The NOESY correlation found between H-7 and H-10
further supported the sugar junction. The negative specific rotation
0
0
0
0
the vicinal coupling constants of J1 ,2 = 7.7 Hz, J2 ,3 = 9.0 Hz, and
0
0
0
0
0
0
J3 ,4 = J4 ,5 = 9.3 Hz indicated that methine protons H-1 to H-5
of 13, [R]23 ꢀ55 (c 0.17, MeOH), was similar to those of the
all occupy axial positions. The intense HMBC correlation from
methoxy protons (δH 3.56, 3H, s) to C-40 (δC 79.1) and the
correlation from H-40 (δH 3.24) to the methoxyl carbon (δC
59.6) demonstrated that the 40-OH group was methylated. The
linkage of the sugar to C-6 (δC 157.8) of the aglycone was evident
D
known co-metabolites 14 and 15.4 Due to the sample shortage,
further analysis to confirm the absolute configuration of the sugar
moiety of 13 was not performed; however, the co-occurrence
suggested that 13 also possesses a 4-O-methyl-D-glucose.
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Journal of Natural Products
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Table 6. Biological Activities of Triterpenes 5ꢀ7 and Bioxanthracenes 9 and 10
P. falciparuma
M. tuberculosisb
HSV-1c
cytotoxicity (IC50, μM)d
compound
(IC50, μM)
(MIC, μM)
(IC50, μM)
KB
10
MCF-7
NCI-H187
Vero
5
>21
9.8
>22
10
>105
52
21
14
28
15
68
47
69
47
6
5.6
>109
4.4
15
7
>109
>75
>75
>109
>75
>75
>109
22
>109
71
>109
>75
>75
9
10
15
37
12
a Antimalarial activity against Plasmodium falciparum K1. Standard antimalarial drug dihydroartemisinin showed an IC50 value of 0.0044 μM.
b Antitubercular activity against Mycobacterium tuberculosis H37Ra. MIC values of standard anti-TB drug isoniazid were 0.17ꢀ0.34 μM. c Anti-HSV-1
activity. Standard antiviral compound Acyclovir showed an IC50 value of 17 μM. d IC50 values of a standard compound, doxorubicin hydrochloride,
against the cancer cell-lines KB, MCF-7, and NCI-H187 were 0.27, 4.9, and 0.23 μM, respectively. The IC50 value of a standard compound, ellipticine,
against Vero cells was 12 μM.
New compounds 1ꢀ7, 9, and 10 were subjected to our biological
activity protocols inclusive of antiplasmodial (Plasmodium falcipar-
um K1), antimycobacterial (Mycobacterium tuberculosis H37Ra),
and antiviral (herpes simplex virus type 1, HSV-1) activities and
cytotoxicity against the human cancer cell lines KB (oral epidermoid
carcinoma), MCF-7 (breast cancer), and NCI-H187 (small cell
lung cancer) and nonmalignant Vero cells (African green monkey
kidney fibroblasts). Conoideocrellide A (1) and linear analogues
2ꢀ4were inactive in these assays. In contrast, paecilodepsipeptide A
(16) was reported to show antimalarial (IC50 4.9 μM) and cytotoxic
activities in the same assays.11 Triterpene 6 and bioxanthracenes 9
and 10 exhibited weak antimalarial activity (Table 6). Triterpenes 6
and 7 also showed anti-HSV-1 and cytotoxic activities. ES-242
derivatives are known to exhibit antimalarial activity.6a,7
The present study demonstrates that the genus Conoideocrella
is a potent source of novel bioactive compounds. Structural
similarity and the occurrence in closely related species of 1 and
16 suggest that the nonribosomal peptide synthetases for these
depsipeptides are almost identical, with the only difference being
the recognition site for D-Ser (for 1) or D-Ala (for 16). Since 16
was also isolated from Gliocladium sp., these depsipeptides are
not specific to Conoideocrella. Other scale insect pathogens,
Hypocrella and Moelleriella and their anamorph Ascherisonia,
are also reported as producers of hopane-type triterpenes.14,15 While
these genera commonly produce a mixture of three hopanoids,
zeorin (8), dustanin (hopane-15R,22-diol), and 3β-acetoxyhopane-
15a,22-diol, with a variety of relative compositions,14 the genus
Conoideocrella has been known to produce only zeorin.2 A unique
structural feature of the new hopanoids 5ꢀ7 is the axial 6-OH, while
6R-OH (equatorial) hopanoids such as zeorin (8) are well known.
