Streptopyrazinones A?D, rare metabolites from marine-derived Streptomyces sp. ZZ446

Article abstract of DOI:10.1016/j.tet.2018.03.028

Secondary metabolites from marine-associated actinomycetes are important source for the discovery of novel bioactive compounds. In this study, an actinomycete Streptomyces sp. ZZ446 was isolated from coastal soils and different media were used to culture this isolated marine actinomycete. It has been found that this actinomycete in the liquid medium of 2216 E with sea salt produced five new compounds of streptopyrazinones A?D (1–4) and N-acetyl-L-isoleucine-L-leucinamide (5) as well as six known diketopiperazines (6–11) and one alkaloid (12). Structures of the new compounds were determined by extensive NMR analyses, HRESIMS data, electronic circular dichroism (ECD) calculation, chemical degradation, Marfey's method, and X-ray diffraction analysis. This type of streptopyrazinones A?D (1–4) is rarely found in the natural resources. New compounds 1–5 showed activity in inhibiting the growth of Candida albicans and methicillin-resistant Staphylococcus aureus.

Full text of DOI:10.1016/j.tet.2018.03.028

Tetrahedron xxx (2018) 1e7
Contents lists available at ScienceDirect
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Streptopyrazinones AꢀD, rare metabolites from marine-derived
Streptomyces sp. ZZ446
a
a
b
a
a, *
Mengxuan Chen , Weiyun Chai , Rongyao Zhu , Tengfei Song , Zhizhen Zhang
Xiao-Yuan Lian
a
,
b, **
Ocean College, Zhoushan Campus, Zhejiang University, Zhoushan 316021, PR China
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, PR China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 January 2018
Received in revised form
Secondary metabolites from marine-associated actinomycetes are important source for the discovery of
novel bioactive compounds. In this study, an actinomycete Streptomyces sp. ZZ446 was isolated from
coastal soils and different media were used to culture this isolated marine actinomycete. It has been
found that this actinomycete in the liquid medium of 2216 E with sea salt produced five new compounds
4
March 2018
Accepted 12 March 2018
Available online xxx
of streptopyrazinones AꢀD (1e4) and N-acetyl-L-isoleucine-L-leucinamide (5) as well as six known
diketopiperazines (6e11) and one alkaloid (12). Structures of the new compounds were determined by
extensive NMR analyses, HRESIMS data, electronic circular dichroism (ECD) calculation, chemical
degradation, Marfey's method, and X-ray diffraction analysis. This type of streptopyrazinones AꢀD (1e4)
is rarely found in the natural resources. New compounds 1e5 showed activity in inhibiting the growth of
Candida albicans and methicillin-resistant Staphylococcus aureus.
Keywords:
Streptomyces sp. ZZ446
Marine actinomycete
Streptopyrazinones
Candida albicans
© 2018 Elsevier Ltd. All rights reserved.
Methicillin-resistant Staphylococcus aureus
1. Introduction
the culture of strain ZZ446 in the liquid medium of 2216 E with sea
salt showed the most antimicrobial activity and contained more
The marine environment has proven to be an important source
of bioactive natural products, which have had nine approved drugs
and 12 current clinical trial agents for the past over 50 years.
metabolites. Chemical investigation into this active crude extract
resulted in the isolation and identification of 12 compounds
including five new ones. In this article, we describe the isolation
and culture of strain ZZ446, the isolation and structural elucidation
of the new compounds, and their activities against the growth of
methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli,
and Candida albicans and the proliferation of different glioma cells.
1
e3
There has been a growing perception that many marine bioactive
natural products found originally from collected biomass of mac-
roorganisms, such as sponges, mollusks, and tunicates, are actually
1
being produced by symbiotic or associated microorganisms. Sec-
ondary metabolites from marine-associated microorganisms,
especially marine actinomycetes, are abundant source for the dis-
2
. Results and discussion
4
e7
covery of novel bioactive compounds and drug leads.
Over the course of our ongoing project for the discovery of novel
Strain ZZ446 (Supplementary Data Fig. S1) was assigned as
8
e15
bioactive compounds from marine microorganisms,
a strain
Streptomyces sp. ZZ446 (Table S1) based on the result (Fig. S2) of its
6S rDNA sequence analysis. Ten different media a-j (Table S2) were
ZZ446 was isolated from a sample of coastal soils. Ten different
media were used to culture this marine actinomycete strain ZZ446
to induce different silent gene clusters to express and then produce
1
used to culture this marine actinomycete and a crude extract pre-
pared from the culture of strain ZZ446 in the liquid medium of
15e17
different novel natural products.
