α-Pyrones and diketopiperazine derivatives from the marine-derived actinomycete nocardiopsis dassonvillei hr10-5

Article abstract of DOI:10.1021/np200597m

Three new α-pyrones, nocapyrones E-G (1-3), and three new diketopiperazine derivatives, nocazines -C (4-6), together with a new oxazoline compound, nocazoline A (7), were isolated from the marine-derived actinomycete Nocardiopsis dassonvillei HR10-5. The

Full text of DOI:10.1021/np200597m

Article
pubs.acs.org/jnp
α-Pyrones and Diketopiperazine Derivatives from the Marine-
Derived Actinomycete Nocardiopsis dassonvillei HR10-5
Peng Fu,† Peipei Liu,† Haijun Qu,‡ Yi Wang,† Dianfeng Chen,† Hui Wang,† Jing Li,† and Weiming Zhu*,†
†Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy, Ocean University of China,
Qingdao 266003, People’s Republic of China
‡Affiliated Hospital of Medical College of Qingdao University, Qingdao 266003, People’s Republic of China
S
* Supporting Information
ABSTRACT: Three new α-pyrones, nocapyrones E−G (1−3), and
three new diketopiperazine derivatives, nocazines A−C (4−6), together
with a new oxazoline compound, nocazoline A (7), were isolated from
the marine-derived actinomycete Nocardiopsis dassonvillei HR10-5. The
new structures of 1−7 were determined by spectroscopic analysis, X-ray
single-crystal diffraction, CD spectra, and modified Mosher and Marfey
methods. Compounds 1−3 showed modest antimicrobial activity
against Bacillus subtilis with MIC values of 26, 14, and 12 μM,
respectively.
ith more than 13 000 bioactive metabolites described,
cultured bacteria have been a prolific resource for drug
three new α-pyrones (1−3), three new diketopiperazine
derivatives (4−6), and a new oxazoline compound (7).
These compounds were inactive against HL-60 and A549
tumor cell lines, but 1−3 display modest antimicrobial activity
against B. subtilis with MIC values of 26, 14, and 12 μM,
respectively.
W
discovery.1 About 70% of these bioactive compounds have been
isolated from cultured actinomycetes. The actinomycetes are
best known as soil bacteria; thus, the majority of microbial drug
discovery research has focused on terrestrial actinomycetes.2
Unfortunately, beginning in the late 1980s, the rate of discovery
of new drug candidates from terrestrial actinomycetes began to
decrease,3 which led many pharmaceutical researchers to
abandon terrestrial actinomycetes in favor of alternate sources
of chemical diversity such as the marine-derived actino-
mycetes.4−9 The Yellow River delta located in the north of
the Shandong peninsula is a typical coastal wetland, which
possesses a complex ecosystem and rich biodiversity due to the
interaction of land and sea.10,11 Although the microorganisms
in this special environment might be an important source for
structurally new and bioactive compounds, very little research
on secondary metabolites from these microbes has been
undertaken. In order to search for new bioactive compounds,
marine sediment samples were collected from the estuary of the
Yellow River. From one sediment sample, the actinomycete
strain HR10-5, which was identified as Nocardiopsis dassonvillei,
was isolated and selected for chemical study because its
secondary metabolites showed antibacterial activity against
Bacillus subtilis at a concentration of 100 μg/mL. Other strains
of N. dassonvillei had been reported to show antimicrobial
activity12 and produce a macrolide antibiotic.13 A series of
metabolites contained in the extract of strain HR10-5 showed
UV absorptions similar to those of α-pyrones such as
gibepyrones A−F by HPLC-UV analysis at 233 and 328
nm.14 Additionally, α-pyrones are known to have antimicrobial
activity.14 Chemical investigation resulted in the isolation of
Nocapyrone E (1) gave an HRESIMS peak at m/z 193.1237
[M + H]+, corresponding to the molecular formula C12H16O2.
Its IR absorption at 1708 cm−l and UV peaks at λmax 233 and
328 nm are in accordance with an extended α-pyrone
chromophore.14 Analysis of the 1D NMR data for 1 revealed
one carbonyl, three quaternary carbons, two methylenes, and
1
three methyls (Table 1). The H NMR spectrum showed two
signals at δH 6.09 (d, J = 6.6 Hz) and 7.10 (d, J = 6.6 Hz), due
to two vicinal protons in a 3,6-disubstituted α-pyrone system.