This is also the first report of the isolation of ES-242 derivatives from
Torrubiella or Conoideocrella. Several compounds possessing a 4-O-
methyl-β-glucopyranoside moiety have been previously isolated
from insect pathogenic fungi.2,4,16ꢀ18 However, isocoumarin agly-
cones are known only from Conoideocrella.4
Fungal Material. Conoideocrella tenuis was isolated from a scale
insect (Hemiptera) in Khao Yai National Park, Nakhon Nayok Province,
and identified on the basis of the morphology of the fungus on the scale
insect. The fungus produced white to cream mycelia surrounding the
host and had conical perithecia. The ITS rDNA of this strain was
sequenced and compared to the in-house database for invertebrate
pathogens and was further subjected to a BLAST search in GenBank.
The molecular data revealed it to be Conoideocrella tenuis and confirmed
the morphological identification.1 This fungus was deposited in the
BIOTEC Culture Collection (BCC) as BCC 18627 on August 30, 2005.
Fermentation, Extraction, and Isolation. The fungus BCC
18627 was maintained on potato dextrose agar at 25 °C. The agar was
cut into small plugs and inoculated into 6 ꢁ 250 mL Erlenmeyer flasks
containing 25 mL of potato dextrose broth (PDB; potato starch 4.0 g,
dextrose 20.0 g, per liter). After incubation at 25 °C for 4 days on a rotary
shaker (200 rpm), each primary culture was transferred into a 1 L
Erlenmeyer flask containing 250 mL of the same liquid medium (PDB)
and incubated at 25 °C for 4 days on a rotary shaker (200 rpm). These
secondary cultures were pooled, and each 25 mL portion was transferred
into 60 ꢁ 1 L Erlenmeyer flasks containing 250 mL of M102 medium
(sucrose 30.0 g, malt extract 20.0 g, bacto-peptone 2.0 g, yeast extract 1.0
g, KCl 0.5 g, MgSO4 7H2O 0.5 g, KH2PO4 0.5 g, per liter; pH was not
3
adjusted), and final fermentation was carried out at 25 °C for 20 days
under static conditions (final pH 5.93). The cultures were filtered to
separate mycelia and filtrate (broth). Culture broth was extracted with
EtOAc (2 ꢁ 15 L), and the combined organic phase was concentrated to
obtain a brown gum (extract A, 3.10 g). Wet mycelia were macerated in
MeOH (1.5 L, rt, 2 days), then filtered. The filtrate was defatted by
partitioning with hexanes (1 L). The aqueous MeOH layer was
concentrated under reduced pressure. The residue was dissolved in
EtOAc (1 L), washed with H2O (200 mL), and concentrated under
reduced pressure to leave a brown solid (extract B, 5.10 g). Extract A was
subjected to column chromatography (CC) on silica gel (5.0 ꢁ 15 cm,
MeOH/CH2Cl2, step gradient elution from 0:100 to 20:80) to obtain
three pooled fractions: fractions A-1 (51 mg), A-2 (732 mg), and A-3
(1.70 g). Fraction A-1 was subjected to preparative HPLC using a
reversed-phase column (LiChroCART, 10 ꢁ 250 mm, 10 μm; mobile
phase MeCN/H2O, 50:50, flow rate 4 mL/min) to furnish 11 (0.8 mg,
tR 10 min) and 12 (10.0 mg, tR 24 min). Fraction A-2 was repeatedly
fractionated by CC on silica gel (MeOH/CH2Cl2, step gradient elution)
to afford 4 (11.2 mg). Fraction A-3 was fractionated by CC on silica gel
(MeOH/CH2Cl2, step gradient elution) and Sephadex LH-20 (2.8 ꢁ
60 cm, MeOH) to give 14 (5.6 mg). Mycelial extract (extract B) was
triturated in MeOH (3 mL, rt, 2 h) and filtered. Residual solid (B1, 2.5 g)
and filtrate (B2) were separately subjected to chromatographic fractio-
nations. The insoluble solid (B1) was fractionated by CC on silica gel
(5.0 ꢁ 15 cm, MeOH/CH2Cl2, step gradient elution from 0:100 to
’ EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were mea-
sured with an Electrothermal IA9100 digital melting point apparatus.