A crude extract prepared from
2
216 E with sea salt (medium h) showed the most activity (Fig. S3)
against the growth of methicillin-resistant Staphylococcus aureus
MRSA), Escherichia coli, and Candida albicans and had more me-
(
tabolites as detected by HPLC (Fig. S4 and Fig. S5). A total of 60 L
culture of strain ZZ446 was conducted in medium h and the crude
extract made from this large culture was separated by column
chromatography, followed by HPLC purification, to afford five new
*
Corresponding author.
* Corresponding author.
E-mail addresses: zzhang88@zju.edu.cn (Z. Zhang), xylian@zju.edu.cn
X.-Y. Lian).
*
(
https://doi.org/10.1016/j.tet.2018.03.028
040-4020/© 2018 Elsevier Ltd. All rights reserved.
0
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2
M. Chen et al. / Tetrahedron xxx (2018) 1e7
compounds 1e5 (Fig. 1) and seven known ones (Fig. S6). Based on
the NMR and HRESIMS data and the results (Figs. S7ꢀS13) from
Marfey's analyses, the known compounds were identified as cyclo-
1
8,19
19
L
-proline-
-proline-
L
-methionine (6),
cyclo-
L
-proline-
L
-valine (7), cyclo-
20
L
L
-leucine (8),18 cyclo-
-phenylalanine (10),
11), and N-acetyl-b-oxotryptamine (12). The C NMR data of
L-proline-
L
-isoleucine (9), cyclo-L-
21
proline-
(
L
cyclo-D-proline-L-phenylalanine
21
22
13
6
ꢀ12 were summarized in Table S3.
Compound 1 was obtained as colorless needle crystals (mp
ꢁ
177e178 C) and has a molecular formula of C13
21
H N
3
O
2
deduced
þ
from its HRESIMS ions at m/z [MþH] 252.1711 (calcd. 252.1712)
þ
13
and [MþNa] 274.1531 (calcd. 274.1531) as well as its C NMR data.
Its UV spectrum showed absorption at 228 and 325 nm, which are
Fig. 2. HMBC correlations of compounds 1, 3, and 5.
2
3e25
very close to the UV absorption of arglecin (1a)
and argvalin
two metabolites with a skeleton of 2(1H)-pyrazinone and
a guanidine group that were previously isolated from Streptomyces
sp. The carbon signals at 156.4 (C, C-3), 155.5 (C, C-2), 138.8 (C, C-
), 120.5 (CH, C-1) and proton signal at 7.08 (1H, s) observed in
the C and H NMR spectra suggested the presence of a skeleton of
(1H)-pyrazinone in 1, which were confirmed by HMBC correla-
tions (Fig. 2) and X-ray diffraction analysis (Fig. 3). The acetyl group
was easily recognized by its NMR signals at 169.0 (C, C-12), 22.6
CH , C-13) and 1.79 (3H, s, H -13) and HMBC correlation (Fig. 2)
of H-13 with C-12; while the isobutyl (2-methylpropyl) group was
indicated by its characteristic NMR signals at 41.0 (CH , C-5),
2.45 (2H, d, 7.1 Hz, H-5),
.05 (1H, m, H-6), 0.85 (6H, d, 6.7 Hz, H-7, H-8). HMBC correlations
7.85, 1H, br t, 5.7 Hz)
2.99, 2H, m) with C-12
2
6,27
(1b),
Consideration of the HRESIMS data, compound 2 must have an
d
C
additional methylene (-CH
quadmethylene group (eCH
2
-) with the partial structure A of a
eCH eCH eCH e). Accordingly,
4
d
H
2
2
2
2
13
1
compound 2 was identified as streptopyrazinone B, a new pyr-
azinone derivative.
Compounds 3 and 2 have the same molecular formula of
14 23 3 2
C H N O and similar UV absorption. Comparison of the NMR
data (Tables 1 and 2) of 3 and 2 proved that they are structurally
different only in the substitute at C-2. In compound 3, the substi-
tute at C-2 was assigned as 1-methylpropyl as indicated by its NMR
2
d
C
(
3
d
H
3
d
C
2
d
C
2
2
6.0 (CH, C-6), 22.5 (CH
3
, C-7, C-8) and
d
H
signals at
C-8) and
d
C
35.6 (CH, C-5), 27.0 (CH
3.05 (1H, m, H-5), 1.37 (1H, m, H-6), 1.67 (1H, m, H-6),
2 3 3
, C-6),11.8 (CH , C-7),17.8 (CH ,
d
H
(Fig. 2) of H-5 with C-2 and C-3, NH-11 (
d
H
0.77 (3H, t, 7.4 Hz, H-7), 1.06 (3H, d, 6.9 Hz, H-8). HMBC correlations
as shown in Fig. 2 also demonstrated the presence of this 1-
methylpropyl group at C-2. The absolute configuration at C-5 of 3
was determined by electronic circular dichroism (ECD) calculation.