Received: July 18, 2011
Published: September 29, 2011
© 2011 American Chemical Society and
American Society of Pharmacognosy
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1
Table 1. H and 13C NMR Data for 1−3 and 7 (600 and 150 MHz, CDCl3, TMS, δ in ppm)
1
2
3
7
position
δC
δH (J, Hz)
δC
δH (J, Hz)
δC
δH (J, Hz)
δC
δH (J, Hz)
1
2
3
4
5
6
7
8
9
110.5, C
159.8, C
163.1, C
128.6, C
163.6, C
123.1, C
162.8, C
129.8, C
116.8, CH 6.99, dd (8.2, 1.1)
133.7, CH 7.36, dt (8.2, 1.1)
118.9, CH 6.86, dt (7.7, 1.1)
128.3, CH 7.64, dd (8.3, 1.6)
166.9, C
138.4, CH 7.10, d (6.6)
100.8, CH 6.09, d (6.6)
159.7, C
140.1, CH 7.12, d (7.1)
100.8, CH 6.06, d (7.1)
159.9, C
138.0, CH 7.11, d (7.1)
102.1, CH 6.17, d (7.1)
158.8, C
125.4, C
125.4, C
126.6, C
135.4, CH 6.54, dt (7.4, 1.1) 135.5, CH 6.54, t (7.4)
135.9, CH 6.50, d (8.2)
21.9, CH2 2.23, dq (7.4,
7.4)
22.0, CH2 2.23, dq (7.4,
7.4)
64.9, CH
4.72, dq (8.1,
6.4)
67.0, CH
4.46, m
10
11
12
13.6, CH3 1.06, t (7.4)
12.4, CH3 1.85, s
13.6, CH3 1.06, t (7.7)
12.3, CH3 1.85, s
23.4, CH3 1.34, d (6.4)
12.7, CH3 1.62, s
68.7, CH2 4.44, dd (9.9, 1.8); 4.33, t (6.1)
23.5, CH2 2.49, q (7.1)
23.6, CH2 2.50, q (7.1)
64.0, CH2 3.86, dd (11.5, 3.3); 3.68, dd (11.6,
3.8)
13
12.2, CH3 1.17, t (7.1)
16.7, CH3 2.09, s
12.4, CH3 1.18, t (7.1)
2-OH
11.96, brs
The 1H−1H COSY and HMBC correlations (Figure 1) further
supported that a α-pyrone nucleus, a pent-2-en-2-yl residue,
were almost the same as those of nocapyrone E (1), except for
differences in the pentenyl unit. The lack of methylene group
signals at δH/δC 2.23/21.9 and an additional oxygenated
methine group signal at δH/δC 4.72/64.9 indicated that
compound 3 was the hydroxylated derivative of 1 at C-9.
This was confirmed by the downfield shifts of CH3-10 and C-7.
The NOESY correlation between H-9 and H-11 revealed the E
configuration of the pentenyl moiety in 3. The absolute
configuration of C-9 was determined as S by the modified
Mosher method.15 When it was reacted with (R)- and (S)-
MTPA chloride, compound 3 gave the corresponding (S)- and
(R)-MTPA esters 3a,b, respectively. The observed chemical
shift differences ΔδS−R (Figure 1) clearly defined the S
configuration. Therefore, nocapyrone G (3) was identified as
(S,E)-3-ethyl-6-(4-hydroxypent-2-en-2-yl)-2H-pyran-2-one.
Nocazine A (4) was obtained as colorless needles with the
molecular formula C22H22N2O4 from the ESIMS peak at m/z
1
379.1665 [M + H]+. Its H and 13C NMR data (Table 2)
Figure 1. Selected 2D NMR correlations for 1 and 5−7 and Δδ (=δS
− δR) values for (S)- and (R)-MTPA esters of 3.
corresponded to one N-methyl group (δH/δC 3.00/35.2), three
methoxy groups (δH/δC 3.83/54.3, 3.85/54.3, and 4.04/53.3),
ten sp2 methines, seven sp2 quaternary carbons, and one
and an ethyl group were present in 1, and the pent-2-en-2-yl
and ethyl residues were linked to C-6 and C-3 of the α-pyrone
nucleus, respectively. The NOESY correlation between H-9 and
H3-11 indicated the E configuration of the pent-2-en-2-yl
residue (Figure 1). Thus, the structure of nocapyrone E (1) was
determined to be (E)-3-ethyl-6-(pent-2-en-2-yl)-2H-pyran-2-
one.
