Cytotoxic piperazine-2,5-dione derivatives from marine fungus Gliocladium sp.

Article abstract of DOI:10.1691/ph.2009.9598

Two new piperazine-2,5-dione derivatives, gliocladride A (1) and B (2), along with deoxymycelianamide (3) were isolated from the mycelial mass of marine fungus Gliocladium sp. Their structures were established on the basis of spectral data, and the stereo

Full text of DOI:10.1691/ph.2009.9598

ORIGINAL ARTICLES
School of Basic Medical Science1, Ningxia Medical University, Yinchuan; School of Traditional Chinese Materia Medica2,
Shenyang Pharmaceutical University, Shenyang; College of Chemical Engineering3, Qingdao University of Science & Tech-
nology, Qingdao; The First Institute of Oceanography4, State Oceanic Administration of China, Qingdao; Research Center of
Medical Science and Technology5, Ningxia Medical University, Yinchman, P.R. China
Cytotoxic piperazine-2,5-dione derivatives
from marine fungus Gliocladium sp.
Yao Yao1, 5, Li Tian3, 4, Juan Li1, Jiaqing Cao2, Yuehu Pei2
Received April 8, 2009, accepted May 8, 2009
Yuehu Pei, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University,
103 Wenhua Road, Shenyang, 110016, P.R. China
peiyueh@vip.163.com
Pharmazie 64: 616–618 (2009)
doi: 10.1691/ph.2009.9598
Two new piperazine-2,5-dione derivatives, gliocladride A (1) and B (2), along with deoxymyceliana-
mide (3) were isolated from the mycelial mass of marine fungus Gliocladium sp. Their structures were
established on the basis of spectral data, and the stereochemical assignments were made by chiral
HPLC analysis of the hydrolyzed compounds and optical rotation comparison with known compound.
Their cytotoxic activity was tested on three cancer cell lines, HL-60, U937 and T47D by MTT assay.
As a result, gliocladride A (1) and B (2) showed moderate cytotoxic activity against the three cell lines
with IC50 values from 11.60 mg/ml to 52.83 mg/ml, while deoxymycelianamide (3) showed strong cyto-
toxic activity against U937 cell line with IC50 values of 0.785 mg/ml.
1. Introduction
phy on the acetone extract afforded three piperazine-2,5-
dione derivatives (1–3). We report here the structure eluci-
dation of the two new compounds (1–2) and the cytotoxic
activities of all compounds on three cancer cell lines, HL-
60, U937 and T47D.
Marine microorganisms are a promising source for novel
antitumor agents (Fenical 1993). Marine derived fungi
also represent potential for the discovery of new cytotoxic
metabolites (Bugni et al. 2004). To discover new cytotoxic
compounds, we have investigated several marine microor-
ganisms (Zhang et al. 2004; Huang et al. 2006a, 2006b;
Sun et al. 2006a, 2006b). In our continuous study, the fun-
gus Gliocladium sp. was isolated from sea mud collected
in Rushan, Shandong province, China, and then be culti-
vated and extracted. The primary bioassay of the acetone
extract from the mycelial mass showed strong inhibition
against Alternaria solani. Repeated column chromatogra-
2. Investigations, results and discussion
Gliocladride A (1) was isolated as a white solid, and it’s
molecular formula was determined to be C22H28N2O4 from
its HR-FAB MS at m/z 385.2125 ([M þ H]þ, calcd. for
385.2127), indicating 10 degrees of unsaturation. The UV
bands at 225, 315 nm and IR absorptions at 3310, 2921,
1688, 1606, 1521, 1442 and 1251 cmꢀ1 suggested the pre-
sence of hydroxyl, amide and aromatic groups. General
analyses of 1H NMR (600 MHz, DMSO-d6), 13C NMR
