New diketopiperazine derivatives from a deep-sea-derived Nocardiopsis alba SCSIO 03039

Article abstract of DOI:10.1038/ja.2012.88

The strain SCSIO 03039 was isolated from a sediment sample in the Indian Ocean and was characterized as a Nocardiopsis alba species on the basis of its 16S rRNA gene sequence. Seven diketopiperazines (DKPs), including two new DKPs nocazines D (2a) and E (2b), and five known DKPs (1, 3-6), were isolated from N. alba SCSIO 03039, along with two known compounds 2-methoxy-1,4-naphthoquinone (7) and 1-hydroxy-4-methoxy-2-naphthoic acid (8). Their structures were elucidated by mass and NMR spectroscopic analyses. The structure of methoxyneihumicin (1), previously proposed in a conference poster lacking publicly available experimental data, was validated for the first time by detailed NMR analyses and X-ray diffraction study. The two enantiomers nocazines D (2a) and E (2b) were isolated as a mixture. Compounds 3 and 4 were only known as synthetic compounds before. Methoxyneihumicin (1) exhibited in vitro cytotoxicities against MCF-7 and SF-268 with IC 50 values of 4.6 and 12.7 μM, respectively, better than those of 6 (22.0 and 20.6 μM). The other compounds showed less pronounced cytotoxities against three tested human cancer cell lines and no compounds displayed antibacterial activities toward four indicator strains.

Full text of DOI:10.1038/ja.2012.88

The Journal of Antibiotics (2013) 66, 31–36
2013 Japan Antibiotics Research Association All rights reserved 0021-8820/13
www.nature.com/ja
&
ORIGINAL ARTICLE
New diketopiperazine derivatives from a
deep-sea-derived Nocardiopsis alba SCSIO 03039
1
,2
1
3
1
1
1
1
Qingbo Zhang , Sumei Li , Yuchan Chen , Xinpeng Tian , Haibo Zhang , Guangtao Zhang , Yiguang Zhu ,
1
3
1
Si Zhang , Weimin Zhang and Changsheng Zhang
The strain SCSIO 03039 was isolated from a sediment sample in the Indian Ocean and was characterized as a Nocardiopsis
alba species on the basis of its 16S rRNA gene sequence. Seven diketopiperazines (DKPs), including two new DKPs nocazines
D (2a) and E (2b), and five known DKPs (1, 3–6), were isolated from N. alba SCSIO 03039, along with two known compounds
2
-methoxy-1,4-naphthoquinone (7) and 1-hydroxy-4-methoxy-2-naphthoic acid (8). Their structures were elucidated by mass
and NMR spectroscopic analyses. The structure of methoxyneihumicin (1), previously proposed in a conference poster lacking
publicly available experimental data, was validated for the first time by detailed NMR analyses and X-ray diffraction study. The
two enantiomers nocazines D (2a) and E (2b) were isolated as a mixture. Compounds 3 and 4 were only known as synthetic
compounds before. Methoxyneihumicin (1) exhibited in vitro cytotoxicities against MCF-7 and SF-268 with IC50 values of 4.6
and 12.7 mM, respectively, better than those of 6 (22.0 and 20.6 mM). The other compounds showed less pronounced
cytotoxities against three tested human cancer cell lines and no compounds displayed antibacterial activities toward four
indicator strains.
The Journal of Antibiotics (2013) 66, 31–36; doi:10.1038/ja.2012.88; published online 24 October 2012
Keywords: cytotoxicity; deep-sea; diketopiperazines; marine actinomycetes; Nocardiopsis
INTRODUCTION
were only known as synthetic compounds before.16 The two known
Marine actinomycetes have been proven as a prolific resource for drug DKPs, (3Z,6Z)-3-(4-methoxybenzylidene)-6-(2-methylpropylidene)-
discovery.1–3 However, reports of natural products derived from deep- piperazine-2,5-dione (5)17,18 and XR334 (6),19 were reisolated from
sea (41000m) culturable actinomycetes are still scarce, probably N. alba SCSIO 03039. In addition, another two known compounds,
because of the difficulty in accessing the deep-sea samples and the 2-methoxy-1,4-naphthoquinone (7)20,21 and 1-hydroxy-4-methoxy-2-
unculturability of deep-sea-derived actinomycetes.4 Our recent naphthoic acid (8),22 were also isolated. We reported herein the
investigations on deep-sea-derived actinomycetes have led to the isolation, structure elucidation and biological activities of these
discovery of culturable new actinobacterial taxa,5–9 and the isolation compounds.
