Pleosporin A, an antimalarial cyclodepsipeptide from an elephant dung fungus (BCC 7069)

Article abstract of DOI:10.1016/j.tetlet.2013.11.063

Cyclodepsipeptides SCH 217048 (1), SCH 218157 (2), and a new analog, pleosporin A (3), were isolated from cultures of an unidentified elephant dung fungus of the family Pleosporaceae. The structure of 3 was elucidated on the basis of detailed spectroscopic interpretation. The absolute configurations of 1-3 were determined by chiral column HPLC analysis and Marfey's method. Cyclodepsipeptides 1-3 exhibited antimalarial activity against Plasmodium falciparum K1 with respective IC₅₀ values of 1.6, 6.4, and 1.6 μg/mL, while they did not show cytotoxicity against KB, MCF-7 and NCI-H187 cell-lines or non-cancerous Vero cells at 50 μg/mL.

Full text of DOI:10.1016/j.tetlet.2013.11.063

Tetrahedron Letters 55 (2014) 469–471
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Pleosporin A, an antimalarial cyclodepsipeptide from an elephant
dung fungus (BCC 7069)
⇑
Masahiko Isaka , Somporn Palasarn, Somjit Komwijit, Sayanh Somrithipol, Sujinda Sommai
National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road, Klong Luang, Pathumthani 12120, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 22 November 2013
Cyclodepsipeptides SCH 217048 (1), SCH 218157 (2), and a new analog, pleosporin A (3), were isolated
from cultures of an unidentified elephant dung fungus of the family Pleosporaceae. The structure of 3
was elucidated on the basis of detailed spectroscopic interpretation. The absolute configurations of 1–
3 were determined by chiral column HPLC analysis and Marfey’s method. Cyclodepsipeptides 1–3 exhib-
ited antimalarial activity against Plasmodium falciparum K1 with respective IC50 values of 1.6, 6.4, and
Keywords:
Coprophilous fungi
Pleosporaceae
SCH 217048
Cyclodepsipeptide
Antimalarial activity
1
.6
l
g/mL, while they did not show cytotoxicity against KB, MCF-7 and NCI-H187 cell-lines or non-can-
g/mL.
cerous Vero cells at 50
l
Ó 2013 Elsevier Ltd. All rights reserved.
The isolation of SCH 217048, a potent cyclodepsipeptide tachy-
kinin (NK ) receptor inhibitor, from an unidentified fungus was re-
ported in 1998 by Puar and co-workers. SCH 217048 exhibited a
highly selective NK antagonist-type activity (K 27.43 nM), while
it showed no activity at 10 M in NK and NK assays. This report
determination of the absolute configurations of 1–3 (Fig. 1), and
the evaluation of their biological activities.
2
1
The fungus BCC 7069 was fermented in peptone yeast glucose
medium (PYGM; 28 ꢀ 250 mL) at 25 °C for 17 days under static
conditions. The cultures were filtered to separate mycelia (residue)
and broth (filtrate). The EtOAc extract from the broth (1.20 g) was
subjected to fractionation using Sephadex LH-20 (MeOH) and silica
2
i
l
1
3
was soon followed by the isolation of the closely related analogs,
SCH 378161, SCH 378167, and SCH 378199 produced by the same
2
fungal strain, and SCH 218157 co-produced with SCH 217048 by
gel (0–30% MeOH in CH
eluted fractions containing cyclodepsipeptides were further sepa-
rated by preparative HPLC (ODS column, MeCN/H O = 50:50) to
2 2
Cl ) column chromatography, and the
another unidentified fungus.3 The planar structures including the
sequences of the amino acids and 2-hydroxy-3-methylpentanoic
acid (Hmp) were elucidated by NMR and MS analyses, while their
absolute configurations have not been determined. The assignment
of the absolute configurations and the accessibility to these mole-
cules would have been key requirements for further biochemical
studies as leads for selective tachykinin receptor inhibitors.
As part of our research program on bioactive secondary metab-
olites from fungal sources in Thailand, we have been investigating
the constituents of several coprophilous fungi isolated from ele-
phant dung samples, primarily due to our interest in this unique
2
furnish 1 (56 mg), 2 (28 mg), and 3 (16 mg). The MeOH extract
from mycelia (245 mg) was also fractionated using similar proce-
dures to give 1 (12 mg), 2 (10 mg), and 3 (5 mg). The structures
of 1 and 2 were elucidated by the analysis of their NMR spectro-
6
,7
A comparison of the 1H and
13
scopic data obtained in CDCl
3
.