1
amidocarbonyl (δC 161.8). Two sets of coupled H NMR
signals at δH 7.18 (d, J = 8.4 Hz), 6.89 (d, J = 8.6 Hz) and δH
8.09 (d, J = 8.8 Hz), 6.93 (d, J = 8.8 Hz) revealed the presence
of two 1,4-disubstituted benzene rings that were further
identified as two 4-OCH3 phenyl units from the HMBC
correlations between 11-OCH3 and C-11 and between 18-
OCH3 and C-18. Those HMBC correlations from the olefinic
proton H-7 to C-9 and C-5, from 5-OCH3 to C-5, and from H-
14 to C-16 and amidocarboyl carbon (C-2) identified the
specific diketopiperazine core formed from two p-MeO-Phe
moieties. The proposed structure was confirmed by X-ray
crystal diffraction analysis, which further revealed the Z
configurations of both Δ6 and Δ3(14) double bonds (Figure
2). The structure of nocazine A (4) was determined as
(3Z,6Z)-5-methoxy-3,6-bis(4-methoxybenzylidene)-1-methyl-
1,6-dihydropyrazin-2(3H)-one.
The molecular formula of nocapyrone F (2) was determined
to be C11H14O2 on the basis of the HRESIMS peak at m/z
179.1073 [M + H]+, with one less CH2 than 1. The 1H and 13
C
NMR data of 2 (Table 1) were similar to those of 1, except that
a methyl signal (δH/δC 2.09/16.7) replaced the 3-ethyl signals
at δH/δC 2.49/23.5 and 1.17/12.2. In addition, C-3 and C-4
were shifted upfield and downfield, respectively. These data
indicated 2 as the C-3 methyl analogue of 1. The NOESY
correlation between H-9 and H3-11 indicated the E
configuration of the pent-2-en-2-yl residue. Nocapyrone F
(2) was accordingly elucidated as (E)-3-methyl-6-(pent-2-en-2-
yl)-2H-pyran-2-one.
Nocazine B (5) was obtained as a yellow, amorphous
powder. Its molecular formula was determined as C21H20N2O3
according to its HRESIMS peak at m/z 349.1546 [M + H]+,
with one less CH2O unit than 4. Its 1D NMR spectra were
similar to those of 4, except for the lack of a methoxy signal and
The molecular formula of nocapyrone G (3) was determined
to be C12H16O3 by HRESIMS (m/z 209.1170 [M + H]+), with
one more oxygen atom than 1. The 1H and 13C NMR data of 3
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1
Table 2. H and 13C NMR Data for 4−6 (600 and 150 MHz for 4 and 6, 500 and 125 MHz for 5, CDCl3, TMS, δ in ppm)
4
5
6
position
δC
δH (J, Hz)
3.00, s
δC
δH (J, Hz)
δC
δH (J, Hz)
1-NCH3
35.2, CH3
161.8, C
129.5, C
36.1, CH3
162.6, C
132.4, C
3.00, s
35.4, CH3
167.5, C
57.3, CH
2.90, s
2
3
4.33, m
4-NH
6.67, br s
5
155.0, C
156.7, C
164.4, C
5-OCH3
6
53.3, CH3
126.2, C
4.04, s
6.79, s
54.4, CH3
128.6, C
4.05, s
6.82, s
129.5, C
7
113.7, CH
125.9, C
115.3, CH
126.8, C
121.3, CH
125.7, C
7.03, s
8
9/13
10/12
11
129.8, CH
112.8, CH
158.3, C
7.18, d (8.4)
6.89, d (8.6)
130.9, CH
113.8, CH
159.5, C
7.19, d (8.4)
6.89, d (8.7)
131.2, CH
113.8, CH
159.9, C
7.11, d (8.8)
6.89, d (8.8)
11-OCH3
14
54.3, CH3
127.3, CH
127.3, C
3.83, s
7.24, s
55.3, CH3
127.3, CH
135.5, C
3.83, s
7.32, s
55.4, CH3
39.8, CH2
135.4, C
3.83, s
3.33, dd (13.8, 3.9); 3.03, dd (13.7, 8.8)
15
16/20
17/19
18
132.1, CH
112.7, CH
159.0, C
8.09, d (8.8)
6.93, d (8.8)
131.4, CH
128.3, CH
131.6, CH
8.10, d (8.6)
129.7, CH
127.6, CH
125.7, CH
7.24, d (7.1)
7.39, dd (7.3, 8.2)
7.31, t (7.3)
7.31, dd (7.2, 7.7)
7.22, t (7.7)
18-OCH3
54.3, CH3
3.85, s
Figure 2. Final X-ray drawing of 4.