Optical rotations were measured with a JASCO P-1030 digital polari-
meter. UV spectra were recorded on a GBC Cintra 404 spectrophot-
ometer. FTIR spectra were taken on a Bruker VECTOR 22
spectrometer. NMR spectra were recorded on Bruker DRX400 and
AV500D spectrometers. ESITOF mass spectra were measured with
Micromass LCT and Bruker micrOTOF mass spectrometers.
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Journal of Natural Products
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50:50) to obtain five pooled fractions: fractions B1-1 (700 mg), B1-2
(250 mg), B1-3 (250 mg), B1-4 (210 mg), and B1-5 (350 mg). Fraction
B1-1 contained mainly fatty acids and a small amount of 8. Fraction B1-2
was further fractionated by CC on silica gel (2.8 ꢁ 15 cm, MeOH/
CH2Cl2, step gradient elution from 0:100 to 20:80) to give four
fractions, B1-2-1ꢀB1-2-4. Fraction B1-2-2 (50 mg) was purified by
CC on silica gel (1.8 ꢁ 15 cm, acetone/CH2Cl2, step gradient elution
from 0:100 to 10:90) to furnish 7 (7.6 mg). Fraction B1-2-3 (150 mg)
was fractionated by CC on Sephadex LH-20 (2.8 ꢁ 60 cm, MeOH),
followed by CC on silica gel (MeOH/CH2Cl2, step gradient elution), to
obtain 8 (8.0 mg), 5 (15.4 mg), and 7 (13.8 mg). Fraction B1-3 was
purified by CC on silica gel (MeOH/CH2Cl2, step gradient elution) to
obtain 6 (126 mg). The filtrate from trituration (B2) was subjected to
preparative HPLC using a reversed-phase column (Phenomenex Luna
10u C18(2) 100A, 21.2 ꢁ 250 mm, 10 μm; mobile phase MeCN/H2O,
50:50, flow rate 15 mL/min) to collect five fractions, B2-1ꢀB2-5. Fraction
B2-1 (739 mg) was further separated by CC on silica gel (MeOH/CH2Cl2,
step gradient elution from 0:100 to 20:80) to furnish 3(12.2 mg) and 2(4.2
mg). Fraction B2-2 (697 mg) was further purified by preparative HPLC
(Phenomenex Luna) to obtain 1 (520 mg). Fraction B2-3 (225 mg) was
further purified by CC on silica gel (MeOH/CH2Cl2, step gradient elution
from 0:100 to 20:80) and preparative HPLC (Phenomenex Luna) to
furnish 9 (4.9 mg, tR 19 min) and 12 (5.0 mg, tR 24 min). Fraction B2-4
(75 mg) was also purified by CC on silica gel (MeOH/CH2Cl2, step
gradient elution from 0:100 to 20:80) and preparative HPLC
(Phenomenex Luna) to furnish 12 (27.1 mg, tR 24 min), while fraction
B2-5 (213 mg) gave 10 (10.8 mg, tR 31 min).
Hopane-6β,11r,22,27-tetraol (6): colorless solid; mp 220ꢀ221 °C;
[R]24D þ7 (c 0.20, MeOH); IR (KBr) νmax 3456, 2941 cmꢀ1; 1H NMR
(500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) data, see
Table 2; HRMS (ESI-TOF) m/z 499.3759 [M þ Na]þ (calcd for
C30H52O4Na, 499.3763).