The conformational analyses were carried out via random search-
ing in the Sybyl-X 2.0 using the MMFF94S force field with an energy
cutoff of 2.5 kcal/mol. The results showed six lowest energy
conformers for compound 3. Subsequently, the two lowest con-
1 2
formers C and C (Table S4 and Fig. S63) weighing for more than
C H
with C-11 (d 37.8) and C-12, and H-11 (d
established that the acetyl group and the isobutyl group were
13
connected to NH-11 and C-2, respectively. The C NMR spectrum of
displayed 13 signals, of which four were assigned to the skeleton
1
of 2(1H)-pyrazinone, four to the isobutyl group, two to the acetyl
group, and the remaining three to a partial structure A of a tri-
2
8
methylene group (eCH
2
eCH
2
eCH
2
e) located between C-4 and
NH-11, which was confirmed by HMBC correlations as depicted in
Fig. 2. The 13C and 1H NMR data (Tables 1 and 2) were assigned
using HSQC and HMBC correlations. Based on the foregoing evi-
dence, compound 1 was elucidated as a new compound, named as
streptopyrazinone A, a pyrazinone derivative.
99% were further re-optimized using DFT at the b3lyp/6-31þg(d)
2
9
level in gas phase by the GAUSSIAN 09 program. The energies,
oscillator strengths, and rotational strengths (velocity) of the first
60 electronic excitations were calculated using the TDDFT meth-
odology at the b3lyp/6-311þþg(d,p) level in vacuum. The ECD
Compounds 2 and 1 have very similar UV spectra, suggesting
that they are analogues. Compound 2 has a molecular formula of
spectra were simulated by the overlapping Gaussian function (half
þ
30
C
2
H
14 23
N
3
O
2
as established by its HRESIMS ions at m/z [MþH]
the bandwidth at 1/e peak height,
s
¼ 0.2). To get the final
þ
66.1864 and [MþNa] 288.1679, 14 mass units higher than 1,
spectra, the simulated spectra of the conformers were averaged
according to the Boltzmann distribution theory and their relative
2
corresponding to a CH group. Detailed comparison of the NMR
data (Tables 1 and 2) of 2 with those of 1 indicated that both 2 and 1
Gibbs free energy (DG). Theoretical ECD spectrum of the corre-
share the common isobutyl and acetyl groups and the skeleton of
sponding enantiomer was obtained through the direct inverse of
the ECD spectrum of the calculated model molecule. By comparing
the experiment spectrum with the calculated ECD spectra (Fig. 4),
the absolute configuration at C-5 of 3 was determined to be R.
Based on the above evidence, compound 3 was determined as
streptopyrazinone C, a new pyrazinone derivative.
2
(1H)-pyrazinone, but have a different partial structure A.
Compounds 4 and 1 share the same molecular formula of
C H N O and similar UV absorption, suggesting that 4 is also an
13 21 3 2
analogue of streptopyrazinones AꢀB (1e3). Further analysis of the
NMR data (Tables 1 and 2) of 4 and 1 demonstrated that the
structural difference between 4 and 1 is only the substitute at C-2.
This means that the isobutyl group at C-2 in 1 is replaced by a 1-
13
methylpropyl group in 4. The similar C chemical shifts for C-1
to C-8 between 4 and 3 also supported that 4 has the 1-
methylpropyl group at C-2. Just like 1 and 2, the structural differ-
ence between 4 and 3 is due to the partial structure A with a tri-
methylene group for 4 and a quadmethylene group for 3. The
absolute configuration at C-5 of 4 was assigned as R also based on
the result of ECD calculation (Fig. 5). Therefore, compound 4 was
Fig. 1. Structures of compounds 1e5 isolated from the culture of Streptomyces sp.
ZZ446.
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M. Chen et al. / Tetrahedron xxx (2018) 1e7
3
Fig. 3. X-ray crystal structure of compound 1.
Table 1
Compound 5, a colorless amorphous powder, has a molecular
1
3C NMR data for compound 1e5 (125 MHz, in DMSO‑d
6
).
formula of C14
H
27
N
3
O
3
, which was deduced from its HRESIMS
þ
þ
13
No.
1
2
3
4
5
[MþH] and [MþNa] ions and C NMR data. The NMR spectra of 5
showed four nitrogenated proton signals at
.83 (1H, d, 8.5 Hz), 7.18 (1H, s), and 6.95 (1H, s) and 14 carbon
signals including three carbonyl carbons ( 174.0,170.9, and 169.4),
five methyls ( 23.0, 22.5, 21.5, 15.4, and 11.0), four methines (
7.1, 50.8, 36.3, and 24.2), and two methylenes ( 40.8 and 24.4).