resonances for a monosubstituted benzene nucleus replaced
those for a corresponding 1,4-disubstituted phenyl ring (Table
2). The HMBC correlations from the vinyl proton H-7 and 5-
OCH3 to C-5, from H-9/13 to C-7 and C-11, and from 11-
and 3.03/39.8, indicating that the Δ3(14) double bond was
hydrogenated. HMBC correlations from H2-14 to C-16 and C-
20 and from H-3 to C-15 established the phenylalanine (Phe)
unit, whose absolute configuration was determined as L by
Marfey’s method.16 The 1-fluoro-2,4-dinitrophenyl-5-L-alanine
amide (FDAA) derivative of the acid hydrolysates of 6 gave the
same retention time as that prepared from an authentic L-Phe
sample in HPLC analysis (Figure S37, Supporting Informa-
tion). The Z configuration of the Δ6 double bond in 6 could be
deduced from the relative downfield shift of H-7 (δ H 7.03) and
comparison with the vinyl proton chemical shifts of the 6-
benzylidene-substituted derivatives of piperazine-2,5-dione in
the literature. The (Z)-vinyl proton is farther downfield than
the (E)-vinyl proton because of the deshielding effect of the 5-
ketone in this kind of compound, such as δH‑7 7.17 in threo-
(Z)-6-benzylidene-3-(α-methoxybenzyl)-1,4-dimethylpipera-
zine-2,5-dione17,18 and δ H‑7 6.54 in (3S,6E)-3-benzyl-6-
benzylidenepiperazine-2,5-dione.19 In addition, there was no
NOESY correlation between H-7 and 1-NCH3, further
suggesting the Z configuration of the double bond in 6.
Therefore, the structure of 6 was assigned as (3S,6Z)-3-benzyl-
6-(4-methoxybenzylidene)-1-methylpiperazine-2,5-dione. It is
noted that the constitution of 6 has been previously proposed,
but without experimental data.20
1
OCH3 to C-11, combined with the H−1H COSY correlations
from H-16/20 (δH 8.10) to H-18 (δH 7.31) through H-17/19
(δH 7.39), indicated that the 18-OCH3 of 4 was missing in 5
(Figure 1). Furthermore, the Z configurations of both Δ6 and
Δ
3(14) double bonds in 5 could be deduced from the similarity
of the chemical shifts of CH-7 and CH-14 to those of 4 (δH/δC
6.82/115.3 and 7.32/127.3 vs 6.79/113.7 and 7.24/127.3,
respectively). Thus, the structure of 5 was elucidated as
(3Z,6Z)-3-benzylidene-5-methoxy-6-(4-methoxybenzylidene)-
1-methyl-1,6-dihydropyrazin-2(3H)-one.
The molecular formula for nocazine C (6) was assigned to be
C20H20N2O3 from the molecular ion peak at m/z 337.1564 [M
+ H]+ in the positive-ion HRESIMS spectrum. Its 1D NMR
spectra displayed two amidocarbonyl carbon signals of a
diketopiperazine at δC 164.4 and 167.5 and one amide proton
1
signal at δH 6.67. Careful comparison of its H and 13C NMR
spectra (Tables 2) with those of 4 showed that two methoxy
signals, 18-OCH3 and 5-OCH3, were absent. Moreover,
resonances for a trisubstituted double bond were replaced by
a methine at δH/δC 4.33/57.3 and a methylene at δH/δC 3.33
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°C. The producing strain was prepared on Gause’s synthetic agar
slants and stored at 4 °C.