(150 MHz, DMSO-d6) and HMQC signals gave some im-
portant information about the structure. Four aromatic pro-
ton signals d7.45 (2 H, d, J ¼ 8.7 Hz, H-10,100), 6.96
(2 H, d, J ¼ 8.7Hz, H-11,110) were typical for a 1,4-disub-
stituted aromatic ring; a vinyl singlet at d6.74 (1 H, s, H-
8) and it’s HMBC correlation with d131.0 (C-10) indi-
cated the presence of a benzylidene unit. Three CH3
groups at d1.71 (3 H, s, H-22), 1.64 (3 H, s, H-20), 1.57
(3 H, s, H-21), three CH2 groups at d4.57 (2 H, d,
J ¼ 6.6 Hz, H-13), 2.09 (2 H, m, H-17), 2.04 (2 H, m, H-
16) and two vinyl groups at d5.43 (1 H, t, J ¼ 6.6 Hz, H-
14), 5.08 (1 H, t, J ¼ 6.6 Hz, H-18) were attributable to a
geranyloxy side chain, of which the three methyl reso-
nance assignments were made by HMBC and NOESY
correlations. The methyl signal at d1.71 (3 H, s, H-22)
was positioned at C-15 on the basis of HMBC correlation
between H-22 and vinyl carbons at d140.5 (C-15) and
O
8
10'
11
4
N
6
HO
9
22
15
21
19
11'
12
5
2
13
17
10
H
N
7
3
1
O
20
16
18
14
O
1
H
O
N
N
O
O
OH
2
O
HN
NH
O
O
3
616
Pharmazie 64 (2009) 9

ORIGINAL ARTICLES
d119.6 (C-14). The signal at d1.64 (3 H, s) was assign-
ment to be H-20 as the NOE correlation between H-20
and vinyl proton signal d5.08 (1 H, t, J ¼ 6.6 Hz, H-18),
while NOE correlation between d1.57 (3 H, s, H-21) and
d5.08 (1 H, t, J ¼ 6.6 Hz, H-18) was not observed. The
H
N
H
O
O
N
1
remaining H NMR (600 MHz, DMSO-d6) resonances at
OH
d9.96 (1 H, s, NH-1), d4.31 (1 H, q, J ¼ 6.8 Hz, H-3),
d10.22 (1 H, s, NH––OH), d1.45 (1 H, d, J ¼ 6.8 Hz, H-7)
and 13C NMR (150 MHz, DMSO-d6) resonances at d166.3
(C-2), d60.0 (C-3), d157.3 (C-5), d123.4 (C-6), d17.1 (C-
7) were attributable to a 3-methyl-2,5-diketonpiperazine
unit, which was supported by the HMBC correlations be-
tween D2O exchangeable proton at d9.96 (1 H, s, NH-1)
and d166.3 (C-2), d123.4 (C-6), and between d4.31 (1 H,
q, J ¼ 6.8 Hz, H-3) and d166.3 (C-2), d157.3 (C-5),
d17.1 (C-7). The other D2O exchangeable proton at
d10.22 (1 H, s, N––OH) had weak correlations with d60.0
(C-3) and d157.3 (C-5). Thus, the remaining one hydroxyl
group accounted from the molecular composition was po-
sitioned on N-4. Then we got three units: a benzylidene
unit, a geranyloxy side chain and a 3-methyl-4-N-hydro-
xyl-2,5-diketonpiperazine unit. The connection of these
three units was based on HMBC correlation. The HMBC
correlation between d6.74 (1 H, s, H-8) and d123.4 (C-6),
125.7 (C-9) indicated that the benzylidene unit was con-
nected with the piperazine-2,5-dione ring by C-6. The cor-
relation from d4.57 (2 H, d, J ¼ 6.6 Hz, H-13) to d158.4
(C-12) showed that the geranyloxy side chain was con-
nected with the benzylidene unit by C-12.
The configuration of double bonds was determined by
NOESY experiment. NOE between aromatic proton at
(7.45 (2 H, d, J ¼ 8.7 Hz, H-10, 100) and proton at (9.96
(1 H, s, NH-1) confirmed the Z-configuration of D6, 8 dou-
ble bond in the benzylidene unit; NOE between methyl
protons at (1.71 (3 H, s, H-22) and methene protons at
(4.57 (2 H, d, J ¼ 6.6 Hz, H-13) confirmed the E-config-
uration of D14, 15 double bond in the geranyl group. To
determine the absolute configuration of C-3, acid hydroly-
sis and chiral HPLC analysis of 1 were done. The Ala in
1 was defined as L-Ala, thus C-3 was S-configuration as
shown in Fig. 1, which was consistent with the optical
rotation comparison. It’s optical rotation ([a]D-150)
showed the same sign as that of 3S-mycelianamide ([a]D-
217) (Brich et al. 1956).