An acetyl group was indicated by a HMBC correlation of 1.85 (3H,
s, H -14) and 169.4 (C-13). Therefore, compound 5 was suggested
H
d 7.90 (1H, d, 8.4 Hz),
1
2
3
4
5
6
7
8
9
120.5, CH
155.5, C
156.4, C
138.8, C
120.5, CH
155.5, C
156.5, C
139.1, C
120.4, CH
159.3, C
156.1, C
138.9, C
35.6, CH
120.5, CH
159.1, C
156.2, C
138.8, C
35.7, CH
27.0, CH
11.8, CH
17.8, CH
27.2, CH
28.1, CH
37.8, CH
169.2, C
22.6, CH
e
174.0, C
50.8, CH
40.8, CH
24.2, CH
21.5 , CH
7
2
d
C
d
C
d
C
a
41.0, CH
26.0, CH
22.5, CH
22.5, CH
27.1, CH
28.1, CH
37.8, CH
169.0, C
22.6, CH
e
2
41.0, CH
26.0, CH
22.5, CH
22.5, CH
29.2, CH
25.6, CH
28.6, CH
38.1, CH
169.0, C
22.6, CH
2
3
3
5
d
C
a
27.0, CH
11.8, CH
17.8, CH
29.2, CH
25.6, CH
28.6, CH
38.0, CH
169.0, C
22.6, CH
2
3
3
2
2
2
2
2
3
3
2
2
2
23.0 , CH
d
H
3
3
2
2
2
3
3
2
2
2
2
170.9, C
57.1, CH
36.3, CH
24.4, CH
11.0, CH
15.4, CH
169.4, C
22.5, CH
3
d
C
to be a dipeptide. Further analyses of the HSQC and HMBC corre-
lations (Fig. 2) demonstrated that compound 5 is composed of N-
acetyl-isoleucine and leucinamide and their sequence was estab-
lished to be N-acetyl-isoleucine-leucinamide. In order to assign the
absolute configuration, compound 5 was hydrolyzed by hydro-
10
11
12
13
14
2
3
3
3
3
3
3
3
a
chloric acid to release two free amino acids of L-isoleucine and L-
The data in each column may be interchangable.
0
31
leucine, which were confirmed by C
standard amino acids ( -isoleucine, L-allo-isoleucine,
isoleucine, D-allo-isoleucine, -leucine) as references and LC-MS
analysis (Figs. S14 and S15). The retention times were found to be
36.06 min for -isoleucine-FDAA and 37.05 min for -leucine-FDAA
(Fig. S16). Therefore, the structure of 5 was determined as N-acetyl-
3
-Marfey s method using
L
L-leucine,
D-
identified as streptopyrazinone D, a new pyrazinone derivative.
Streptopyrazinones AꢀD (1e4) are new compounds with a
skeleton of 2(1H)-pyrazinone, to the best of our knowledge,
arglecin (1a) and argvalin (1b) are the only two additional exam-
ples of this type of compounds that have been found from the
natural resources. The possible biosynthetic origin of streptopyr-
azinones AꢀD (1e4) has been proposed in Fig. 6. Two different
D
L
L
13
1
L-isoleucine-
L-leucinamide, a new linear peptide. Its C and H
NMR data are listed in Tables 1 and 2.
New compounds 1e5 were assayed for their activity in inhib-
iting the growth of methicillin-resistant Staphylococcus aureus
(MRSA), Escherichia coli, and Candida albicans by using micro broth
amino acids of N-acetyl-
leucine (or -alloisoleucine) cyclized to form the cyclic dipeptide,
which further transformed to one compound of streptopyrazinones
AꢀD (1e4).
L-lysine (or N-acetyl-L-ornithine) and L-
L
10
dilution method. Gentamicin (an antibiotic against both Gram-
positive and Gram-negative bacteria) and amphotericin B (an
Table 2
1
H NMR data for compound 1e5 (500 MHz, in DMSO‑d
6
, J ¼ Hz).
No.
1
2
3
4
5
1
2
3
4
5
6
7
8
9
7.08, s
e
7.05, s
e
7.08, s
e
7.09, s
e
e
4.19, m
1.44, m
1.55, m
0.82 , d (6.5)
0.87 , d (6.5)
e
e
e
e
e
e
e
e
a
2.45, d (7.1)
2.05, m
0.85, d (6.7)
0.85, d (6.7)
2.37, t (7.6)
1.64, m
2.99, m
e
2.44, d (7.1)
2.03, m
0.83, d (6.7)
0.83, d (6.7)
2.36, t (7.6)
1.51, m
1.33, m
3.00, m
e
3.05, m
1.37, m; 1.67, m
0.77, t (7.4)
1.06, d (6.9)
2.37, t (7.6)
1.53, m
1.35, m
3.00, m
3.05, m
1.36, m; 1.67, m
0.77, t (7.4)
1.06, d (6.9)
2.37, t (7.5)
1.64, m
3.00, m
e
a
e
4.09, t (8.0/7.6)
1.69, m
1.06, m; 1.40, m
0.79, t (7.5)
0.81, d (6.6)
e
1
1
1
1
1
1
2
8
1
1
0
1
2
3
1.79, s
e
e
1.79, s
e
4
1.77, s
e
1.77 s
e
1.85, s
-NH
-NH
-NH
1-NH
2-NH
2
e
e
6.95, s; 7.18, s
7.83, d (8.5)
7.90, d (8.4)
e
e
e
e
e
e
e
e
e
7.85, br t (5.7)
e
e
e
7.87, br t (5.6)
e
7.81, br t (5.6)
7.82, br t (5.3)
e
a
The data in each column may be interchangable.