Nocazoline A (7) was isolated as a yellow oil and assigned
the molecular formula C10H11NO3 on the basis of the
HRESIMS peak at m/z 194.0810 [M + H]+. The IR spectrum
showed the presence of a hydroxy group (3329 cm−1), an
aromatic ring (1617, 1492 cm−1), and an imine group (1642
Fermentation and Extraction. Spores were directly inoculated
into 500 mL Erlenmeyer flasks containing 150 mL of fermentation
media (glucose 20 g, beef extract 3 g, yeast extract 10 g, soluble starch
10 g, peptone 10 g, K2HPO4 0.5 g, MgSO4 0.5 g, CaCO3 2 g, and
marinum salt 33 g, dissolved in 1 L of tap water, pH 7.0). The flasks
were incubated on a rotatory shaker at 180 rpm at 28 °C for 8 days. At
harvest, 60 L of whole broth was extracted three times with EtOAc (90
L each). The EtOAc extract was concentrated under reduced pressure
to give a dark brown gum (30 g).
1
cm−1). The H NMR signals at δH 6.99 (dd, J = 8.2, 1.1 Hz),
7.36 (dt, J = 8.2, 1.1 Hz), 6.86 (dt, J = 7.7, 1.1 Hz), and 7.64
(dd, J = 8.3, 1.6 Hz) indicated that a 1,2-disubstituted phenyl
nucleus was present in 7. A survey of the literature revealed that
the 1H and 13C NMR spectra of 7 were very similar to those of
aerugine (8),21 except for downfield shifts of C-7 and C-10
(Table 1), showing that the sulfur of the aerugine thiazoline
was replaced by oxygen. This deduction was confirmed by
1H−1H COSY correlations of H-10/H-9/H-12 and the key
HMBC connections from H-6, H-9, and H-10 to C-7 (Figure
1). The R configuration of 7 was deduced from CD Cotton
effects similar to those for aerugine22 at 304 nm (Δεmax −0.8)
Purification. The EtOAc extract (30 g) was separated into seven
fractions on a silica gel VLC column using step gradient elution with
CH2Cl2−petroleum ether (50%−100%) and then with MeOH−
CH2Cl2 (0−50%). Fraction 1 (3.5 g) was further separated into two
subfractions by Sephadex LH-20, with MeOH−CH2Cl2 (1:2) as
eluent. Subfraction 1-2 (1.1 g) was further purified by semipreparative
HPLC (80% MeOH−H2O) to yield 2 (52 mg, tR = 7.2 min) and 1
(310 mg, tR = 8.6 min). Fraction 2 (2.4 g) was separated into four
subfractions by Sephadex LH-20, with MeOH−CH2Cl2 (1:2) as
eluent. Subfraction 2-2 (720 mg) was additionally separated by
semipreparative HPLC (85% MeOH−H2O) to yield 5 (3.9 mg, tR =
11.6 min) and 4 (14.3 mg, tR = 15.2 min). Fraction 3 (2.1 g) was
separated into three subfractions by Sephadex LH-20, with MeOH−
CH2Cl2 (1:1) as eluent. Subfraction 3-2 (670 mg) was separated by
semipreparative HPLC (65% MeOH−H2O, 4.0 mL/min) to yield 3
(12.5 mg, tR = 7.8 min) and 6 (41.0 mg, tR = 12.2 min). Subfraction 3-
3 (520 mg) was further separated by semipreparative HPLC (60%
MeOH−H2O) to yield 7 (30.3 mg, tR = 9.5 min).
and 248 nm (Δεmax +1.5). The specific rotation of 7 ([α]25
=
D
+15°) is similar to that of (R)-4-hydroxymethyl-2-phenyl-4,5-
dihydrooxazole ([α]25 = +35°)23 and has the opposite sign
D
compared to the rotation of (S)-4-hydroxymethyl-2-phenyl-4,5-
dihydrooxazole ([α]25 = −82°),24 which further supports the
D
R configuration of 7. Thus, nocazoline A (7) was identified as
(R)-2-(2-hydroxyphenyl)-4-hydroxymethyl-4,5-dihydrooxazole.
Compounds 1−7 were tested for cytotoxic effects on the
HL-60 cell line using the MTT method25 and on the A549 cell
line using the SRB method.26 These compounds did not show
significant cytotoxic activities (IC50 ≥ 100 μM). The
antimicrobial activities against Escherichia coli, Aerobacter
aerogenes, Pseudomonas aeruginosa, Bacillus subtilis, Staph-
ylococcus aureus, and Candida albicans were also evaluated by
an agar dilution method.27 Compounds 1−3 showed modest
antimicrobial activity against Bacillus subtilis with MIC values of
26, 14, and 12 μM, respectively.