NOESY
Fig. 2: Selective NOESY correlation of gliocladride B (2)
Table 1: 1H and 13C NMR data of compounds 1 and 2 in
a
DMSO-d6
Position
1
2
dC
dH (JHz
)
dC
dH (JHz)
1
2
3
4
5
6
7
8
9
9.96 (s)
9.62 (s)
166.3
60.0
162.0
59.3
4.31(q, 6.8)
10.22 (s)
4.40 (q, 6.8)
10.49 (s)
157.3
123.4
17.1
115.2
125.7
131.0
114.9
158.4
64.5
157.8
126.0
16.8
117.9
125.1
132.5
113.7
158.7
64.5
1.45 (d, 6.8)
6.74 (s)
1.45 (d, 6.8)
6.56 (s)
10, 100
11, 110
12
13
14
15
16
17
18
19
20
21
22
7.45 (d, 8.7)
6.96 (d, 8.7)
7.36 (d, 8.7)
6.84 (d, 8.7)
4.57 (d, 6.6)
5.43 (t, 6.6)
4.56 (d, 6.6)
5.42 (t, 6.6)
119.6
140.5
38.9
119.7
140.5
38.9
2.04 (m)
2.09 (m)
5.08 (t)
2.04 (m)
2.08 (m)
5.08 (t)
25.9
25.9
123.9
131.2
25.6
17.7
16.5
123.9
131.1
25.6
1.64 (s)
1.57 (s)
1.71 (s)
1.63 (s)
1.57 (s)
1.71 (s)
17.7
16.5
a All assignments are based on HSQC and HMBC experiments
configuration. The configuration of C-3 was assumed to be
S due to its optical rotation ([a]D-126) which had the same
direction as 1, thus gliocladride B was decided to be a cis-
trans-isomer of gliocladride A as shown in Fig. 2.
Gliocladride B (2) was also a white solid, which had the
same molecular formula as gliocladride A (1) from its HR-
FAB MS at m/z 385.2122 ([M þ H]þ, calcd. for 385.2127).
The UV bands at 224, 315 nm, IR absorptions at 3310,
2920, 1687, 1605, 1521, 1442 and 1250 cmꢀ1 and its NMR
spectrums were quite similar with those of 1 (Table 1).
Main difference was in the NOESY spectrum: NOE correla-
tion between (d6.56 (1 H, s, H-8) and (d9.62 (1 H, s, NH-1)
was observed instead of the correlations between (d7.36
(2 H, d, J ¼ 8.7 Hz, H-10, 100) and (9.62 (1 H, s, NH-1).
Thus the D6, 8 double bond in 2 was determined to be E-
Compound 3 was found to be identical to deoxymycelia-
namide by spectroscopic data analysis and comparison of
its chemical and physical properties with those reported in
the literature (Gallina et al. 1966, 1968). This is the first
time to report its cytotoxic activity.
The cytotoxicity of compounds 1–3 was evaluated against
three cancer cell lines, HL-60, U937 and T47D, in an
MTT assay (Mosmann 1983). Vincristin was tested as a
reference. Structure-function relationships of these com-
pounds from the activity test results (Table 2) need to be
discussed. The exist of N––OH may decrease the cytotoxic
activity, while the configuration of D6, 8 double bond
seems to have no significient effect.
O
Table 2: The results of cytotoxic activity testa
HO
N
HL-60
U937
T-47D
NH
O
1
2
3
17.87
19.86
2.02
12.80
11.60
0.79
42.80
52.83
30.51
12.57
O
HMBC
NOESY
Vincristin
2.46
1.67
a
Fig. 1: Selective HMBC and NOESY correlations of gliocladride A (1)
IC50 values, mM, cells were treated for 72 h
Pharmazie 64 (2009) 9
617

ORIGINAL ARTICLES
1442, 1250 cmꢀ1 1H NMR and 13C NMR data, see Table 1; EI-MS m/z:
;
3. Experimental
384 [M]þ, 368, 248, 217, 133, 81, 69 (base); HR-FAB MS m/z 385.2122
([M þ H]þ, calcd. for 385.2127).
3.1. General Procedures
Optical rotations were measured on a Perkin-Elmer 241MC polarimeter.
UV spectra were performed with a Shimadzu UV260 spectrometer. IR
spectra were recorded on a Thermo Nicolet Nexus 470 FT-IR spectrometer.