Please cite this article in press as: Chen M, et al., Streptopyrazinones AꢀD, rare metabolites from marine-derived Streptomyces sp. ZZ446,
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4
M. Chen et al. / Tetrahedron xxx (2018) 1e7
Table 3
Activity of compounds 1e5 against the growth of C. albicans and MRSA (MIC:
mg/mL).
Microorganisms Compounds
1
2
3
4
5
Gentamicin Amphotericin
C. albicans
35.0 38.0 45.0 45.0 60.0
e
0.2
MRSA
58.0 60.0 62.0 65.0 65.0 1.0
e
against E. coli at concentration of 100.0
mg/mL. Compounds 1e5
were also tested for their activity in suppressing the proliferation of
glioma U87MG, U251, SHG44, and C6 cells. Unfortunately, all of
them were inactive.
3. Conclusion
Marine Streptomyces sp. ZZ446 was isolated from a sample of
coastal soils. The medium of 2216 E with sea salt was used to largely
culture the actinomycete strain ZZ446, which produced five new
compounds and seven known ones. The discovery of new com-
1
5e17
Fig. 4. Experimental ECD spectra (205e400 nm) of 3 in MeOH and the calculated ECD
spectra of the model molecules of 3 at the B3LYP/6-311þþG (d, p) level.
pounds was the result of an OSMAC strategy,
which uses
different cultural media to activate the metabolic pathways to
obtain different types of secondary metabolites. Based on the
extensive NMR analyses, HRESIMS data, ECD calculation, chemical
degradation, Marfey's method, and X-ray diffraction, the structures
of the new compounds were determined as streptopyrazinones
AꢀD (1e4) and N-acetyl-
L-isoleucine-L-leucinamide (5). Strepto-
pyrazinones AꢀD are rare compounds obtained from the natural
resources. New compounds 1e5 showed activity in inhibiting the
growth of C. albicans and MRSA.
4. Experimental section
4.1. General experimental procedures
Melting point was measured with a WRX-4 microscope appa-
ratus (Shanghai Yice Apparatus & Equipment Co. Ltd., China) and
are uncorrected. UV spectra were recorded on a METASH UV-8000
(Shanghai METASH Instruments Co. Ltd., China). Optical rotation
and ECD spectra were measured on a JASCO DIP-370 digital polar-
imeter and a JASCO J715 spectropolarimeter (JASCO, Japan),
respectively. NMR spectra were acquired on a Bruker 500 spec-
trometer using standard pulse programs and acquisition parame-
ters. Chemical shifts were expressed in d (ppm) and referred to the
Fig. 5. Experimental ECD spectra (205e380 nm) of 4 in MeOH and the calculated ECD
spectra of the model molecules of 4 at the B3LYP/6-311þþG (d, p) level.
NMR solvent used. HRESIMS data were acquired on an Agilent 6230
TOF LC/MS spectrometer. Octadecyl-functionalized silica gel (ODS,
Cosmosil 75C18-Prep, Nacalai Tesque Inc., Japan) and Diaion HP-20
antifungal drug) were used as positive controls. The results
(Mitsubishi Chemical, Japan) were used for column chromatog-
(
Table 3) showed that compounds 1e5 had antimicrobial activity
with MIC values of 35.0e60.0 g/mL against C. albicans and
8.0e65.0 g/mL against MRSA. None of them showed activity
raphy. HPLC separation was performed on a CXTH 3000 prepared
HPLC system (Beijing Chuangxintongheng Science & Technology
Co. Ltd., China) using a CT-30 column (Fuji-C18, 280 ꢂ 30 mm,
m
5
m
Fig. 6. Possible biosynthetic origin of streptopyrazinones AꢀD (1e4).