C
NMR spectroscopic data of 1 and 2 in DMSO-d
6
with those reported
in the literature confirmed that these were SCH 217048 and SCH
218157, respectively.
8
Pleosporin A (3) was obtained as a colorless solid. The molec-
4
ecological niche. One of the unidentified coprophilous strains,
ular formula of
3 was established by HRMS (ESI-TOF) as
5
BCC 7069 of the family Pleosporaceae, was chemically explored
C H N O13, which suggested the presence of two N-methylglu-
56 87 11
1
13
in detail as a culture extract exhibited antimalarial activity against
tamine units (N-Me-Gln) as in 2. The H and C NMR spectroscopic
data in CDCl were similar to those of 1 and 2, and were indicative
of a closely related structure. Specifically, the presence of four
broad singlets due to carboxamide protons (d 5.62, 5.59, 5.56,
Plasmodium falciparum K1 with an IC50 value of 29
displayed unique H NMR spectroscopic data suggesting the pres-
l
g/mL, and it
3
1
ence of a mixture of peptides. The study led to the isolation of SCH
H
1
2
17048 (1), SCH 218157 (2), and a new analog named pleosporin A
and 5.49) in the H NMR spectrum was consistent with the pres-
ence of two N-Me-Gln residues. Nine amino acids were assigned
by the analysis of COSY, HMQC, and HMBC data as two N-methyl-
glutamine (N-Me-Gln-1, N-Me-Gln-2), two proline (Pro-1, Pro-2),
phenylalanine (Phe), N-methylvaline (N-Me-Val), valine (Val), and
isoleucine (Ile) (Table 1). The Hmp residue was also assigned on
(3). We report herein the structure elucidation of 3, and the
⇑
Corresponding author. Tel.: +66 25646700/3554; fax: +66 25646707.
E-mail address: isaka@biotec.or.th (M. Isaka).
0
040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.11.063

4
70
M. Isaka et al. / Tetrahedron Letters 55 (2014) 469–471
the basis of the 2D NMR data. The 1H and 13C NMR spectroscopic
H
N
data for Hmp were similar to those of 1 and 2, which suggested
that these compounds share the same absolute configuration.
The sequence of nine amino acids and Hmp was assigned to be
the same as in 1 and 2 by analysis of HMBC and NOESY correlations
N
O
O
O
N
O
N
O
O
O
R
NH
N
NH2
(
Fig. 2). Thus, a HMBC correlation from the NH of Phe to the ester
carbonyl (C-1) of Hmp indicated the Hmp–Phe amide linkage. An
intense NOESY correlation between H-5 of Pro-1 (d 3.78, m) and
O
HN
O
H
O
O
O
H-2 of Phe revealed the amide bonding of Phe–Pro-1. The linkage
of Pro-1–Gly–N-Me-Val–N-Me-Gln-1–Ile was established by fol-
lowing HMBC correlations: from NH of Gly to C-1 of Pro-1, from
N
N
H
O
3
N-CH of N-Me-Val to C-1 of Gly and C-2 of N-Me-Val, from N-
N-Me-L-Glu
L-Pip
CH of N-Me-Gln-1 to C-1 of N-Me-Val and C-2 of N-Me-Gln-1,
3
N-Me-L-Gln-2
and from NH of Ile to C-1 of N-Me-Gln-1. NOESY correlation be-
1
2
R = OH : SCH 217048
R = NH : SCH 218157
2
Pro-1
Gly
L-Pro-1
Gly
N-Me-Val
N-Me-D-Val
H
H
N
N
Phe
N
N
L-Phe
O
O
O
O
N
O
N
O
N
O
O
O
N
O
N
O
O
O
NH
O
N
O
NH
NH2
NH2
N-Me-Gln-1
N-Me-L-Gln-1
HN
O
O
HN
O
Hmp
O
O
(
2R,3S)-Hmp
O
O
O
O
Ile
N
N
L-Ile
N
H
N
H
H N
2
H2N
O
O
Pro-2
L-Pro-2
N-Me-Gln-2
Val
L-Val
HMBC
N-Me-L-Gln-2
NOESY
3
Figure 2. Selected HMBC and NOESY correlations for 3, indicating the sequence of
Figure 1. Structures of the cyclodepsipeptides isolated from BCC 7069.
nine amino acids and Hmp.
Table 1
NMR spectroscopic data for 3 in CDCl
3
(500 MHz for 1H and 125 MHz for 13C)
Position
d
C
, Mult.
d
H
, Mult. (J in Hz)
Position
d
C
, Mult.
d
H
, Mult. (J in Hz)
Position
d
C
, Mult.