NMR spectra were recorded on a Bruker-ARX-600 spectrometer (1H at
600 MHz and 13C at 150 MHz). HR-FAB MS spectra were taken with a
Q-trap LC-MS-MS system using turbo ionspray source. Colume chromato-
graphy was carried with silica gel (200–300 mesh) obtained from Qingdao
Marine Chemistry Co. Ltd., Qingdao, P.R. China.
3.7. Acid hydrolysis and chiral amino acid analysis
Compound 1 (2.0 mg) was hydrolyzed by heating the sample in a seal vial
at 120 ꢁC for 22 h in 6 N HCl, and then dried under vacuum. The hydro-
lysate was eluted from a C18 column (Dikma) using MeOH/H2O (10 : 90).
The elute was dried under vacuum and reconstituented with 100 mL of
H2O prior to analysis [CHIRAL PAK CR(þ), 4.6ꢃ150 mm; detection: UV
200 nm; injected amount: 5 nmol; mobile phase: pH 1.5 HClO4 in H2O,
flow rate 0.4 ml/min]. The hydrolysate was chromatographed alone and co-
injected with standards to confirm assignment. Retention time of the in 1
was 4.66 min, which was identical to the authentic l-Ala. The standard
retention time of the d-Ala was 3.54 min.
3.2. Fungus material
The fungus strain was isolated from sea mud collected in Rushan, Shan-
dong province, China, in May of 2004, and identified as Gliocladium sp.
by Prof. Li Tian. A voucher specimen (No.CAAN045011) is deposited in
the key laboratory of Marine Biology of State Oceanography Administra-
tion, China.
3.8. Cytotoxic activity test
The three cancer cell lines, HL-60, U937 and T47D, were obtained from
the American Type Culture Collection (ATCC), and cultured in RPMI-
1640 medium supplemented with 10% fetal bovine serum (FBS), in a hu-
midified atmosphere of 5% CO2 in air, at 37 ꢁC. Cell inhibition were meas-
ured by MTT assay (Mosmann 1983), and all experiments were performed
five times.
3.3. Cultivation and extraction
The strain was cultured on seed medium at 24ꢁ on a rotary shaker for
9 days. The culture medium contained potato decoction 200 ml, sea mud
extract 20 ml, peptone 2 g, dextrose 15 g, NaCl 12 g, MgCl2 ꢂ 6 H2O 1.1 g,
KCl 0.1 g, and distilled water 1000 ml. On the tenth day, the fermentation
broth, including cells, was harvested and then centrifugated to separate
mycelial mass from aqueous layer. The mycelial mass was exhaustively
extracted with acetone (six times) to get a crude extract (26 g).
Acknowledgements: This work was supported by 863 Hi-Tech Research
and Development Program of China (Grant No. 2001AA624020) and Spe-
cial Talent Research Project of Ningxia Medical University (Grant
No. XT200704).
3.4. Isolation and characterization
The extract was subjected to gradient elution in petroleum ether/acetone
(100 : 1 to 1 : 1) on a silica gel column to give a series of fractions. The
seventh fraction (5 : 1) was chromatographed over Sephadex LH-20 column
(Pharmadex, CHCl3/MeOH 1 : 1) and further purified on reversed-phase
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C18, MeOH/H2O 7 : 3), to give compound 3
(42.0 mg). The eighth fraction (10 : 3) was chromatographed over Sepha-
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afford compounds 1 (30.2 mg) and 2 (12.6 mg).
3.5. Gliocladride A (1)
White solid; [a]D20-150ꢁ(c 0.05, CH3OH); UV lmax (CH3OH, log e) nm
225 (3.94), 315 (4.02); IR (KBr) nmax cmꢀ1 3310, 2921, 1688, 1606,
1521, 1442, 1251 cmꢀ1 1H NMR and 13C NMR data, see Table 1; EI-MS
;
m/z: 384 [M]þ, 368, 248, 232, 217, 133, 81, 69 (base); HR-FAB MS m/z
385.2125 ([M þ H]þ, calcd. for 385.2127).
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3.6. Gliocladride B (2)
White solid; [a]D20-66ꢁ(c 0.05, CH3OH); UV lmax (CH3OH, log e) nm 224
(3.92), 315 (4.06); IR (KBr) nmax cmꢀ1 3310, 2920, 1687, 1605, 1521,
618
Pharmazie 64 (2009) 9