Please cite this article in press as: Chen M, et al., Streptopyrazinones AꢀD, rare metabolites from marine-derived Streptomyces sp. ZZ446,
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M. Chen et al. / Tetrahedron xxx (2018) 1e7
5
1
0
m
m) or a Welch-20 column (XB-C18, 250 ꢂ 21.2 mm, 5
m
m). All
Diaion HP-20 eluting with water and then 100% MeOH to give a
MeOH elution (part A). The mycelia were extracted with MeOH to
give a MeOH solution (part B). The mixture of part A and part B was
concentrated under reduced pressure to give a crude extract, which
was redissolved in DMSO to prepare crude sample of 5.0 mg/mL for
bioactive assay and HPLC analysis.
solvents used for this study were purchased from the J & K Scien-
tific Ltd. (Shanghai, China). Methicillin-resistant Staphylococcus
aureus (MRSA) ATCC 43300, Escherichia coli ATCC 25922, and
Candida albicans were gifts from Drs. Zhongjun Ma, Pinmei Wang,
and Bin Wu, respectively. Human glioma U87MG (JDS-2568), U251
(
XB-0439), SHG44 (RX-J150) cells, and rat glioma C6 (XB-003) cells
were ordered from the Cell Bank of the Chinese Academy of Sci-
ences. Amphotericin B (>95.0%) and gentamicin (99.6%) were ob-
tained from Meilune Biotechnology Co. Ltd. (Dalian, China), L-allo-
isoleucine and D-allo-isoleucine (>98.0%) from Shanghai Yuanyei
Bio-Chem Technology Co., Ltd., other standard amino acids
4
.6. Large culture of strain ZZ446 in 2216 E medium with sea salt
The seed broth (5 mL) of strain ZZ446 was inoculated into an
Erlenmeyer flask (500 mL), containing 250 mL of 2216 E liquid
medium with sea salt. All flasks were incubated at 28 C for 13 days
on a rotary shaker at 180 rpm. A total of 60 L culture was prepared
for this study.
ꢁ
(>98.0%) from Shanghai Aladdin Bio-Chem Technology Co., Ltd.,
and Doxorubicin (DOX, >98.0%) from Sigma-Aldrich.
4.2. Isolation of strain ZZ446
4.7. Extraction and isolation of compounds 1ꢀ12
Strain ZZ446 was isolated from a sample of coastal soil, which
The 60 L culture of strain ZZ446 was filtered into a filtrate and
was collected from Zhoushan Islands (Zhejiang, China) in August
mycelia. The filtrate was applied to a HP-20 column eluting with
water and then 100% MeOH to obtain MeOH fraction. The mycelia
were extracted with MeOH three times to give MeOH extract. The
mixture of MeOH fraction and MeOH extract was dried in vacuo to a
crude extract. This crude extract was successively partitioned with
cyclohexane, ethyl acetate (EtOAc), and n-butanol (BuOH) to give
part A (2.0 g), part B (4.5 g), and part C (10 g) after removal of the
organic solvents. Both EtOAc and BuOH extracts showed activity
against C. albicans and only BuOH extract had activity against MRSA
and E. coli (Fig. S64).
ꢁ
2
016. The soil was air dried at 28 C for eight days and then the
ꢁ
dried soil (1.0 g) was pretreated by heating at 120 C for 1 h and
3
2,33
added to sterile water (9.0 ml).
The soil solution was incubated
at 40 C on a rotary shaker at 180 rpm for 20 min and then diluted
to be 10 , 10 , 10 g/mL, stepwisely. Each diluted suspension
ꢁ
ꢀ3
ꢀ4
ꢀ5
(
200
m
L) was covered on the surface of Gauze's medium in petri
ꢁ
dish and incubated at 28 C for five days. The single colony was
picked with sterile needles and transferred to a Gauze's agar plate.
ꢁ
After another five days of growth at 28 C, the single colonies of
ꢀ3
strain ZZ446 from 10 g/mL suspension that grew well were
transferred onto Gauze's agar slants, which were stored at 4 C until
Part B was fractioned by ODS column with gradient elution of
ꢁ
4
0e100% MeOH to give fractions B
analysis. Fraction B was separated by preparative HPLC using a CT-
0 column (mobile phase: MeOH/H O, 30/70; flow rate: 10 mL/
min) to give 7 (33.5 mg, 0.20%, t 36.77 min); while 6 (22 mg, 0.13%,
34.18 min) was obtained from fraction B by preparative HPLC
purification using the same CT-30 column (mobile phase: MeOH/
O, 35/65; flow rate: 10 mL/min). Similarly, by HPLC purification
using the same CT-30 column and the same flow rate, compound 8
10.7 mg, 0.06%, t 67.38 min, MeOH/H O: 32/68) and 9 (38.6 mg,
59.11 min) were obtained from fraction B ; 11 (44.2 mg,
42.30 min, MeOH/H O: 40/60) from fraction B ,12 (9.4 mg,
50.47 min, MeOH/H O: 40/60), 10 (61.7 mg, 0.37%, t
6.68 min) and 1 (19.6 mg, 0.12%, t 66.43 min) from fraction B ; 4
55.75 min, MeOH/H O: 43/57), 5 (6 mg, 0.04%, t
2.50 min) and 2 (84 mg, 0.51%, t 67.26 min) from fraction B
was separated by preparative HPLC using a Welch-20
column (mobile phase: ACN/H O, 20/80; flow rate: 10 mL/min) to
give 3 (7.6 mg, 0.05%, t 31.45 min).