Hopane-6β,7β,22-triol (7): colorless solid; mp 245ꢀ246 °C;
1
[R]24 þ12 (c 0.20, MeOH); IR (KBr) νmax 3423, 2946 cmꢀ1; H
D
NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6)
data, see Table 2; HRMS (ESI-TOF) m/z 483.3821 [M þ Na]þ (calcd
for C30H52O3Na, 483.3809).
Compound 9 (atropisomer of ES-242-2): yellow powder;
[R]25 ꢀ26 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 238 (4.74),
D
312 (3.95), 341 (3.78), 356 (3.82) nm; IR (KBr) νmax 3406, 1735, 1625,
1365, 1233, 1096 cmꢀ1; 1H NMR (500 MHz, CDCl3) and 13C NMR
(125 MHz, CDCl3) data, see Table 3; HRMS (ESI-TOF) m/z 685.2264
[M þ Na]þ (calcd for C36H38O12Na, 685.2261).
Compound 10: yellow powder; [R]25D þ66 (c 0.15, MeOH); UV
(MeOH) λmax (logε) 239 (4.75), 311 (3.90), 354 (3.82) nm; IR (KBr) νmax
3384, 1730 sh, 1726, 1626, 1361, 1236, 1095 cmꢀ1; 1H NMR (500 MHz,
CDCl3) and 13C NMR (125 MHz, CDCl3) data, see Table 4; HRMS (ESI-
TOF) m/z 685.2256 [M þ Na]þ (calcd for C36H38O12Na, 685.2261).
6,8-Dihydroxy-5-methoxy-3-methylisocoumarin 6-O-(4-
O-methyl-β-D-glucopyranoside) (13): colorless, amorphous solid;
[R]23D ꢀ55 (c 0.17, MeOH); UV (MeOH) λmax (log ε) 235 (4.44), 242
(4.44), 263 (3.94), 279 sh (3.70), 338 (3.69) nm; IR (KBr) νmax 3442,
1694, 1649, 1619, 1488, 1107, 980 cmꢀ1; 1H NMR (500 MHz, acetone-
d6) and 13C NMR (125 MHz, acetone-d6) data, see Table 5; HRMS
(ESI-TOF) m/z 421.1108 [M þ Na]þ (calcd for C18H22O10Na,
421.1105).
Another fermentation batch (M102 medium, 40 ꢁ 250 mL), which
was examined prior to the isolation described above, gave 1 (20 mg), 8
(not purified), 9 (8.3 mg), 10 (4.0 mg), 12 (8.1 mg), and 15 (70.5 mg).
Mass fermentation (40 ꢁ 250 mL) in yeast extract sucrose medium
(sucrose 150 g, yeast extract 20 g, per liter) was also performed. The
extracts provided 1 (293 mg), 3 (9.0 mg), and 13 (2.1 mg), whereas
several other compounds were not purified.
Acid Hydrolysis of 1. Conoideocrellide A (1, 20 mg) was
hydrolyzed by heating in 6 M HCl (6 mL)/MeOH (2 mL) at 110 °C
for 15 h. After cooling, the aqueous solution was extracted with Et2O
(3 ꢁ 5 mL), and the combined organic phase was dried over MgSO4,
evaporated, and dried in vacuo. The residual solid was purified by CC on
Conoideocrellide A (1): colorless solid; mp 141ꢀ142 °C; [R]23
D
Sephadex LH-20 (1.5 ꢁ 30 cm, MeOH) to furnish (2S)-L-3-phenyllactic
1
acid (3.7 mg): colorless solid; [R]26 ꢀ22 (c 0.18, EtOH); H NMR
þ29 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 227 (4.31), 277 (3.24),
284 sh (3.18) nm; IR (KBr) νmax 3493, 3405, 3368, 1738, 1697, 1673,
1650, 1513, 1237 cmꢀ1; 1H NMR (500 MHz, DMSO-d6) and 13C NMR
(125 MHz, DMSO-d6) data, see Table 1; HRMS (ESI-TOF) m/z
758.3397 [M þ H]þ (calcd for C40H48N5O10, 758.3396), 780.3216 [M
þ Na]þ (calcd for C40H47N5O10Na, 780.3215).