1
0
of novel natural products, including spirotetronates, b-carboline
1
1
spiro-bisindole alkaloids,12 diaza-
and indolactam alkaloids,
1
3
14
anthraquinone derivatives and angucycline glycosides, which RESULTS AND DISCUSSION
exhibit antibacterial, anti-malarial and antitumor properties. From Taxonomy of the producing strain
our numerous actinomycetes derived from deep-sea sediments, the The almost full 16S rRNA gene sequence (1447 bp) of the producing
isolate SCSIO 03039, identified as a Nocardiopsis alba, was found to strain SCSIO 03039 was submitted to GenBank under the accession
produce seven diketopiperazines (DKPs), including two new DKPs number JX195181, which showed the highest similarity (99.3%) to
nocazines D (2a) and E (2b), and five known DKPs (1, 3–6). that of N. alba DSM 43377T (X97883). The phylogenetic tree
Compound 1, methoxyneihumicin, previously reported in
a
generated by a neighbor-joining method clearly revealed the evolu-
1
5
conference poster without available experimental data, was struc- tionary relationship of the strain SCSIO 03039 with a group of
turally validated herein for the first time by NMR analyses and the X-ray Nocardiopsis species (Supplementary Figure S1). On the basis of the
diffraction study. Compound 3 (S,Z)-3-benzylidene-6-methylpiperazine- 16S rRNA gene sequence analysis,23 the strain SCSIO 03039 was
2,5-dione and 4 (S,Z)-3-benzylidene -6-isopropylpiperazine-2,5-dione designated as a species of N. alba.
1CAS Key Laboratory of Marine Bio-resources Sustainable Utilization, RNAM Center for Marine Microbiology, Guangdong Key Laboratory of Marine Materia Medica, South China
Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China; 2Graduate University of the Chinese Academy of Sciences, Beijing, China and 3Guangdong
Institute of Microbiology, Guangzhou, China
Correspondence: Professor C Zhang, CAS Key Laboratory of Marine Bio-resources Sustainable Utilization, RNAM Center for Marine Microbiology, Guangdong Key Laboratory of
Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road, Guangzhou 510301, China.
E-mail: czhang2006@gmail.com
Received 2 July 2012; revised 17 September 2012; accepted 20 September 2012; published online 24 October 2012

New diketopiperazine derivatives
Q Zhang et al
3
2
Isolation of compounds 1-8
The comparison of 1D NMR data of 2 with those of nocazine C24
The fermentation broths of 20l culture of N. alba SCSIO 03039 were showed that 2 only differed from nocazine C by lacking signals for
extracted with butanone and the mycelia cakes were extracted with N-4 methyl group (N-1 methyl group in nocazine C).
methanol, respectively. The combined crude extracts were subjected to Correspondingly, C-2, C-3, C-5 and C-14 (C-5, C-6, C-2 and C-7,
2
4
various chromatographic methods (such as silica gel column, respectively, in nocarzine C ) shifted upfield for 4.5, 4.7, 2.5 and
Sephadex LH-20 chromatography and semipreparative HPLC) and 5.0 p.p.m., respectively. The planar structure of 2 (Figure 1) was
led to the isolation of two new DKPs nocazines D (2a) and E (2b), further confirmed by HMBC correlations from H-6 to C-2/C-5, from
five known DKPs (1, 3–6), along with two known compounds 2- H-14 to C-2/C-5 and from H-7 to C-5/C-6 (Figure 2). The D3
methoxy-1,4-naphthoquinone (7) and 1-hydroxy-4-methoxy-2- double bond in 2 was deduced to be Z configuration from the relative
,14
naphthoic acid (8).
downfield shift of H-14 (dH 6.81), in comparison with the vinyl
2
4
proton chemical shifts of H-7 (d 7.03) in nocazine C, and H-7 (dH
H
6
.54) in (3S,6E)-3-benzyl-6-benzylidenepiperazine-2,5-dione,25 based
on previous reports that the (Z)-vinyl proton was farther downfield
Structure elucidation
Compounds 5–8 (Figure 1) were found to be known compounds
than the (E)-vinyl proton because of the deshielding effect of the
24–27
(3Z,6Z)-3-(4-methoxybenzylidene)-6-(2-methylpropylidene)-pipera-
zine-2,5-dione (5),
5
-ketone in DKP compounds.
3,14
Consistent with the Z configuration
1
7,18
XR334 (6),19 2-methoxy-1,4-naphthoquinone
of D , the NOESY correlation was observed between 4-NH (d 9.65
H
(
7)20,21 and 1-hydroxy-4-methoxy-2-naphthoic acid (8),22 respectively,
in DMSO-d ) and H-20 (d 7.18 in DMSO-d ), and no NOESY
6
H
6
by comparing their NMR data with those previously reported
Supplementary Figures S6–S9).
