A new curvularin glycoside and its cytotoxic and antibacterial analogues from marine actinomycete Pseudonocardia sp. HS7

Article abstract of DOI:10.1080/14786419.2015.1047775

Five curvularin macrolides (1-5) were isolated from the cultured broth of marine actinomycete Pseudonocardia sp. HS7 that was obtained from the cloacal aperture of sea cucumber Holothuria moebii. The structures of these isolates were characterized as (11S,15R)-11-hydroxycurvularin (1), (11R,15R)-11-hydroxycurvularin (2), curvularin-7-O-α-D-glucopyranoside (3), trans-dehydrocurvularin (4) and curvularin (5) based on their NMR and HRESIMS data as well as chemical degradation. Compound 3 is a new macrolide with a rare α-D-glucopyranose substituent. Compounds 1-4, 5a and 5c (the acyl products of 5), suppressed the proliferation of all six tested cancer cell lines and 4 is the most active compound with IC₅₀ values ranging from 0.59 to 3.39 M. The 11-hydroxycurvularins 1 and 2 also showed antibacterial activity inhibiting the growth of Escherichia coli.

Full text of DOI:10.1080/14786419.2015.1047775

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A new curvularin glycoside and
its cytotoxic and antibacterial
analogues from marine actinomycete
Pseudonocardia sp. HS7
a
a
a
a
Xuewei Ye , Komal Anjum , Tengfei Song , Wenling Wang , Siran
a
a
b
a
Yu , Haocai Huang , Xiao-Yuan Lian & Zhizhen Zhang
a
Ocean College, Zhejiang University, Hangzhou 310058, P.R. China
College of Pharmaceutical Sciences, Zhejiang University,,
b
Hangzhou 310058, P.R. China
Published online: 29 Jun 2015.
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To cite this article: Xuewei Ye, Komal Anjum, Tengfei Song, Wenling Wang, Siran Yu, Haocai
Huang, Xiao-Yuan Lian & Zhizhen Zhang (2015): A new curvularin glycoside and its cytotoxic and
antibacterial analogues from marine actinomycete Pseudonocardia sp. HS7, Natural Product
Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1047775
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Natural Product Research, 2015
http://dx.doi.org/10.1080/14786419.2015.1047775
A new curvularin glycoside and its cytotoxic and antibacterial analogues
from marine actinomycete Pseudonocardia sp. HS7
a
a
a
a
a
a
Xuewei Ye , Komal Anjum , Tengfei Song , Wenling Wang , Siran Yu , Haocai Huang ,
b
Xiao-Yuan Lian * and Zhizhen Zhang *
a
a
b
Ocean College, Zhejiang University, Hangzhou 310058, P.R. China; College of Pharmaceutical
Sciences, Zhejiang University, Hangzhou 310058, P.R. China
(
Received 22 December 2014; final version received 23 April 2015)
Five curvularin macrolides (1–5) were isolated from the cultured broth of marine
actinomycete Pseudonocardia sp. HS7 that was obtained from the cloacal aperture of
sea cucumber Holothuria moebii. The structures of these isolates were characterized as
(
7
11S,15R)-11-hydroxycurvularin (1), (11R,15R)-11-hydroxycurvularin (2), curvularin-
-O-a-D-glucopyranoside (3), trans-dehydrocurvularin (4) and curvularin (5) based on
their NMR and HRESIMS data as well as chemical degradation. Compound 3 is a new
macrolide with a rare a-D-glucopyranose substituent. Compounds 1–4, 5a and 5c (the
acyl products of 5), suppressed the proliferation of all six tested cancer cell lines and 4
is the most active compound with IC50 values ranging from 0.59 to 3.39 mM. The 11-
hydroxycurvularins 1 and 2 also showed antibacterial activity inhibiting the growth of
Escherichia coli.
Keywords: Pseudonocardia sp. HS7; curvularin glycoside; cytotoxic; antibacterial
activity
1
. Introduction
Gliomas are one of the most challenging cancers to treat and account for 80% of all malignant
brain tumours. Chemotherapy is an important adjunctive therapy for treating gliomas and
temozolomide (TMZ) is the only drug that has been independently used for the treatment
of gliomas. The efficacy of the currently used anticancer drugs including TMZ is limited
(
Chamberlain 2010; Patil et al. 2013). There is therefore a need to discover lead compounds for
the development of novel anti-glioma drugs. Natural products are highly significant sources of
new drug leads (Newman & Cragg 2012).
*
Corresponding authors. Email: xylian@zju.edu.cn; zzhang88@zju.edu.cn
q 2015 Taylor & Francis

