Journal of Asian Natural Products Research

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A new α-pyrone from the deep-sea actinomycete

Nocardiopsis dassonvillei subsp. dassonvillei DSM
43111(T)

Ge Zou, Xiao-Jian Liao, Qi Peng, Guo-Dong Chen, Fang-Ying Wei, Zheng-Xiong

Xu, Bing-Xin Zhao & Shi-Hai Xu

To cite this article: Ge Zou, Xiao-Jian Liao, Qi Peng, Guo-Dong Chen, Fang-Ying Wei, Zheng-
Xiong Xu, Bing-Xin Zhao & Shi-Hai Xu (2017): A new α-pyrone from the deep-sea actinomycete
Nocardiopsis dassonvillei subsp. dassonvillei DSM 43111(T), Journal of Asian Natural Products
Research, DOI: 10.1080/10286020.2017.1307186

To link to this article: http://dx.doi.org/10.1080/10286020.2017.1307186

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Journal of Asian Natural Products Research, 2017
http://dx.doi.org/10.1080/10286020.2017.1307186

A new α-pyrone from the deep-sea actinomycete Nocardiopsis

dassonvillei subsp. dassonvillei DSM 43111(T)
Ge Zoua,b#, Xiao-Jian Liaoa#, Qi Penga, Guo-Dong Chenb, Fang-Ying Weia,
Zheng-Xiong Xua, Bing-Xin Zhaoa and Shi-Hai Xua,b
a
Department of Chemistry, Jinan University, Guangzhou 510632, China; bCollege of Pharmacy, Jinan University,
Guangzhou 510632, China

ABSTRACT ARTICLE HISTORY

A new α-pyrone, nocapyrone S (1), together with five known Received 3 November 2016
compounds (2-6), were isolated from the deep-sea actinomycete Accepted 13 March 2017
Nocardiopsis dassonvillei subsp. dassonvillei DSM 43111(T). Their KEYWORDS
structures were determined by spectroscopic analyses. The absolute α-pyrone; deep-sea
configuration of 1 was established by quantum approaches. Cytotoxic actinomycete; Nocardiopsis
activity of 1 was evaluated against K562, MCF-7, SGC7901, A375, Hela, dassonvillei
and HepG2 cell lines.

1. Introduction
In recent decades, a lot of new biologically active metabolites had originated from actino-
mycetes, which had attracted great interest as significant resources in drug discovery [1].
Nocardiopsis, a non-Streptomycete genus of actinomycetes, had been reclassified into a genus
distinct from Actinomadura and Nocardia in 1976 [2]. The genus Nocardiopsis was widely
distributed in terrestrial soil, marine sediments, or associated with marine organisms, such
as corals and sponges [3]. In addition, a variety of bioactive compounds had been isolated
from Nocardiopsis, such as pyrones, macrolides, diketopiperazines, phenazines, indolactams,
and peptides, most of which exhibited a wide range of activities including cytotoxicity,
antibiosis and anti-angiogenesis, etc. [3–7].
As a part of our continuing chemical investigations on the deep-sea actinomycetes
Nocardiopsis dassonvillei subsp. dassonvillei DSM 43111(T), a new α-pyrone, nocapyrone

CONTACT  Bing-Xin Zhao  zbx840622@163.com; Shi-Hai Xu  txush@jnu.edu.cn

#
These authors contributed equally to this work.
 Supplemental data for this article can be accessed http://dx.doi.org/10.1080/10286020.2017.1307186.
© 2017 Informa UK Limited, trading as Taylor & Francis Group
2   G. ZOU ET AL.

Figure 1. Chemical structure of 1.

S (1), along with five known compounds (2-6), have been isolated (Figure 1). Herein, we
report the isolation, structural elucidation, and biological activities of 1.