The molecular formula of compound 1 was determined to be
correlation between 4-NH (dH 9.65 in DMSO-d ) and H-14 (dH 6.30
6
(
in DMSO-d ) was observed (Figure 2, Supplementary Figure S3).
6
Marfey’s method was applied to determine the absolute configuration
of the phenylalanine moiety in 2.28 Unexpectedly, both L- and
D-phenylalanine derivatives were found in HPLC analysis of the
1-fluoro-2,4-dinitrophenyl-5-L-alnaine amide (FDAA) derivatives
of the acid hydrolysates of 2, when compared with those
C H N O on the basis of high-resolution fast atom bombardment
2
0 18 2 3
þ
MS (HRFABMS) (m/z 335.1393 [Mþ H] , calcd. for 335.1396). The
1
13
24
H and C NMR data of 1 were very similar to those of nocazine A,
metabolite isolated from the marine-derived Nocardiopsis
a
dassonvillei HR10-5. The comparison of 1H and 13C NMR data
between 1 and nocazine A revealed the loss of signals for one N-Me
prepared from authentic samples of both L- and D-phenylalanine
(FDAA tR ¼ 11.9min; L-phenylalanine derivative tR ¼ 14.5min;
1
and one methoxy group in 1. The sets of coupled H NMR signals at
dH 7.43 (2H, ddd, J ¼ 8.0, 7.5, 1.8 H ), 7.37 (2H, brd, J ¼ 7.5 H ) and
Z
Z
7
1
1
.32 (1H, tt, J ¼ 7.8, 1.5 H ) revealed the presence of one
Z
-substituted benzene in 1. HMBC correlations from H-7 to C-9/C-
3 located the 1-substituted benzene at C-7 (Figure 2). Thus, these
comparisons indicated that 1 differed from nocazine A by losing N-1
methyl group and C-11 methoxy group (Figure 1). The X-ray crystal
diffraction analysis of 1 also supports this conclusion (Figure 3), and
further determined the Z configuration of both D6,7 and D3,14 double
bonds. Thus, compound 1 was identified as (3Z, 6Z)-6-benzylidene-
5-methoxy-3-(4-methoxybenzylidene)-1,6-dihydropyrazin-2(3H)-one
(methoxyneihumicin, Figure 1). It should be noted that the structure
of 1 was previously proposed in a conference poster without publicly
available experimental data.15
The molecular formula of compound 2 was determined to be
þ
C H N O on the basis of HRFABMS (m/z 345.1215 [M þ Na] ,
1
9 18 2 3
1
13
calcd for C H N O Na 345.1215). The H and C NMR data of 2
9 18 2 3
1
were classified into one methoxy group (d /d 3.83/55.4), ten sp2
methines, four sp quaternary carbons, one sp methylene, one sp
H
C
2
2
3
Figure 2 Selected key 1 H- H COSY and HMBC correlations for compounds
1
methine and two amidocarbonyl carbons (d 159.9, 165.0) (Table 1). 1–4.
C
Figure 1 Chemical structures of compounds 1–8.
The Journal of Antibiotics

New diketopiperazine derivatives
Q Zhang et al
3
3
Figure 3 X-ray crystal structure of methoxyneihumicin (1).