2
X. Ye et al.
OH
OH
OH
OH
OH
O
5
HO
OH
O
HO
O
H
O
O
HO
O
O
HO
HO
7
OH
O
O
O
O
O
OH
O
1
O
O
O
1
5
O
R
11
3a
4
1
2
.R=α-OH
.R=β-OH
3
OH
OH
O
O
O
O
O
O
O
O
O
O
HO
O
O
O
HO
O
O
O
O
O
O
O
5
5a
5b
5c
Figure 1. Structures of compounds 1–5, 3a and 5a–5c.
During the course of our ongoing programme for the discovery of novel anti-glioma agents
from natural sources (Xin et al. 2012; Ye et al. 2014; Yu, Ye, Chen et al. 2014; Yu et al. 2015;
Yu, Ye, Xin et al. 2014), the cultured broth of marine actinomycete strain HS7 isolated from the
cloacal aperture of sea cucumber Holothuria moebii was found to be active against the
proliferation of glioma cells. The strain HS7 was identified by 16S rDNA sequence analysis and
its top16S rDNA sequence was 100% sequence match to Pseudonocardia antitumoralis strain
SCSIO 01299. This actinomycete strain SCSIO 01299 was recently isolated as a new species
from deep-sea sediment collected from the northern South China (Tian et al. 2013). Previously
chemical investigation indicated that strain SCSIO 01299 produced deoxynyboquinone and
three new diazaanthraquinone derivatives of pseudonocardians A, B and C. These
diazaanthraquinones were shown to have potent cytotoxic and antibacterial activities (Li et al.
2
011).
In this study, chromatographic separation of an active EtOAc extract prepared from the
cultured actinomycete strain HS7 afforded five curvularin analogues (1–5, Figure 1). Three acyl
products (5a–5c) of curvularin (5) were also prepared for bioactivity assay. Compound 3 is a
new curvularin macrolide glycoside with a rare a-D-glucopyranose substituent and 5a is a new
synthetic compound. We herein report the isolation and structural elucidation of these
compounds and their activities inhibiting the proliferation of cancer cells and the growth of
Staphylococcus aureus and Escherichia coli.
2
. Results and discussion
Marine actinomycete strain HS7 was isolated from the cloacal aperture of sea cucumber
H. moebii. The taxonomic identity of this isolated actinomycete was determined by 16S rDNA
sequence analysis. The top sequence of HS7 was 100.0% sequence similarity to P. antitumoralis
strain SCSIO 01299 (accession number: NR_109460.1) and 99% sequence similarity to other
nine Pseudonocardia sp. strains Table S1) in the GenBank database. Therefore, the taxonomy of
actinomycete HS7 was proposed to be Pseudonocardia sp. HS-7.
An EtOAc extract obtained from the cultured broth of strain HS7 showed inhibitory activity
against glioma cells. This active EtOAc crude was separated by HPLC to afford compounds 1–5.