D
(400 MHz, acetone-d6) and MS data were identical to those of the
commercial sample (Sigma-Aldrich).
Preparation and Analysis of Marfey Derivatives. Conoideo-
crellide A (1, 3.0 mg) was hydrolyzed by heating in 6 M HCl (1 mL) at
110 °C for 14 h. After cooling, the solution was evaporated and dried in
vacuo. The hydrolysate was redissolved in H2O (150 μL). To this
solution were added 1% (w/v) FDAA (Marfey’s reagent, NR-(2,4-
dinitro-5-fluorophenyl)-L-alaninamide) in acetone (300 μL) and 1 M
NaHCO3 solution (70 μL), and the mixture was incubated at 40 °C for 1
h. The reaction was quenched by addition of 1 M HCl (70 μL), and the
resulting homogeneous solution was diluted with MeOH (1.0 mL).
Standard L- and D-amino acids were also derivatized with FDAA in the
same manner as that for the hydrolysate of 1. O-Prenyl-L-Tyr and O-
prenyl-D-Tyr were previously synthesized for structure elucidation of
paecilodepsipeptide A.9 HPLC analysis was performed with the follow-
ing conditions: NovaPak C18 (3.9 ꢁ 150 mm, 4 μm), mobile phase
MeCN/(0.05% TFA or 20 mM ammonium phosphate in H2O), flow
rate 0.5 mL/min, UV detection at 340 nm. Three mobile phase
conditions were employed for polarity and separation reasons: (1)
MeCN/(20 mM ammonium phosphate in H2O) = 20:80, L-Ser (tR 4.6
min), D-Ser (tR 5.3 min); (2) MeCN/(0.05% TFA in H2O) = 25:75, Gly
(tR 9.8 min), L-Ala (tR 12.2 min), D-Ala (tR 18.3 min), L-Tyr (tR 19.8
min), D-Tyr (tR 27.6 min); (3) MeCN/(0.05% TFA in H2O) = 45:55, L-
Tyr (tR 6.6 min), D-Tyr (tR 8.7 min), O-prenyl-L-Tyr (tR 9.1 min), O-
prenyl-D-Tyr (tR 12.2 min). The derivatized hydrolysate of 1 contained
D-Ser, Gly, L-Ala, D-Tyr, and O-prenyl-D-Tyr.
Conoideocrellide B (2): colorless, amorphous solid; [R]25D ꢀ20
(c 0.18, MeOH); UV (MeOH) λmax (log ε) 226 (4.05), 277 (3.22), 284
sh (3.14) nm; IR (KBr) νmax 3418, 1638, 1544, 1515, 1241 cmꢀ1; 1H
NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6)
data, see Supporting Information; HRMS (ESI-TOF) m/z 774.3356
[M ꢀ H]ꢀ (calcd for C40H48N5O11, 774.3358).
Conoideocrellide C (3): colorless solid; mp 163ꢀ164 °C; [R]25D ꢀ20
(c 0.15, MeOH); UV (MeOH) λmax (log ε) 227 (4.27), 278 (3.43), 284 sh
(3.37) nm; IR (KBr) νmax 3423, 3283, 1741, 1633, 1544, 1514, 1237 cmꢀ1
;
1H NMR (500 MHz, DMSO-d6) and13C NMR (125 MHz, DMSO-d6) data,
see Supporting Information; HRMS (ESI-TOF) m/z 812.3467 [M þ Na]þ
(calcd for C41H51N5O11Na, 812.3477).
Conoideocrellide D (4): colorless solid; mp 181ꢀ182 °C; [R]25D ꢀ20
(c 0.20, MeOH); UV (MeOH) λmax (log ε) 226 (4.36), 277 (3.55), 284 sh
(3.47) nm; IR (KBr) νmax 3371, 3281, 1739, 1634, 1514, 1235 cmꢀ1; 1H
NMR (500 MHz, DMSO-d6) and13C NMR (125 MHz, DMSO-d6) data, see
Supporting Information; HRMS (ESI-TOF) m/z 828.3414 [M þ Na]þ
(calcd for C41H51N5O12Na, 828.3426).