Table 1 1H and 13C NMR assignment of compound 1–4 (J in Hz within parentheses) (500MHz for 1H and 125 MHz for 13C, CDCl and
3
tetramethylsilane)
1
2
3
4
Position
dC
dH (multi, J in Hz)
dC
dH (multi, J in Hz)
dC
dH (multi, J in Hz)
dC
dH (multi, J in Hz)
1
2
3
5
5
6
7
-NH
6.39 br s
160.4 C
129.7 C
153.6 C
55.3 CH3
124.5 C
110.1 CH
159.9 C
123.8 C
165.0 C
159.7 C
125.4 C
166.2 C
160.1 C
125.2 C
165.3 C
-OCH3
3.85 (s)
6.56 (s)
57.1 CH
4.40 (m)
51.6 CH
4.30 (m)
6.99 (s)
61.3 CH
4.07 (dd, 3.0, 2.5)
7.00 (s)
41.0 CH2
3.05 (dd,13.8, 8.8) 116.2 CH
116.0 CH
3
.39 (dd 13.8, 3.8)
8
9
1
1
1
1
1
1
1
1
133.6 C
134.7 C
132.8 C
132.8 C
, 13
0, 12
1
128.3 CH
7.37 (brd, 7.5)
129.7 CH
7.24 overlapped
7.35 (t, 7.3)
7.28 (d, 7.0)
6.81 (s)
128.4 CH
7.35 (d, 8.0)
128.8 CH
7.36 (d, 7.5)
7.44 (dd, 8.5, 7.5)
7.34 (m, overlapped)
2.43 (ddd, 7.0, 6.8, 3.0)
1.01 (d, 6.8)
129.5 CH 7.43 (ddd, 8.0, 7.5, 1.8) 129.1 CH
129.5 CH 7.45 (dd, 8.0, 7.5) 129.5 CH
128.3 CH
128.6 CH
128.0 C
7.32 (tt, 7.8, 1.5)
7.27 (s)
127.8 CH
116.3 CH
125.0 C
128.8 CH
21.9 CH3
7.36 overlapped
1.60 (d, 7.0)
128.4 CH
33.3 CH
16.2 CH3
18.5 CH3
4
5
6, 20
7, 19
8
133.4 CH
113.9 CH
160.4 C
8.12 (d, 9.0)
6.93 (d, 9.0)
129.9 CH
114.9 CH
159.8 C
7.22 (d, 8.5)
6.95 (d, 8.5)
1.10 (d, 7.0)
8-OCH3
54.5 CH3
4.06 (s)
55.4 CH3
3.83 (s)
D-phenylalanine derivative t ¼ 15.3min) (Supplementary Figure the HMBC correlations from H-14 to C-5/C-6 located the methyl
R
S10A). Further HPLC analysis revealed that the two isomers were group (d /d 1.60/21.9) at C-6. Therefore, the planar structure of 3
H
C
separable on an analytical chiral column (Supplementary Figure was determined to be 3-benzylidene-6-methylpiperazine-2,5-dione
S10B). These data indicated that 2 was actually a mixture of two (Figure 1). The Z configuration of D3,7 double bond in 3 was
enantiomers of 2a (6R, containing a D-phenylalanine) and 2b (6S, deduced from the chemical shift of H-7 (dH 6.99), similar to that of 2
containing an L-phenylalanine) (Figure 1). However, compound 2 was (dH 6.81, H-14) (Table 1). The absolute configuration of the alanine
only isolated in minor amounts, preventing further efforts to obtain moiety in 3 was determined as L by Marfey’s method (FDAA t ¼ 15.9
R
pure 2a and 2b by preparative chiral HPLC. Thus, the structures of min; L-alanine derivative t ¼ 14.5min; D-alanine derivative t ¼ 16.2
R
R
enantiomers 2a and 2b were determined as (R, Z)-3-benzylpiper- min) (Supplementary Figure S11A). Therefore, the structure of
azine-6-(4-methoxybenzylidene)-2,5-dione and (S, Z)-3-benzylpiper- compound 3 was elucidated as (S, Z)-3-benzylidene-6-methylpiper-
azine-6-(4-methoxybenzylidene)-2,5-dione, designated nocazines D azine-2,5-dione. The compound 3 was chemically synthesized
1
16
(
2a) and E (2b), respectively (Figure 1).
Compound 3 was obtained as white powder. The inspection of the
before,16 however, only H NMR data were available.
The molecular formula of compound 4 was assigned to be
1H and 13C NMR data of 3 revealed the presence of one methyl group C H N O by HRFABMS (m/z 245.1294 [Mþ H] , calcd. for
þ
1
4 16 2 2
13
(
d /d 1.60/21.9), six sp2 methines, one sp3 methine, two sp2 C H N O 245.1290). The 1H and C NMR data of 4 was
H
C
14 17 2 2
quaternary carbons and two amidocarbonyl carbons (dC 159.7, consistent with those of the synthetic compound 6-benzylidene-3-
66.2) (Table 1). The comparison of its NMR data with that of 2 isopropylpiperazine-2,5-dione. (Supplementary Figure S5). The Z
1
revealed the existence of 3-benzylidenepiperazine-2,5-dione moiety, configuration of D3,7 double bond in 4 could be deduced from the
which were further identified by the HMBC correlations from H-7 to similarity of the chemical shift of H-7 (d 7.00) to that of 3 (d 6.99).
C-2/C-9, from H-9 to C-7/C-11 and from H-10 to C-8. In addition, The absolute configuration of the valine moiety in 4 was determined
H
H
The Journal of Antibiotics

New diketopiperazine derivatives
Q Zhang et al
3
4
as L by Marfey’s method (FDAA t ¼ 11.9min; L-valine derivative (40–70 mm; Amersham Pharmacia Biotech AB, Uppsala, Sweden) and YMC*-
R
t ¼ 12.9min; D-valine derivative t ¼ 14.1min) (Supplementary GEL ODS-A (12 nm S-50mm; YMC Company Ltd., Kyoto, Japan). TLC (0.1–
R
R
0
.2mm or 0.3–0.4mm) was conducted with precoated glass plates (silica gel
Figure S11B). Thus, compound 4 was elucidated as (S, Z)-3-
benzylidene-6-isopropylpiperazine-2,5-dione (Figure 1).