1
-B14 based on the results of TLC
use.
2
3
2
4
.3. Taxonomic identity of strain ZZ446
R
t
R
3
The 16S rDNA analysis of strain ZZ446 was performed by Leg-
enomics (Hangzhou, China) and its DNA sequence using BLAST
nucleotide sequence comparison) was compared to the GenBank.
The 16S rDNA sequence of strain ZZ446 has been deposited in
GenBank (accession number: MG266891). The voucher strain of
Streptomyces sp. ZZ446 was preserved at the Laboratory of Institute
of Marine Biology, Ocean College, Zhoushan campus, Zhejiang
University, Zhoushan, China.
H
2
(
(
R
2
0
0
0
5
(
6
.23%, t
.27%, t
.06%, t
R
4
R
2
5
R
2
R
R
6
10.2 mg, 0.06%, t
R
2
R
4
.4. Culture of strain ZZ446 in different media
R
7
.
Fraction B
8
Strain ZZ446 from the Gauze's agar slant was refreshed on the
ꢁ
2
plates of Gauze's agar at 28 C for six days. The pure colonies of
R
ZZ446 were inoculated in an Erlenmeyer flask (500 mL) containing
ꢁ
2
50 mL 2216 E liquid medium and then incubated at 28 C for four
4
.7.1. Streptopyrazinone A (1)
Colorless needle crystals from a mixture solvent of MeOH and
days on a rotary shaker at 180 rpm to produce seed broth. The seed
broth (5 mL) was inoculated into an Erlenmeyer flask (500 mL),
containing 250 mL different liquid media of Gauze's medium (a),
potato medium (b), 2216 E medium (c), starch casein medium (d),
glycerol arginine medium (e), Gauze's medium with sea salt (f),
potato medium with sea salt (g), 2216 E medium with sea salt (h),
soluble starch casein medium with sea salt (i), and glycerol arginine
medium with sea salt (j). The ingredients of each liquid medium
were list in Table S2. A total of 1 L culture for each medium was
prepared for this study.
ꢁ
H
(
(
2
O (5: 1); molecular formula C13
H
21
N
3
O
2
; mp 177e178 C; UV
13
MeOH)
125 MHz, in DMSO‑d
lmax (log ε) 228 (3.96), 325 (3.98) nm; C NMR data
1
6
), see Table 1, H NMR data (500 MHz, in
þ
DMSO‑d
6
), see Table 2; HRESIMS m/z [MþH] 252.1711 (calcd for
C
C
13
H
H
22
N
3
O
NaO
2
,
252.1712) and [MþNa]þ 274.1531 (calcd for
13
21
N
3
2
, 274.1531).
4.7.2. Streptopyrazinone B (2)
23 3 2
Colorless amorphous powder; molecular formula C14H N O ;
1
3
4
.5. Preparation of crude extract for bioactive assay and HPLC
UV (MeOH)
(125 MHz, in DMSO‑d
lmax (log ε) 227 (3.97), 325 (4.21) nm; C NMR data
1
analysis
6
), see Table 1, H NMR data (500 MHz, in
þ
DMSO‑d
6
3
3
), see Table 2; HRESIMS m/z [MþH] 266.1864 (calcd for
Each culture from the ten different liquid media was filtered to
give filtrate and mycelia. The filtrate was applied to a column of
C
C
14
H
H
24
N
N
O
2
,
266.1869) and [MþNa]þ 288.1679 (calcd for
14
23
NaO
2
, 288.1688).
Please cite this article in press as: Chen M, et al., Streptopyrazinones AꢀD, rare metabolites from marine-derived Streptomyces sp. ZZ446,
Tetrahedron (2018), https://doi.org/10.1016/j.tet.2018.03.028

6
M. Chen et al. / Tetrahedron xxx (2018) 1e7
4.7.3. Streptopyrazinone C (3)
4.12. Antimicrobial assay
Colorless amorphous powder; molecular formula C14
H
23
N
3
O
2
;
2
5
ꢁ
Spot method10 was initially used to evaluate the antimicrobial
activity of the crude extracts and new compounds 1e5 against the
growth of MRSA, E. coli, and C. albicans. Micro broth dilution
method was further applied to determine the antimicrobial ac-
tivity of pure active compounds to obtain the MIC values. Genta-
micin and amphotericin B were used as positive controls.