H
d , Mult. (J in Hz)
Hmp
Gly
1
2
Pro-2
1
2
3
4
5
Val
1
2
1
2
3
4
5
169.2, qC
76.0, CH
36.3, CH
170.4,a qC
41.1, CH
2.30, d (16.0)
172.9,e qC
59.5, CH
5.20, d (2.1)
2.15, m
1.19, m; 1.18, m
0.84, t (7.5)
0.76, d (6.8)
2
4.54, dd (17.0, 8.4)
3.99, dd (17.0, 2.5)
7.12, dd (8.4, 2.5)
4.67, m
29.8, CH
25.1,d CH
2
2
2.01, m; 1.77, m
2.14, m; 1.88, m
3.70, m; 3.68, m
26.4, CH
11.6, CH
14.8, CH
2
3
3
NH
N-Me-Val
48.0, CH
2
3
-CH
3
1
2
3
4
170.4,a qC
57.9, CH
27.6, CH
e
Phe
1
2
5.18, d (10.7)
2.40, m
0.92, m
0.89, m
3.03, s
172.8, qC
54.9,d CH
31.3, CH
171.5, qC
52.6, CH
4.66, m
2.16, m
0.94, m
0.89, m
4.88, m
3.12, dd (14.0, 9.9)
19.7, CH
18.5, CH
29.1, CH
3
3
3
3
0
3
37.6, CH
2
4
4
4
18.2, CH
19.4, CH
3
0
3
.05, dd (14.0, 4.6)
N-CH
3
3
4
137.0, qC
129.3, CH
128.4, CH
126.8, CH
N-Me-Gln-1
NH
N-Me-Gln-2
1
2
3
4
9.02, d (10.1)
5
6
7
, 9
, 8
7.29, m
7.28, m
7.22, m
7.87, d (8.8)
1
2
3
4
168.9, qC
59.0, CH
25.0,b CH
5.31, m
169.39,c qC
62.4, CH
2
2.35, m; 1.86, m
2.27, m; 2.17, m
5.56, br s; 5.49, br s
2.79, s
3.91, dd (7.4, 6.4)
2.53, m; 2.28, m
2.51, m; 2.33, m
5.62, br s; 5.59, br s
3.29, s
NH
Pro-1
31.7, CH
174.2, qC
2
24.5, CH
31.1, CH
2
2
5 (CONH )
2
1
2
3
4
5
170.9, qC
60.4, CH
N-CH
3
29.4, CH
3
5 (CONH
N-CH
2
)
174.4, qC
38.7, CH
3
4.38, dd (8.0, 3.6)
2.12, m; 2.03, m
2.28, m; 1.99, m
3.78, m; 3.50, m
Ile
1
2
3
c
28.6, CH
25.3, CH
47.3, CH
2
2
2
169.36, qC
55.0,d CH
38.9, CH
4.72, dd (7.9, 3.5)
1.79, m
3
4
5
3
23.9, CH
11.7, CH
15.8, CH
2
3
3
1.42, m; 0.99, m
0.86, m
0.89, m
-CH
3
NH
6.42, d (7.9)
a
The carbon resonances were superimposed.
Carbon chemical shifts may be interchanged.
b–e

M. Isaka et al. / Tetrahedron Letters 55 (2014) 469–471
471
tween H-5 of Pro-2 and H-2 of Ile suggested the Ile–Pro-2 connec-
tion. HMBC correlations from NH of Val to C-1 of Pro-2, and from
N-CH of N-Me-Gln-2 to C-1 of Val and C-2 of N-Me-Gln indicated
the connections of Pro-2–Val–N-Me-Gln. Finally, the ester linkage
of N-Me-Gln–Hmp to form a cyclodecadepsipetide was revealed
by the HMBC correlation from H-2 of Hmp to C-1 of N-Me-Gln-2.
The structure of 3 is most closely related to SCH 378161, which
possesses Pro instead of pipecolic acid (Pip) in 1.
Supplementary data
3
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.
2013.11.063.
References and notes
1
2
3
4
.
.
.
.
Hegde, V. R.; Puar, M. S.; Chan, T. M.; Dai, P.; Das, P. R.; Patel, M. J. Org. Chem.
998, 63, 9584–9586.
Hegde, V. R.; Puar, M. S.; Dai, P.; Pu, H.; Patel, M.; Anthes, J. C.; Richard, C.;
Terracciano, J.; Das, P. R.; Gullo, V. J. Antibiot. 2001, 54, 125–135.