Nocapyrone E (1): colorless oil; UV (MeOH) λmax (log ε) 233
(3.62), 328 (3.60) nm; IR (KBr) νmax 2973, 2937, 2879, 1708, 1638,
1
1577, 1459, 1379, 1273, 1122, 1092, 1025, 960 cm−1; H NMR and
13C NMR data, Table 1; HRESIMS m/z 193.1237 [M + H]+ (calcd
for C12H17O2 193.1229).
Nocapyrone F (2): colorless oil; UV (MeOH) λmax (log ε) 233
(3.54), 328 (3.51) nm; IR (KBr) νmax 2928, 1706, 1641, 1579, 1456,
1
1377, 1345, 1278, 1122, 1090, 994 cm−1; H NMR and 13C NMR
data, Table 1; HRESIMS m/z 179.1073 [M + H]+ (calcd for
C11H15O2 179.1072).
Nocapyrone G (3): colorless oil; [α]25 = −37° (c 0.4, CHCl3);
EXPERIMENTAL SECTION
D
■
UV (MeOH) λmax (log ε) 229 (3.89), 321 (3.84) nm; IR (KBr) νmax
General Experimental Procedures. Specific rotations were
obtained on a JASCO P-1020 digital polarimeter. UV spectra were
recorded on a Beckman DU 640 spectrophotometer. CD spectra were
measured on a JASCO J-715 spectropolarimeter. IR spectra were taken
on a Nicolet Nexus 470 spectrophotometer as KBr disks. NMR data of
1−4, 6, and 7 were recorded on a JEOL JNM-ECP 600 spectrometer
using TMS as internal standard, and chemical shifts were recorded as δ
values. NMR spectra of 5 were recorded on a Bruker Avance 500
spectrometer. ESIMS measurements were carried out on a Q-TOF
ULTIMA GLOBAL GAA076 LC mass spectrometer. Semipreparative
HPLC was performed using an ODS column (YMC-pack ODS-A, 10
× 250 mm, 5 μm, 4.0 mL/min). TLC and column chromatography
(CC) were performed on plates precoated with silica gel GF254 (10−
40 μm) and over silica gel (200−300 mesh, Qingdao Marine Chemical
Factory), and Sephadex LH-20 (Amersham Biosciences), respectively.
Vacuum-liquid chromatography (VLC) utilized silica gel H (Qingdao
Marine Chemical Factory). The marinum salt used was made from the
evaporation of seawater collected in Laizhou Bay (Weifang Haisheng
Chemical Factory).
3426, 2968, 2928, 2868, 1686, 1645, 1559, 1449, 1252, 1121, 1062,
1
1024 cm−1; H NMR and 13C NMR data, Table 1; HRESIMS m/z
209.1170 [M + H]+ (calcd for C12H17O3 209.1178).
Nocazine A (4): colorless needles (MeOH); mp 120 °C; UV
(MeOH) λmax (log ε) 242 (3.18), 371 (3.62) nm; IR (KBr) νmax 2921,
1
2851, 1664, 1601, 1508, 1463, 1253, 1175, 1102, 1033, 800 cm−1; H
NMR and 13C NMR data, Table 2; HRESIMS m/z 379.1665 [M +
H]+ (calcd for C22H23N2O4 379.1658).
Nocazine B (5): yellow, amorphous powder; UV (MeOH) λmax
(log ε) 242 (3.35), 367 (3.85) nm; IR (KBr) νmax 3409, 2919, 2851,
1
1665, 1604, 1465, 1347, 1252, 1175, 1106, 1033 cm−1; H NMR and
13C NMR data, Table 2; HRESIMS m/z 349.1546 [M + H]+ (calcd
for C21H21N2O3 349.1552).
Nocazine C (6): yellow, amorphous powder; [α]25 = −307° (c
D
1.4, CHCl3); UV (MeOH) λmax (log ε) 221 (3.66), 306 (3.83) nm; IR
(KBr) νmax 3438, 2953, 1684, 1627, 1501, 1370, 1253, 1176, 1110,
1029, 832 cm−1; 1H NMR and 13C NMR data, Table 2; HRESIMS m/
z 337.1564 [M + H]+ (calcd for C20H21N2O3 337.1552).