[
(
(
a
]
D
ꢀ1.90 (c 0.25, MeOH); ECD (10 mg/L, MeOH)
max
l (Dε) 222
max (log ε) 227 (3.89), 324
ꢀ6.15), 328 (ꢀ0.62) nm; UV (MeOH)
l
3.90) nm; 13C NMR data (125 MHz, in DMSO‑d
1
6
), see Table 1, H
10
NMR data (500 MHz, in DMSO‑d
6
), see Table 2; HRESIMS m/z
þ
, 266.1869) and [MþNa]þ
[
2
24 3 2
MþH] 266.1863 (calcd for C14H N O
88.1679 (calcd for C14
23 3 2
H N NaO , 288.1688).
4.13. Culture of glioma cells
4.7.4. Streptopyrazinone D (4)
Colorless amorphous powder; molecular formula C13
H
21
N
3
O
2
;
2
5
ꢁ
Glioma U87MG cells were cultured in MEM (Minimum Essential
[
(
(
a
]
D
ꢀ1.58 (c 0.25, MeOH); ECD (10 mg/L, MeOH)
max
l (Dε) 225
lmax (log ε) 227 (4.53), 323
Medium, Gibco) with 10% FBS (Fetal Bovine Serum, PAA Labora-
tories Inc.), U251 and C6 cells in DMEM (Dulbecco's Modified Eagle
Medium, Gibco), and SHG44 cells in RPMI-1640 Medium (Roswell
ꢀ7.55), 330 (ꢀ0.70) nm; UV (MeOH)
4.52) nm; 1 C NMR data (125 MHz, in DMSO‑d
3
1
6
), see Table 1, H
NMR data (500 MHz, in DMSO‑d
6
), see Table 2; HRESIMS m/z
þ
, 252.1712) and [MþNa]þ
Park Memorial Institution 1640 Medium, Gibco). All cells were
[
2
MþH] 252.1708 (calcd for C13
22 3 2
H N O
2
, 274.1531).
ꢁ
incubated at 37 C in a humidified incubator with 5% CO
2
. Cells after
74.1525 (calcd for C13
H
21
N
3
NaO
the third generation were used for experiments.
4
.7.5. N-acetyl- -isoleucine-L
L
-leucinamide (5)
4
.14. Sulforhodamine B (SRB) assay
Colorless amorphous powder; molecular formula C14
H N
27 3
O
3
;
2
5
ꢁ
[
a
]
D
ꢀ9.92 (c 0.25, MeOH); UV (MeOH)
lmax (log ε) 201 (4.07) nm;
The SRB assay12,13 was used to evaluate the activity of new
1
3
1
C NMR data (125 MHz, in DMSO‑d
6
), see Table 1, H NMR data
þ
compounds 1e5 supressing the proliferation of glioma U87MG,
U251, SHG44, and C6 cells. Doxorubicin (DOX) was used as a posi-
tive control.
(
2
(
500 MHz, in DMSO‑d
86.2124 (calcd for C14
calcd for C14 NaO
, 324.1689).
6
), see Table 2; HRESIMS m/z [MþH]
þ
H
28
N
3
O
3
, 286.2131), [MþNa] 308.1946
þ
H
27
N
3
3
, 308.1950), and [MþK] 324.1685 (calcd
3 3
for C14H27KN O
Conflict of interest
4.8. Acid hydrolysis of 5ꢀ11
The authors declare no conflict of interest.
Each (3.0 mg) of the compounds 5e11 was dissolved in 1.2 mL
N HCl and heated at 110 C in a 25 mL round flask for 24 h. The
Acknowledgments
ꢁ
6
hydrolysate was concentrated in vacuo and then used for Marfey's
analysis.
This study was supported by the National Natural Science
Foundation of China (81273428, 81773769, and 81773587). We
appreciate Mrs. Jianyang Pan (Pharmaceutical Informatics Institute
of Zhejiang University) for performing the NMR spectrometry and
Dr. Jiyong Liu (Department of Chemistry of Zhejiang University) for
performing the X-ray diffraction analysis.
4.9. Marfey's or C3-Marfey's analysis
0
31
The Marfey's and C3-Marfey s methods were used for the
analysis of amino acids. Detailed procedure and results were pro-
vided as supplementary data.
Appendix A. Supplementary data
4.10. X-ray crystallographic analysis of streptopyrazinone A (1)
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.tet.2018.03.028.
X-ray diffraction analysis was carried out on an Xcalibur Atlas
Gemini Ultra diffractometer (Agilent Technologies) with Mo K
radiation (
a
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Please cite this article in press as: Chen M, et al., Streptopyrazinones AꢀD, rare metabolites from marine-derived Streptomyces sp. ZZ446,
Tetrahedron (2018), https://doi.org/10.1016/j.tet.2018.03.028