The absolute configurations of Hmp and the amino acid resi-
dues of 1–3 were determined by HPLC analysis of the acid hydro-
lyzate using a ligand-exchange-type chiral column. Thus, SCH
1
Chu, M.; Chan, T. M.; Das, P.; Mierzwa, R.; Patel, M.; Puar, M. S. J. Antibiot. 2000,
2
17048 (1) was hydrolyzed in 6 M HCl at 110 °C for 15 h. After
5
3, 736–738.
cooling, the aqueous solution was extracted with Et O. The Et O
2
2
Isaka, M.; Palasarn, S.; Chinthanom, P.; Thongtan, J.; Sappan, M.; Somrithipol, S.
solution and the aqueous layer were concentrated in vacuo and
separately subjected to HPLC analysis. For the determination of
the absolute configuration of the Hmp residue, standard samples
Tetrahedron Lett. 2012, 53, 4848–4851.
5. The fungus used in this study was isolated from an elephant dung sample
collected from the Sakaerat Environmental Research Station, Nakhon
Ratchasima Province, Thailand, by Dr. Nigel L. Hywel-Jones. The living
culture was deposited in the BIOTEC Culture Collection as BCC 7069 on
September 21, 1999. Results from the LSU and ITS gene sequences indicated
that this fungus belongs to the family Pleosporaceae of the order Pleosporales,
but it is not assignable to any genera. These sequencing data were deposited in
GenBank (accession No. KF486914 for LSU gene, and KF482067 for ITS gene).
of four Hmp isomers were prepared from
L
- and
, 400 MHz) spectroscopic data of
O extract were identical with those of (2S,3R)-Hmp/
D-Ile, and L- and
9
1
D
-allo-Ile. The H NMR (CDCl
3
the Et
2
(
(
2R,3S)-Hmp, and they were different from those of (2S,3S)-Hmp/
2R,3R)-Hmp. HPLC analysis using the chiral column established
2
6
6. SCH 217048 (1): Colorless solid; mp 199–201 °C; ½
a
ꢁ
ꢂ112 (c 0.20, MeOH); UV
D
10
(MeOH) k (log ) 214 (4.20) nm; IR (ATR) 3407, 3328, 1750, 1676, 1631,
e
m
max
max
ꢂ1
that the hydrolyzate contained (2R,3S)-Hmp. The analysis of
the aqueous layer of the hydrolyzate, employing standard - and
-amino acids, revealed the presence of Gly, -Pip, -Pro, N-Me-
Val, -Val, (2S,3S)- -Ile, and
-Phe.11 By acid hydrolysis, the N-Me-
Gln residue should be converted into N-Me-Glu. Due to the signif-
icant retention time flipping of standard N-Me- -Glu and N-Me-
+
1
1
447 cm ; HRESI MS m/z 1159.6386 [M+Na] (calcd for C57
H
88
N
10
O
14Na,
L
159.6374); 1H NMR (500 MHz, DMSO-d
13
6
) and C NMR (125 MHz, DMSO-d )
6
D
L
L
D
-
spectroscopic data were consistent with those reported in the literature.
26
D
7.
SCH 218157 (2): Colorless solid; mp 187–189 °C; ½
a
ꢁ
ꢂ116 (c 0.21, MeOH); UV
L
L
L
(
1
MeOH) kmax (log
ꢂ1
e) 214 (4.20) nm; IR (ATR) mmax 3421, 1749, 1670 sh, 1631,
+
449 cm ; HRESI MS m/z 1136.6719 [M+H] (calcd for C H N O ,
57 90 11 13
1
1
3
L
D
-
6
1136.6714); H NMR (500 MHz, DMSO-d ) and C NMR (125 MHz, DMSO-
d ) spectroscopic data were consistent with those reported in the literature.
6
Glu, they could not be assigned during the analysis. The absolute
configurations of N-Me-Glu and N-Me-Gln in 1 were determined
by application of Marfey’s method.12 The acid hydrolyzate of 1
26
D
8
9
.
.
Pleosporin A (3): Colorless solid; mp 124–126 °C; ½
a
m
+
ꢁ
ꢂ125 (c 0.20, MeOH); UV
(
MeOH) kmax (log ) 214 (4.23) nm; IR (ATR) max 3423, 1750, 1671, 1631,
e
1447 cmꢂ1
HRESI MS m/z 1122.6555 [M+H] (calcd for C56 88N11O13
1122.6558); see Table
125 MHz, CDCl ) data.
Cohen, S. G.; Weinstein, S. Y. J. Am. Chem. Soc. 1964, 86, 5326–5330.