Natural Product Research
3
Based on NMR and HRESIMS spectral analyses and comparison with published NMR data,
compounds 1, 2, 4 and 5 were identified as (11S,15R)-11-hydroxycurvularin (1), (11R,15R)-11-
hydroxycurvularin (2), trans-dehydrocurvularin (4) and curvularin (5) (Lai et al. 1989; Greve
et al. 2008; Dai et al. 2010).
Compound 3 had a molecular formula of C H O deduced from its HRESIMS at m/z
2
2 30 10
þ
13
[M þ Na] 477.1730 (calcd for C H NaO , 477.1737). The C NMR spectrum of 3
exhibited 22 carbon signals, of which 16 were assigned to the aglycone and the remaining six
2
2
30
10
to a sugar moiety. The 16 carbons (in acetone-d ) of aglycone included two carbonyls (d 207.0,
6
C-9; d 171.1, C-1), six aromatic carbons (d 160.0, C-5; d 157.5, C-7; d 136.1, C-3; d 124.3,
C-8; d 113.7, C-4; d 102.6, C-6), one oxymethine (d 73.0, C-15), six methylenes (d 44.7, C-10;
d 39.2, C-2; d 33.2, C-14; d 27.9, C-12; d 25.2, C-13; d 23.6, C-11) and one methyl (d 20.8,
C-16). These carbon signals were almost the same as those (Table S2) of the aglycone of
compound 3a (curvularin-7-O-b-D-glucopyranoside) (Zhan & Gunatilaka 2005), implying that
3
and 3a shared a same aglycone and had a same glycosylated position at C-7, which was
further supported by big different chemical shifts (Dd 1.5 ppm for C-7 and Dd 3.0 ppm for C-8)
between 3 and 5. Acid hydrolysis of 3 produced its aglycone. The aglycone and curvularin (5)
had a same HPLC retention time and very close negative optical rotation values, suggesting
that both compounds had a same configuration at C-15 (Dai et al. 2010; Lai et al. 1989).
Therefore, the aglycone of 3 was proved to be curvularin (5). Further comparison of the NMR
data of 3 with those of 3a included that the structural difference between 3 and 3a was their
3
sugar part. The anomeric proton signal of sugar in 3 resonated at d 5.49 with a small J
H
H1,H2
coupling constant (3.1 Hz), which was quite different from its counterpart at d 4.94 with a
H
1
larger coupling constant (7.6 Hz). The C NMR data of the sugar in 3 resonated at d 99.5
3
0 0 0 0
(
C-1 ), 73.1 (C-2 ), 74.8 (C-3 ) and 75.0 (C-5 ), which were also quite different from their
0
0
0
counterparts of 3a at d 102.3 (C-1 ), 74.6 (C-2 ), 77.8 (C-3 ) and 78.2 (C-5) (Table S2). The
foregoing evidence suggested the present of a-glucopyranose in 3 (Bock & Pederson 1983;
Yamamoto et al. 2002). Acid hydrolysis of 3 furnished D-glucose as detected by GC analysis.
The structure of 3 was thus assigned as curvularin-7-O-a-D-glucopyranoside, a new macrolide
glycoside.
Three synthetic derivatives of 7-acetyl-curvularin (5a), 5-acetyl-curvularin (5b) and 5,7-
diacetyl-curvularin (5c) (Elzner et al. 2008) were made for bioactive assay. The activity of
compounds 1–5 and 5a–5c inhibiting the proliferation of six cell lines of glioma C6, U87-MG,
SHG-44, U251 and colorectal cancer HCT-15 and SW620 was determined by Sulforhodamine B
(
SRB) assay (Xin et al. 2012; Yu, Ye, Chen et al. 2014; Yu et al. 2015; Yu, Ye, Xin et al. 2014).
Doxorubicin (DOX, one of the most potent of the chemotherapeutic drugs) (Tacara et al. 2013)
was used as positive control. The data (Table 1) indicated that compounds 1, 2 and4 had good
activity with IC50 , 11.0 mM for most tested cell lines with 4 being the most active compound
Table 1. Activity of compounds 1–5 and 5a–5b inhibiting the proliferation of cancer cells (IC50: mM).
Compounds
C6
U87-MG
SHG-44
U251
HCT-15
SW620
1
2
3
4
5
5.95 ^ 0.96 3.04 ^ 0.14 20.30 ^ 0.48 5.25 ^ 0.19 2.66 ^ 0.14 2.20 ^ 0.13
3.16 ^ 0.10 3.27 ^ 0.03 31.90 ^ 1.92 10.86 ^ 0.28 2.02 ^ 0.09 2.39 ^ 0.20
44.47 ^ 4.36 81.01 ^ 4.54 43.66 ^ 0.10 32.15 ^ 2.41 23.29 ^ 0.96 20.84 ^ 0.28
0.59 ^ 0.12 2.40 ^ 0.19 1.99 ^ 0.20 3.39 ^ 0.73 2.47 ^ 0.34 0.85 ^ 0.12
.100
4.21 ^ 0.47 9.98 ^ 0.60 73.74 ^ 7.05 13.47 ^ 0.48 17.67 ^ 0.28 2.58 ^ 0.09
2.44 ^ 0.25 47.92 ^ 2.10 .100 .100 20.86 ^ 0.33 6.97 ^ 0.51
.100
.100
.100
.100
71.30 ^ 2.89
5a
5b
5c
49.61 ^ 1.00 24.18 ^ 1.79 16.32 ^ 1.48 25.15 ^ 2.31 21.99 ^ 0.95 8.35 ^ 0.52
0.96 ^ 0.07 1.35 ^ 0.01 4.64 ^ 0.16 2.26 ^ 0.07 1.13 ^ 0.08 1.33 ^ 0.06
DOX