GF254, 10–40nm; Jiangyou Silica Gel Development, Inc.). Medium-pressure
liquid chromatography was performed on automatic flash chromatography
(EZ Purifier III; Leisure Science Co., Ltd., Shanghai, China). ESI-MS was
acquired on a MDS SCIEX API 2000 liquid chromatography/tandem MS (LC/
Biological activity
Eight compounds (1–8) were evaluated for in vitro cytotoxic activities MS/MS) instrument (Applied Biosystems, Inc., Forster, CA, USA). HRFABMS
against three human cancer cell lines SF-268, MCF-7 and NCIH460, data were obtained on a quadrupole-time-of-flight MS (Waters, Milford, MA,
and antibacterial activities against four indicator strains Escherichia USA). The optical rotation was recorded on a 341 Polarimeter (PerkinElmer,
Inc., Norwalk, CT, USA). UV spectra were recorded on an U-2900 spectro-
photometer (Hitachi, Tokyo, Japan). The analytical chiral packed column
coli ATCC 25922, Staphylococcus aureus ATCC 29213, Bacillus subtilis
SCSIO BS01 and Bacillus thuringiensis SCSIO BT01. Methoxyneihu-
2
Chiralpak AD-H (250 ꢁ 4.6 mm ID, 5 mm) (Daicel Chemicals, Tokyo, Japan),
micin (1) showed moderate activities against human tumor cell lines
MCF-7 (IC50 4.6 mM) and SF-268 (IC50 12.7mM). Compound 6
XR334), lacking an O-methyl group at C-5 (Figure 2), exhibited
and an Agilent 1200 series HPLC system (Agilent, Santa Clara, CA, USA) were
used for the chirality analysis. 1H, 13C NMR and 2D NMR spectra were
recorded on a Bruker AV-500 NMR spectrometer (Bruker Biospin GmbH,
(
6
,7
less cytotoxic activities than 1. However, when the D double bond
in compound 6 was reduced to afford a mixture of 2a and 2b, the
Rheinstetten, Germany) with tetramethylsilane (d 0.0 p.p.m.) as the internal
1
standard. H NMR data were reported as follows: chemical shift (multiplicity:
latter compounds showed little cytotoxic activities (Table 2). The singlet (s), doublet (d), triplet (t) and multiplet (m)), coupling constants (Hz);
DKPs 3-5 and the compounds 7 and 8 exhibited little antiproliferative 13C NMR data were reported as follows: chemical shift (quaternary carbon (C),
activities against the three tested cancer cell lines (Table 2). And no methine (CH), methylene (CH2), methyl (CH3)). Deuterated NMR solvents
compounds displayed antibacterial activities (MIC4128 mg ml 1)
ꢀ
were purchased from Cambridge Isotopes (Andover, MA, USA)
against four indicator strains.
DKPs are the smallest cyclic peptides known and exhibit a wide Isolation and taxonomic studies of the strain SCSIO 03039
spectrum of biological activities with various therapeutic possibili- A sediment sample was collected aseptically with a grab-bucket collection
ties.29,30 Recently, marine actinomycetes have been emerged as sampler from the location (E 87159.7 , N 9159.3 ) at the depth of 3412m in the
0
0
producers for novel DKPs, such as cyclomarazines,31 dimeric DKP
naseseazines,32 neihumicins,33 nocardioazines (containing a novel
bridged DKP scaffold),34 nocazines,24 and cyclo(2-hydroxy-Pro-R-
Indian Ocean. The surface layer (0–10 cm) of the sediment sample was
obtained as a subsample. The wet subsample was first air-dried aseptically in a
laminar flow hood. Then, 2 g air-dried sample was suspended in 18ml sterile
seawater. Finally, 0.1 ml of such seawater suspensions was spread on isolation
media plates containing Gauze’s medium No.1 (soluble starch 2%, KNO3
Leu).35 However, none of the described producers were isolated from
a deep-sea environment greater than the depth of 1000m. In contrast,
the strain N. alba SCSIO 03039 was isolated from the abyssal deep
sedimental sample ( ꢀ3412m) in the Indian Ocean. In this study, we
0.1%, K2HPO4 0.05%, MgSO4 ꢂ 7 H2O 0.05%, FeSO4 ꢂ 7 H2O 0.01%, pH 7.4).