Hopan-27-al-6β,11r,22-triol (5): colorless solid; mp 234ꢀ235 °C;
[R]25D ꢀ10 (c0.20, MeOH); IR (KBr) νmax 3531, 3484, 2945, 1697 cmꢀ1
;
1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6)
data, see Table 2; HRMS (ESI-TOF) m/z 497.3605 [M þ Na]þ (calcd for
C30H50O4Na, 497.3601).
Alkaline Hydrolysis of 1. To a solution of 1 (3.0 mg) in dioxane
(1 mL) was added 2 M NaOH (0.2 mL), and the mixture was stirred at
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Journal of Natural Products
ARTICLE
room temperature for 2 h. The mixture was evaporated, and the residue
was diluted with H2O, acidified with 1 M HCl (0.45 mL), and extracted
with EtOAc. The organic layer was dried over MgSO4 and concentrated
in vacuo to leave a colorless, amorphous solid (3.2 mg), whose MS and
1H NMR (DMSO-d6) data were identical to those of 2.
Biological Assays. The assay for activity against Plasmodium
falciparum (K1, multidrug-resistant strain) was performed using the
microculture radioisotope technique.19 Growth inhibitory activity
against Mycobacterium tuberculosis H37Ra, antiviral activity against
herpes simplex virus type-1, and cytotoxicity to Vero cells were assessed
using the green fluorescent protein microplate assay.20 Cytotoxic
activities against KB, MCF-7, and NCI-H187 cells were evaluated using
the resazurin microplate assay.21
confirmed by asymmetric synthesis: Yang, M. J.; Wu, J.; Yang, Z. G.;
Zhang, Y. M. Chin. Chem. Lett. 2009, 20, 527–530.
(14) Isaka, M.; Hywel-Jones, N. L.; Sappan, M.; Mongkolsamrit, S.;
Saidaengkham, S. Mycol. Res. 2009, 113, 491–497.
(15) Boonphong, S.; Kittakoop, P.; Isaka, M.; Palittapongarnpim, P.;
Jaturapat, A.; Danwisetkanjana, K.; Tanticharoen, M.; Thebtaranonth, Y.
Planta Med. 2001, 67, 279–281.
(16) Seephonkai, P.; Isaka, M.; Kittakoop, P.; Boonudomlap, U.;
Thebtaranonth, Y. J. Antibiot. 2004, 57, 10–16.
(17) Bunyapaiboonsri, T.; Yoiprommarat, S.; Intereya, K.; Kocharin,
K. Chem. Pharm. Bull. 2007, 55, 304–307.
(18) Zhang, S.-W.; Xuan, L.-J. Helv. Chim. Acta 2007, 90, 404–410.
(19) Desjardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chulay, J. D.
Antimicrob. Agents Chemother. 1979, 16, 710–718.
(20) Changsen, C.; Franzblau, S. G.; Palittapongarnpim, P. Anti-
microb. Agents Chemother. 2003, 47, 3682–3687.
(21) O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. Eur. J. Biochem.
2000, 267, 5421–5426.
’ ASSOCIATED CONTENT
S
Supporting Information. NMR spectra of 1ꢀ7, 9, 10,
b
and 13. This material is available free of charge via the Internet at
http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: þ66-25646700, ext 3554. Fax: þ66-25646707. E-mail:
isaka@biotec.or.th.
’ ACKNOWLEDGMENT
Financial support from the Bioresources Research Network,
National Center for Genetic Engineering and Biotechnology
(BIOTEC), is gratefully acknowledged.
’ REFERENCES
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(13) After reporting paecilodepsipeptide A,2,11 we noticed the
advanced report on gliotide,12 wherein the location of L- and D-alanine
1
residues was not assigned. Our later acquisition of H and 13C NMR
spectra of paecilodepsipeptide A using the same solvent (CD3OD) as
used for gliotide analysis confirmed that they are identical. The location
of L- and D-Ala as shown in the structural formula (16) was recently
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