2.  Results and discussion

Nocapyrone S (1) was obtained as light yellow oil, with [𝛼]25 D −31.9 (c 0.1, MeOH). The
molecular formula of 1 was established as C13H20O4 by a quasi-molecular ion peak at m/z
241.1432 [M  +  H]+ in its HR-ESI-MS. The UV absorption maxima at 220 and 300  nm
corresponded to an α-pyrone chromophore [3]. The IR bands indicated the presences of
hydroxyl (3462  cm−1) and carbonyl (1716  cm−1) groups. The 1H and 13C NMR spectra
exhibited the presence of a carbonyl (δC 163.0), two trisubstituted double bonds [δH 7.09
(1H, d, J = 6.6 Hz) and 6.36 (1H, d, J = 6.6 Hz); δC 164.6, 140.1, 126.2, and 101.8], an oxygen
quaternary carbon (δC 75.3), two methines [δH 4.00 (1H, q, J = 6.3 Hz) and 1.96 (1H, m);
δC 72.2 and 26.8], a methylene [δH 2.29 (2H, d, J = 7.2 Hz); δC 39.7], and four methyls [δH
1.54 (3H, s), 1.12 (3H, d, J = 6.3 Hz), and 0.91 (6H, d, J = 6.6 Hz); δC 23.8, 22.3, 22.3, and
17.8]. All above information suggested that 1 should possess an α-pyrone moiety. The
1
H-1H COSY, HSQC, and HMBC spectra of 1 allowed the full assignment of all protons
and carbon signals (Table 1).
The 1H-1H COSY data of 1 revealed the presence of three spin coupling systems in bold
as shown in Figure 2. Among them, the spin system (C-11 to C-13/C-14) revealed the
existence of an isobutyl group (1b). In the HMBC spectrum, correlations between H-4
and C-2/C-6 as well as between H-5 and C-3 allowed the establishment of 2-pyrone moiety
(1a). Furthermore, the HMBC correlations between H-9 and C-7 as well as between H-8
and C-10 verified the skeleton of sec-butyl group. Additionally, according to the molecular
formula information and the obvious downfield shifts at C-7 and C-8, the remaining two
hydroxyl groups should be attached to C-7 and C-8, respectively. Thus, the planar structure
of 1c was deduced. Moreover, the HMBC correlations of H-11 to C-2/C-4 and H-8/H-10
to C-6 suggested that the fragments 1b and 1c were linked to C-3 and C-6 of the 2-pyrone
nucleus (1a), respectively.
Though the NOE correlations between H-8/H-9 and H-10 had been observed in the
NOESY spectrum, the relative stereochemistry of 1 could not be confirmed as C-7 and C-8
were ortho-position. As two chiral centers (C-7 and C-8) were found in 1, 1 should possess
two possible relative configurations, 1A (7S*, 8R*) and 1B (7S*, 8S*) (Figure 3). In order to
determine the relative stereochemistry of 1, we calculated the theoretical 13C NMR data of
1A and 1B using Gaussian 09 software, respectively [8,9], which were compared with the
experimental one. According to the results (Figure 4), 1A should be excluded due to the
great relative chemical shift error (up to 14.2 ppm) at C-6. Thus, the structure with relative
configuration of 1 was determined to be 1B, which contained two potential absolute config-
urations, 7S, 8S and 7R, 8R. In order to further confirm the absolute configuration of 1, the
 JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH   3

Table 1. 1H and 13C NMR spectral data of 1 (in CDCl3, δ, J in Hz).
No. δH δC
2 – 163.0
3 – 126.2
4 7.09 d (6.6) 140.1
5 6.36 d (6.6) 101.8
6 – 164.6
7 – 75.3
8 4.00 q (6.3) 72.2
9 1.12 d (6.3) 17.8
10 1.54 s 23.8
11 2.29 d (7.2) 39.7
12 1.96 m 26.8
13 0.91 d (6.6) 22.3
14 0.91 d (6.6) 22.3

Figure 2. Key 1H-1H COSY and HMBC correlations of 1.

Figure 3. Two possible relative configurations of 1.

quantum chemical CD calculation method was used. The overall predicted CD spectra of
(7S, 8S)-1 and (7R, 8R)-1 were subsequently compared with the experimental one (Figure 5).
The calculated CD curve of (7S, 8S)-1 revealed a good agreement with the measured one.
Consequently, the absolute structure of 1 was deduced to be 7S and 8S.
The five known compounds were identified as (4-aminophenyl)acetic acid (2), N-(2-
hydroxyphenyl)-acetamide (3), cyclo-(L-Pro-L-Val) (4), cyclo-(L-Pro-L-Leu) (5), and
4   G. ZOU ET AL.

Figure 4.  The relative errors between the calculated 13C chemical shifts of two potential structures
(1A and 1B) and recorded 13C NMR data of 1.

6
exptl for 1
calcd for (7R,8R)-1
4 calcd for(7S,8S)-1

-2

-4

-6
200 300 400
Wavelength(nm)

Figure 5. Calculated and experimental CD spectra of 1.

cyclo-(L-Pro-L-Ile) (6) by comparing their physical and spectroscopic data with those
reported in literatures [10–13]. In addition, cytotoxic activity of 1 was evaluated against
K562, MCF-7, SGC7901, A375, Hela, and HepG2 cell lines. However, no inhibitory activities
were found on the growth of these cell lines.

3. Experimental
3.1.  General experimental procedures
Optical rotation was measured using Anton Paar MCP-500 polarimeter (Anton Paar, Graz,
Austria). UV spectrum was obtained on Shimadzu UV-2401PC spectrometer (Shimadzu,
Kyoto, Japan). IR spectrum was measured on IR Affinity-1 spectrometer (Shimadzu, Kyoto,
Japan). HR-ESI-MS was acquired from Agilent 6210 LC/MSD TOF mass spectrometer.
 JOURNAL OF ASIAN NATURAL PRODUCTS RESEARCH   5

NMR spectra were measured on Bruker Av 300 MHz NMR spectrometer (Bruker, Fällanden,
Switzerland). CD spectrum was measured on Chirascan circular dichroism spectrometer
(JASCO International Co. Ltd., Hachioji, Tokyo, Japan). Semi-preparative HPLC [Rp-C18:
10 × 250 mm i.d, 4 μm (Phenomenex, America)] was performed on an Agilent 1200 series
apparatus (Agilent, Palo Alto, American). Column chromatography was performed with
silica gel (200–300 mesh, Qingdao Haiyang Chemical Co., Qingdao, China) and Sephadex
LH-20 (GE Healthcare, Uppsala, Sweden). Thin-layer chromatography was carried out with
precoated silica gel plates (GF-254, Jiangyou Silica Gel Development, Inc., Yantai, China).