After incubation at 28 1C for 2 weeks, one gray white colony, designated
SCSIO 03039, was isolated and deposited in the type culture collection of
showed that this deep-sea-derived actinomycete was capable of Center for Marine Microbiology, Research Network of Applied Microbiology,
producing seven DKPs. Nocazines D and E (2a and 2b) were new. South China Sea Institute of Oceanology, Chinese Academy of Sciences,
Methoxyneihumicin (1) was structurally validated for the first time by Guangzhou, China. Isolation of genomic DNA, amplification of 16S rRNA
gene by PCR, sequence comparison and phylogenetic tree construction of the
NMR and X-ray analyses. Compounds 3 and 4, previously known
strain SCSIO 03039 (Supplementary Figure S1) were performed as described
only as synthetic compounds, were isolated for the first time from a
natural source. Interestingly, methoxyneihumicin (1) displayed
previously.5
cytotoxicities against MCF-7 and SF-268 with IC50 values of 4.6
and 12.7mM, significantly better than other DKPs isolated in this
study.
Fermentation of the strain SCSIO 03039, extraction and isolation
of secondary metabolites
A single colony of SCSIO 03039 on ISP 4 medium (soluble starch 1%, K HPO4
2
0
.1%, MgSO4 ꢂ 7 H2O 0.1%, (NH4)2SO4 0.2%, CaCO3 0.2%, sea salt 3%, pH
METHODS
General experimental procedures
Materials for column chromatography were silica gel (100–200 mesh; 300–400
mesh; Jiangyou Silica Gel Development, Inc., Yantai, China), Sephadex LH-20
7.0-7.4 before sterilization) was inoculated into a 250-ml Erlenmeyer flask
containing 30ml of the seed medium (soluble starch 2%, glucose 1%, bacterial
protein 0.5%, yeast exact 0.5%, CaCO3 0.2%, sea salt 3%, pH 7.0 before
sterilization). After incubating on a rotary shaker (200r.p.m.) for 3 days at
28 1C, the pre-cultures were transferred to 1000-ml Erlenmeyer flask contain-
Table 2 Cytotoxicities of compounds 1–8
ing 200 ml of the seed medium. A total of 20l cultures were made by this way.
Fermentations were done on a rotary shaker (200r.p.m.) for 7 days at 28 1C.
The 20-l fermentation broths were extracted four times with 20l butanone,
and the mycelia cakes were extracted four times with 4 l methanol. After
removing solvents, both residues from fermentation broths and mycelia cakes
were combined to afford 11.86 g crude extracts.
IC50 (mM)
Compounds
SF-268
MCF-7
NCI-H460
1
12.7
4100
4100
4100
4100
20.6
4.6
87.7
4100
4100
4100
99.4
The crude extracts were subjected to normal phase silica gel column (100–
200 mesh, 90g) and eluted with CHCl /CH OH (100/0, 100/1, 100/2, 100/4,
2
4100
4100
4100
75.0
3
3
3
100/8, 100/16, 100/32 and 0/100, v/v, each of 360 ml) to yield eight fractions
Fr.1–Fr.8). Fr.1 and Fr.2 were combined and subjected to Sephadex LH-20
(
4
chromatography and were eluted with CHCl3/CH3OH (1:1) to give six
subfractions (Fr.1-1–Fr.1-6). Fr.1-2 (23.7 mg) was further purified by semi-
preparative HPLC on a Varian Star Workstation, using a reversed phase
5
6
22.0
97.0
7
93.2
95.0
97.6
2
column C18 (YMC*GEL ODS-A, 120 A S-5 mm, 250 ꢁ 10mm ; solvent A,
8
4100
3.0
4100
13.9
4100
6.7
0.1% formic acid in water; solvent B, 90% acetonitrile in water; eluted with
constant 70% solvent B) to obtain compounds 7 (2.9mg) and 8 (3.0mg).
Cisplatin
The Journal of Antibiotics

New diketopiperazine derivatives
Q Zhang et al
3
5
˚
ꢀ3
Fr.1-5 (1.42 g) was subjected to silica gel column (100–200 mesh) and was and largest difference peak and hole, 0.206 and ꢀ0.230 e.A . Crystallographic
eluted with petroleum ether/ethyl acetate (100/0, 100/10, 100/20, v/v, each of data (excluding structure factors) for compound 1 in this paper have been
1
00ml) to give three subfractions: Fr.1-5-1 (100/0, v/v), Fr.1-5-2 (100/10, v/v) deposited with the Cambridge Crystallographic Data Center as supplementary
and Fr.1-5-3 (100/20, v/v). The elutions from petroleum ether/ethyl acetate publication number CCDC 888963. Copies of the data can be obtained free of
100/30, 100 ml) were collected with 10ml each and combined on the basis of charge on application to CCDC.