;
H
,
1
13
was derivatized with N
a
-(2,4-dinitro-5-fluorophenyl)-
L-alanina-
1
for
H
NMR (500 MHz, CDCl
3
)
and
C NMR
(
3
mide (FDAA). HPLC analysis using a reverse-phase column revealed
1
3
the presence of N-Me-L-Glu and the absence of N-Me-D-Glu.
1
0. Chiral column: Phenomenex Chirex 3126 (D)-penicillamine, 4.6 ꢀ 250 mm;
flow rate 0.5 mL/min, detection UV 235 nm. The Et O extract from the acid
hydrolysis solution was analyzed using 15% 2-propanol in 2 mM aqueous
CuSO as the mobile phase. The retention times of (2S,3R)-Hmp and (2R,3S)-
The HPLC chromatogram (chiral column) of the acid hydroly-
zate (aqueous layer) of SCH 218157 (2) was indistinguishable from
that of 1, which indicated that both hydrolyzates possessed the
same amino acids. Chiral column HPLC analysis of the hydrolyzate
2
4
Hmp were 107 and 176 min, respectively. The Et
Hmp.
2
O extract contained (2R,3S)-
1
1. Three mobile phase conditions were employed due to the large retention time
differences of the standard amino acids: (1) 5% MeCN in 2 mM aqueous CuSO
Gly (t 12.6 min), -Pip (t 16.7 min), -Pip (t 25.4 min), -Pro (t 18.9 min),
Pro (t 45 min), N-Me- -Val (t 19.6 min), N-Me- -Val (t 34 min), and
3 min); (2) 5% 2-propanol in 2 mM aqueous CuSO -Val (t 25 min),
0 min), -allo-Ile (t 44 min), -Ile (t 52 min), -allo-Ile (t
88 min); (3) 15% 2-propanol in 2 mM aqueous CuSO -Ile (t
(tR 61 min), -Phe (tR 94 min).
2. Marfey, P. Carlsberg Res. Commun. 1984, 49, 591–596.
m), mobile phase MeCN/(0.05% TFA
O) = 45:55, flow rate 0.5 mL/min, UV detection at 340 nm. Retention times
of the FDAA-derivatized standard amino acids, N-Me- -Glu and N-Me- -Glu,
were 8.1 and 7.4 min, respectively. The derivatized hydrolyzate contained N-
Me- -Glu.
of 3 indicated the absence of
L-Pip, and relatively higher peak
4
,
intensity for -Pro than other amino acids when compared with
L
R
L
R
D
R
L
R
D-
the chromatograms for 1 and 2.
R
L
R
D
R
L-Val (t
R
8
4
4
,
L
R
D
-Val (t
R
Compounds 1–3 exhibited antimalarial activity against Plasmo-
L
R
L
R
D
R
71 min), and
D-Ile
dium falciparum K1 with IC50 values of 1.6, 6.4, and 1.6 lg/mL,
(t
R
4
,
L
R
29 min), L-Phe
1
4
respectively. These compounds were also subjected to other bio-
D
1
logical assays: antitubercular activity (Mycobacterium tuberculosis
1
5
15
13. Column: NovaPak C18 (3.9 ꢀ 150 mm, 4
l
H37Ra), antifungal activity (Candida albicans),
antibacterial
in H
2
15
activity (Bacillus cereus), and cytotoxicity to cancer cell-lines
L
D
15
(
KB, MCF-7, and NCI-H187)16 and nonmalignant Vero cells. They
L
were inactive in these assays at 50 g/mL.
l
1
4. Desjardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chulay, J. D. Antimicrob. Agents
In conclusion, a new antimalarial cyclodepsipeptide, pleosporin
A (3), was isolated from an elephant dung fungus and its structure
was elucidated. The absolute configurations of 3 and the known
analogs 1 and 2 were determined by chiral column HPLC analysis
and Marfey’s method. Although the antimalarial activities of 1–3
were much weaker than the standard drug, dihydroartemisinin
Chemother. 1979, 16, 710–718.
15. Changsen, C.; Franzblau, S. G.; Palittapongarnpim, P. Antimicrob. Agents
Chemother. 2003, 47, 3682–3687.
1
6. O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. Eur. J. Biochem. 2000, 267, 5421–
5426.
(
IC50 0.0044 lg/mL in our assay system), their good selectivity in-
dex suggested that they could serve as new antimalarial leads.
Acknowledgment
Financial support from the National Center for Genetic Engi-
neering and Biotechnology (BIOTEC) is gratefully acknowledged.