3.2.  Actinomycete material

The marine actinomycete Nocardiopsis dassonvillei subsp. dassonvillei DSM 43111(T) was
isolated from the deep ocean sediment taken at the Arctic ocean (75º00.507′N, 162º01.744′W,
−2042 m in depth), provided by professor Yong Yu (Polar Research Institute of China).
A voucher strain of this actinomycete (No. S02F) was preserved at the Department of
Chemistry, Jinan University.

3.3.  Fermentation, extraction, and isolation

Spores were directly inoculated into 500 ml Erlenmeyer flasks containing 150 ml of fer-
mentation media (yeast extract 1.0 g, soluble starch 1.0 g, malt extract 15.0 g, and marinum
salt 5.0 g, dissolved in 500 ml of tap water, pH 7.2). The flasks were incubated at 18°C on a
rotatory shaker at 180 rpm for 13 days. The 15 L of whole broth was extracted three times
with EtOAc (20 L each) and evaporated to give the extract (8.5 g) The EtOAc extract (8.5 g)
was subjected to silica gel column chromatography eluting with the gradient solution of
PE (petroleum ether)/EtOAc (90:10–0:100) to give five fractions (Fr. 1-5). Fr. 4 (420 mg)
was then subjected to Sephadex LH-20 (CHCl3/MeOH, 1:1) to provide three subfractions
(Fr. 4-1–Fr. 4-3). Fr. 4-2 (60 mg) was further purified by semi-preparative reversed-phase
HPLC (2 ml/min, MeOH/H2O = 30:70) to give 1 (4.5 mg) (tR = 12.4 min). Fr. 4-3 (80 mg)
was purified by semi-preparative reversed-phase HPLC (2 ml/min, MeOH/H2O = 25:75)
to give 4 (5.0 mg) (tR = 18.5 min) and 5 (3.0 mg) (tR = 28.4 min). Fr. 3 (600 mg) was further
purified by silica gel column chromatography eluting with PE/EtOAc in gradient eluent
(90:10 – 0:100) to give four subfractions (Fr. 3-1–Fr. 3-4). Fr. 3-1 (75 mg) was purified by
semi-preparative reversed-phase HPLC (2 ml/min, MeOH/H2O = 35:65) to give 2 (5.0 mg)
(tR = 9.7 min). Fr. 3-2 (50 mg) was applied to Sephadex LH-20 (CHCl3/MeOH, 1:1) to yield
6 (3.0 mg). Fr. 3-4 (45 mg) was applied to Sephadex LH-20 (CHCl3/MeOH, 1:1) to yield
3 (4.0 mg).

3.3.1. Compound 1
D −31.9 (c 0.1, MeOH); UV (MeOH) λmax: 220, 300  nm; IR (KBr)
Light yellow oil; [𝛼]25
νmax: 3462, 2957, 2870, 1716, 1576, 1464, and 1084  cm−1; CD (MeOH): Δε227  nm  +1.22,
Δε303 nm −4.64 nm; 1H and 13C NMR spectral data, see Table 1; HR-ESI-MS: m/z 241.1432
[M + H]+ (calcd. for C13H21O4, 241.1434).
6   G. ZOU ET AL.

3.4.  Cytotoxic activity

Cytotoxic activity of 1 was evaluated against K562, MCF-7, SGC7901, A375, Hela, and
HepG2 cell lines by using MTT method [14–16]. Briefly, fresh K562, MCF-7, SGC7901,
A375, Hela, and HepG2 cells (2.5 × 104 cells/ml) were cultured in 96-well plates and incu-
bated at 37°C for 12 h in a humidified atmosphere at 5% CO2 and 95% air. The solutions
of 1 were added to the wells and the plates were successively incubated for 48 h under the
same conditions. Then, 20 μl of MTT was added to each well. After incubation at 37 °C for
4 h, all the supernatant was removed and 100 μl of dimethyl sulfoxide (DMSO) was added to
each well and incubated for another 20 min. The absorbance of each well was then measured
at 570 nm using a Thermo scientifc Multiskan FC multiplate photometer (Waltham, MA,
uSA). The IC50 values were calculated based on the dose-response curves.

Acknowledgments
The Super-Computational-Center in CAS (KIB) is also appreciated.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was financially supported by the National Natural Science Foundation of China [grant
number 41376155, grant number 21672084, grant number 41506156, grant number 81302665]; the
Natural Science Foundation of Guangdong Province [grant number 2016A030310095]; the Project
of Pearl River Nova Program of Guangzhou [grant number 201710010088]; the Scientific Research
Cultivation and Innovation Fund Research Project of Jinan University [grant number 21616113];
and the Fundamental Research Funds for the Central Universities [grant number 11616113, grant
number 11615403].

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