Actinomycete Material. The actinomycete Nocardiopsis dasson-
villei HR10-5 was isolated from marine sediments collected from the
estuary of Yellow River, Dongying, People's Republic of China. The
marine sediments (2 g) were air-dried for 15 days in a 45 mL sterile
centrifuge tube. The dried sediments were diluted to 10−3 g/mL, 100
μL of which was dispersed across a solid-phase agar plate (Gause’s
synthetic agar media) and incubated at 28 °C for 10 days. A single
colony was transferred to Gause’s synthetic agar media and was
identified according to its morphological characteristics and 16S rRNA
gene sequences (Supporting Information, GenBank accession No.
JN253591). A reference culture is maintained in our laboratory at −80
Nocazoline A (7): yellow oil; [α]25 = +15° (c 0.1, CHCl3); UV
D
(MeOH) λmax (log ε) 207 (4.05), 234 (3.98), 295 (3.70) nm; CD
(MeOH) λmax (Δε) 304 (−0.8), 248 (+1.5), 233 (+0.5), 219 (+2.6),
210 (−0.7), 199 (+2.9) nm; IR (KBr) νmax 3329, 2930, 1642, 1617,
1492, 1368, 1309, 1260, 1233, 1159, 1130, 1063, 958, 829, 756 cm−1
;
1H NMR and 13C NMR data, Table 1; HRESIMS m/z 194.0810 [M +
H]+ (calcd for C10H12NO3 194.0817).
Preparation of the (S)-and (R)-MTPA Esters of 3 by the
Modified Mosher Method. Compound 3 (2 mg) was dissolved in
500 μL of pyridine, and dimethylaminopyridine (3 mg) and (R)-
MTPACl (8 μL) were then added in sequence. The reaction mixture
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dx.doi.org/10.1021/np200597m|J. Nat. Prod. 2011, 74, 2219−2223

Journal of Natural Products
Article
was stirred for 24 h at room temperature, and 1 mL of H2O was then
added. The solution was extracted by 5 mL of CH2Cl2 and the organic
phase was concentrated under reduced pressure. Then the residue was
purified by semipreparative HPLC (83% MeOH−H2O) to yield (S)-
MTPA ester 3a (1.0 mg, tR = 9.2 min). By the same procedure, (R)-
MTPA ester 3b (1.7 mg, tR 9.2 min) was obtained from the reaction of
ACKNOWLEDGMENTS
■
This work was supported by grants from the National Natural
Science Foundation of China (Nos. 21172204, 30670219, and
30770235), from the National Basic Research Program of
China (No. 2010CB833800), from the Major Program for
Technique Development Research of New Drugs in China
(No. 2009ZX09103-046), and from the Shandong Natural
Science Foundation (No. ZR2009CQ030).
1
3 (2 mg) with (S)-MTPACl (8 μL). (S)-MTPA ester (3a): H NMR
(CDCl3, 600 MHz) δ 7.12 (1H, d, J = 7.1 Hz, H-4), 6.47 (1H, d, J =
9.4 Hz, H-8), 6.22 (1H, d, J = 7.1 Hz, H-5), 5.94 (1H, m, H-9), 2.51
(2H, q, J = 7.7 Hz, H-12), 2.00 (3H, s, H-11), 1.40 (3H, d, J = 6.6 Hz,
H-10), 1.18 (3H, t, J = 7.7 Hz, H-13); ESIMS m/z 425 [M + H]+.
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(R)-MTPA ester (3b): H NMR (CDCl3, 600 MHz) δ 7.11 (1H, d, J
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ASSOCIATED CONTENT
■
S
* Supporting Information
Text giving bioassay protocols used and 16S rRNA gene
sequences of Nocardiopsis dassonvillei HR10-5, figures giving
NMR spectra of compounds 1−7 and HPLC profiles of acidic
hydrolysate of 6, and a CIF file giving crystallographic data for
4. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +86-532-82031268. Fax: +86-532-82031268. E-mail:
weimingzhu@ouc.edu.cn.
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dx.doi.org/10.1021/np200597m|J. Nat. Prod. 2011, 74, 2219−2223