TLC analysis to give four subfractions Fr.1-5-4–Fr.1-5-7. Fr.1-5-4 (87.2 mg) 3-Benzylpiperazine-6-(4-methoxybenzylidene)-2,5-dione (a mixture of
was purified by semipreparative HPLC (YMC*GEL ODS-A, 120 A S-5 mm, nocazine D (2a, 6R) and nocazine E (2b, 6S)): white powder, UV/vis (in
(
2
2
50 ꢁ 10mm ; solvent A, 0.1% formic acid in water; solvent B, 90%
CH3OH) lmax nm (log e): 204 (3.85), 311 (3.44)nm, ESI-MS m/z 345.1
þ
ꢀ
acetonitrile in water; eluted with constant 85% solvent B) to obtain compound [Mþ Na]
,
321.3 [MꢀH] , HRFABMS m/z 345.1215 (calcd. for
þ
1
(8.2mg). Fr.1-5-7 (185.5mg) was purified by semipreparative HPLC C H N O Na [M þ Na]
, 345.1215; see Table 1 and Supplementary
1
9 18 2 3
2
1
13
(YMC*GEL ODS-A, 120 A S-5 mm, 250 ꢁ 10mm ; solvent A, 0.1% formic Figure S3 for H and C NMR data.
acid in water; solvent B, 90% acetonitrile in water, eluted with constant 70%
(S, Z)-3-Benzylidene-6-methylpiperazine-2,5-dione (3): white powder,
nm (log e): 219 (4.52), 292 (3.75)nm, ESI-MS m/
solvent B) to obtain the compounds 5 (10.1 mg) and 6 (3.6mg). Fr.3 was UV/vis (in CH OH) l
3
max
þ
ꢀ
subjected to Sephadex LH-20 chromatography and was eluted with CHCl3/ z 217.4 [M þ H] , 215.1 [MꢀH] ; HRFABMS m/z 215.0817 (calcd. for
ꢀ
CH3OH (1:1) to give two subfractions (Fr.3-1 and Fr.3-2). Fr.3-2 (1.79 g) was
separated by silica gel column chromatography (100–200 mesh) and was eluted Table 1 and Supplementary Figure S4 for H and C NMR data.
C12H11N2O2 [MꢀH] , 215.0815); [a]21D¼ ꢀ2391 (c ¼ 0.14, CH3OH); see
1
13
with CHCl /CH OH (30:1) to give two subfractions (Fr.3-1-1 and Fr.3-1-2).
3
(S, Z)-3-Benzylidene-6-isopropylpiperazine-2,5-dione (4): white needle
crystal, UV/vis (in CH3OH) lmax nm (log e): 224 (3.65), 296 (3.74)nm,
3
Fr.3-1-2 (531 mg) was separated by normal phase silica gel column chromato-
graphy (100–200 mesh) and was eluted with petroleum ether/ethyl acetate ESI-MS m/z 267.3 [M þ Na] , 245.2 [Mþ H] , 243.2 [MꢀH] , HRFABMS
þ
þ
ꢀ
þ
21
(100/0, 100/10, 100/20, 100/30) to give four subfractions (Fr.3-1-2-1–Fr.3-1-2-4).
m/z 245.1294 (calcd. for C14H17N2O2 [Mþ H] , 245.1290), 267.1115 (calcd.
þ
Fr.3-1-2-2 (90.2 mg), Fr.3-1-2-3 (111 mg) and Fr.3-1-2-4 (18.1 mg) were further for C H N O Na, [M þ Na] , 267.1109); [a] ¼ ꢀ2.51 (c ¼ 0.08, CH OH);
14
16
2
2
D
3
1
purified by semipreparative HPLC (YMC*GEL ODS-A, 120 A S-5mm, see Supplementary Figure S5 for H NMR data.
2
2
50 ꢁ 10mm ; solvent A, 0.1% formic acid in water; solvent B, 90%
(3Z, 6Z)-3-(4-Methoxybenzylidene)-6-(2-methylpropylidene)piperazine-
þ
acetonitrile in water) to yield compounds 2 (3.4mg) from Fr.3-1-2-2 (eluted 2,5-dione (5): white needle crystal, ESI-MS m/z 309.2 [Mþ Na] , 285.3
ꢀ
1
13
with constant 50% solvent B), 3 (6.7mg) from Fr.3-1-2-3 (eluted with constant [MꢀH] ; see Supplementary Figure S6 for H and C NMR data.