4
X. Ye et al.
(
IC : 0.59–3.39 mM). The new compound 3 and the synthetic compounds 5a and 5c also
5
0
displayed activity against all the six cancer cell lines with IC50 values ranging from 2.58 to
1.01 mM. Compound 5b only showed activity against C6, U87-MG, HCT-15 and SW620,
8
while curvularin (5) had no activity against the tested cell lines. The positive control DOX had
activity against all six ed tumor cells wtestith IC50 values of 0.96–4.64 mM. It was noted that,
compared to curvularin (5), the activity of compounds 1–4 and 5a–5c was significantly
enhanced because of the presence of a hydroxyl group at C-11 (1 and 2), a sugar group at C-7 (3)
and the acetyl groups at C-5 and C-7 (5a–5c). The result from this study supports the previous
reports that acyl groups are for the activity of some cytotoxic compounds (Chan 2007; Zhang &
Li 2007; Wang et al. 2010; Ye et al. 2014).
The compounds (1–5, 5a–5c) were also assayed for their activity against S. aureus and
E. coli. The antibacterial activity of compounds was initially tested at 100 mg/mL by spot
method (Supplementary material). Only compounds 1 and 2 showed activity inhibiting the
growth of E. coli. None of tested compounds was active against S. aureus. The MIC (minimum
inhibitory concentration) and MBC (minimum bactericidal concentration) of the active
compounds 1 and 2 were further determined by micro broth dilution method (Supplementary
material) and norfloxacin was used as positive control. The results indicated that both 1 and 2
had activity against E. coli with an MIC value of 20 mg/mL and an MBC value of 30 mg/mL. The
positive control drug norfloxacin had activity against E. coli with MIC 1.2 mg/mL.
Curvularin macrolides were previously found in fungi mainly from genus Curvularia (Greve
et al. 2008) and Penicillium (Meng et al. 2013). This type of macrolides was reported to be
cytotoxic towards tumour cells (He et al. 2004; Greve et al. 2008; Meng et al. 2013) and also
inhibit the growth of fungal and bacterial organisms (Dai et al. 2010). In the current study,
several curvularin macrolides including a new curvularin macrolide glycoside (3) were isolated
from marine actinomycete strain HS7. This new macrolide and other isolates were shown to be
active against the proliferation of different cancer cell lines, further confirming the antitumour
property of this type of macrolides. (11S,15R)-11-hydroxycurvularin (1) and (11R,15R)-11-
hydroxycurvularin (2) were found to have antibacterial activity inhibiting the growth E. coli.
To the best of our knowledge, this type of curvularin macrolides is isolated from a bacterial
source for the first time.
3
. Experimental
Experimental section was supplied as online Supplementary material.
4
. Conclusions
A marine actinomycete strain HS7 was isolated from the cloacal aperture of sea cucumber
H. moebii. This strain was identified by 16S rDNA sequence analysis as Pseudonocardia
sp. HS7. Five curvularin macrolides (1–5) and three curvularin acyl products (5a–5c) of
compound 5 were obtained in this study. The structures of these compounds were assigned as
(
11S,15R)-11-hydroxycurvularin (1), (11R,15R)-11-hydroxycurvularin (2), curvularin-7-O-a-D-
glucopyranoside (3), trans-dehydrocurvularin (4), curvularin (5), 7-acetyl-curvularin (5a), 5-
acetyl-curvularin (5b), 5,7-diacetyl-curvularin (5c) mainly based on their NMR and HRESIMS
spectral analyses as well as the published NMR data comparison. Compound 3 is a new
macrolide glycoside with a rare a-D-glucopyranose substituent and 5a is a new synthetic acetyl-
curvularin. New compounds (3 and 5a) and others (1, 2, 4, and 5c) showed to inhibit the
proliferation of all tested cancer cell lines, confirming the antitumour property of this type of
macrolides. (11S,15R)-11-hydroxycurvularin (1) and (11R,15R)-11-hydroxycurvularin (2) were
found to have activity against the growth of E. coli for the first time.

Natural Product Research
5
Supplementary material
Supplementary material relating to this paper is available online.
Acknowledgements
Authors also thank Mrs. Jianyang Pan at Pharmaceutical Informatics Institute of Zhejiang University for
performing NMR spectrometry for structure elucidation.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by grants from the National Science Foundation of China [grant number
8
2
1273428] and the Cross-Disciplinary Research for Ocean Science of Zhejiang University [grant number
012HY018B]. We greatly appreciate Drs. Zhongjun Ma and Pinmei Wang at Ocean College of Zhejiang
University for donating samples of Staphylococcus aureus and Escherichia coli.
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