þ
50% solvent B) and 4 (2.6mg) from Fr.3-1-2-4 (eluted with constant 55%
solvent B), respectively.
XR334 (6): yellow powder, ESI-MS m/z 321.1 [M þ H] , 343.1 [Mþ
þ
1
13
Na] ; see Supplementary Figure S7 for H and C NMR data.
-Methoxynaphthalene-1,4-dione (7): yellow powder, ESI-MS m/z 211.2
2
Mþ H] , 243.2 [Mþ Na] þ ; see Supplementary Figure S8 for H and 13C
þ
1
[
NMR data.
Determining the absolute configuration of amino acids in
compounds 2–4 by Marfey’s method
1
-Hydroxy-4-methoxy-2-naphthoic acid (8): brown powder, ESI-MS m/z
þ
The FDAA derivatized hydrolysates of compounds 2 (0.50 mg), 3 (0.50 mg)
and 4 (0.58 mg), and the standard amino acids were prepared according to the
published method.24 The FDAA derivatives were then subjected to HPLC
ꢀ
1
2
41.0 [Mþ Na] , 217.1 [MꢀH] ; see Supplementary Figure S9 for H and
1
3
C NMR data.
2
analysis (Luna C18 column; 5 mm, 4.6 ꢁ 150 mm ) using the following solvent
system: solvent A, 10% acetonitrile in water supplementing with 0.1% formic Biological activity
acid; solvent B, 90% acetonitrile in water. The HPLC program for detecting Antibacterial activities were evaluated against bacterial strains S. aureus ATCC
FDAA derivatives of compounds 2 and 4 was set as: 5–100% solvent B 29213, Enterococcus faecalis ATCC 29212, B. subtilis SCSIO BS01 and
(
(
0–20min, linear gradient), 100% solvent B (20–23min), 100–5% solvent B B. thuringiensis SCSIO BT01 according to previously described methods.36
23–24 min) and 5% solvent B (24–30 min). The HPLC program for detecting Cytotoxic activities were determined against three tumor cell lines, including
FDAA derivatives of compound 3 was set as: 5–55% solvent B (0–20 min, MCF-7 cells (human breast adenocarcinoma cell line), NCI-H460 (human
linear gradient), 55–100% solvent B (20–24 min), 100% solvent B (24–25 min), non-small cell lung cancer cell line) and SF-268 (human glioma cell line),
5
–100% solvent B (25–26 min) and 5% solvent B (26–30min). The flow rate according to published protocols.37
ꢀ1
was set at 1 ml min with UV detection at l ¼ 340 nm.
ACKNOWLEDGEMENTS
Chiral analysis of compound 2
The solution of 2 in methanol was subjected to HPLC analysis (Chiralpak
This study was supported in part by the Chinese Academy of Sciences for Key
Topics in Innovation Engineering (KZCX2-YW-JC202), the Ministry of
Science and Technology of China (2012AA092104, 2010CB833805) and the
National Science Foundation of China (41006089, 41106143, 31125001). CZ is
a scholar of the ‘100 Talents Project’ of the Chinese Academy of Sciences
2
AD-H, 250ꢁ 4.6 mm , ID 5 mm) (Daicel Chemicals) on an Agilent 1200
series HPLC system, eluted with 20% 2-propanol in n-hexane, UV detection
at l ¼ 254 nm.
Methoxyneihumicin (1): yellow needle crystal, UV/vis (in CH3OH) lmax
nm (log e): 202 (3.96), 227 (3.76), 310 (3.68)nm, ESI-MS m/z 357.0 [Mþ
(
08SL111002). This study was funded by the Natural Science Funds of South
China Sea Institute of Oceanology for Young Scholar (SQ200903). We are
grateful to analytical facilities in the South China Sea Institute of Oceanology.
þ
ꢀ
Na] , 333.1 [MꢀH] , HRFABMS m/z 335.1393 (calcd. for C20H19N2O3
þ
1
[
Mþ H] , 335.1396), see Table 1 and Supplementary Figure S2 for H NMR
and 13C NMR. X-ray crystal structure analysis of compound 1: Yellow needle
˚
crystal, C H N O , M ¼ 334.36, T ¼ 150(2) K, l ¼ 1.54178 A; crystal system,
20 18 2 3
˚
˚
monoclinic, space group P 1 21/c 1, a ¼ 6.39230(10) A, b ¼ 14.9704(3)A,
1
2
3
4
5
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The Journal of Antibiotics