Aspernigrins with anti-HIV-1 activities from the marine-derived fungus Aspergillus Niger SCSIO Jcsw6F30

Article abstract of DOI:10.1016/j.bmcl.2015.12.005

Two new 2-benzylpyridin-4-one containing metabolites, aspernigrins C (3) and D (4), together with six known compounds (1, 2, and 5-8), were isolated from the marine-derived fungus Aspergillus Niger SCSIO Jcsw6F30. The structures of the new compounds were determined by NMR, MS, and optical rotation analyses. All the isolated compounds were evaluated for their inhibitory activities against infection with HIV-1 SF162 in TZM-bl cells. Malformin C (5) showed the strongest anti-HIV-1 activity with IC₅₀ of 1.4 ± 0.06 μM (selectivity index, 11.4), meanwhile aspernigrin C (3) also exhibited potent activity with IC₅₀ of 4.7 ± 0.4 μM (selectivity index, 7.5).

Full text of DOI:10.1016/j.bmcl.2015.12.005

Accepted Manuscript
Aspernigrins with Anti-HIV-1 Activities from the Marine-Derived Fungus As-
pergillus niger SCSIO Jcsw6F30
Xuefeng Zhou, Wei Fang, Suiyi Tan, Xiuping Lin, Tianrong Xun, Bingjie Yang,
Shuwen Liu, Yonghong Liu
PII:
S0960-894X(15)30324-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.12.005
BMCL 23366
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
7 September 2015
11 November 2015
4 December 2015
Please cite this article as: Zhou, X., Fang, W., Tan, S., Lin, X., Xun, T., Yang, B., Liu, S., Liu, Y., Aspernigrins
with Anti-HIV-1 Activities from the Marine-Derived Fungus Aspergillus niger SCSIO Jcsw6F30, Bioorganic &
Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.12.005
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Aspernigrins with Anti-HIV-1 Activities from the
Marine-Derived Fungus Aspergillus niger SCSIO Jcsw6F30
a,†
b,†
c,†
a
c
Xuefeng Zhou , Wei Fang , Suiyi Tan , Xiuping Lin , Tianrong Xun , Bingjie
a,c
c,
a,
Yang , Shuwen Liu * and Yonghong Liu *
a
CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong
Key Laboratory of Marine Materia Medica, South China Sea Institute of
Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China;
b
Center of Bio-pesticide Engineering Research, Hubei Academy of Agricultural
Science, Wuhan 430064, China;
c
Guangdong Provincial Key Laboratory of New Drug Screening, School of
Pharmaceutical Science, South Medical University, Guangzhou 510515, China.
†
These authors contributed equally to this paper.
*
Authors to whom correspondence should be addressed; E-Mails:
yonghongliu@scsio.ac.cn (Y. L.); liusw@smu.edu.cn (S. L.); Tel./Fax:
+86-20-89023244 (Y. L.); +86-20-61648538 (S. L.).
1

Abstract:
Two new 2-benzylpyridin-4-one containing metabolites, aspernigrins C (3) and D (4),
together with six known compounds (1, 2, and 5-8), were isolated from the
marine-derived fungus Aspergillus niger SCSIO Jcsw6F30. The structures of the new
compounds were determined by NMR, MS, and optical rotation analyses. All the
isolated compounds were evaluated for their inhibitory activities against infection with
HIV-1 SF162 in TZM-bl cells. Malformin C (5) showed the strongest anti-HIV-1
activity with IC of 1.4 ± 0.06 μM (selectivity index, 11.4), meanwhile aspernigrin C
5
0
(
3) also exhibited potent activity with IC of 4.7 ± 0.4 μM (selectivity index, 7.5).
50
Keywords: Aspergillus niger; aspernigrins; malformin C; HIV-1 SF162
2

Despite the success of current antiretroviral treatment (ART), infections with the
human immunodeficiency virus 1 (HIV-1) remain a global threat to public health with
more than 33 million infected individuals and 2.3 million new infections in 2012.
Existence of several shortcomings of the current ART, including the emergence of
resistant virus, severe side effects and the high cost, has posed an urgent need for the
1
discovery and development of potent anti-HIV-1 drugs with novel modes of action.
Marine organisms have been proven to be excellent sources of biologically active
compounds against HIV-1. Discovery of anti-HIV-1agents, especially with novel
2
modes of action, from marine organisms is becoming more attractive and promising.
During our continuous chemical study of marine-derived fungi, four
2
-benzylpyridin-4-one-containing metabolites (1-4), including two structurally new
aspernigrins C (3) and D (4), and other four known compounds (5-8) were isolated
from the marine-derived black aspergilli, Aspergillus niger SCSIO Jcsw6F30 (Fig. 1).
All of the compounds we obtained were tested for their inhibitory activities against
chemokine receptor subtype 5 (CCR5) tropic HIV-1 SF162 infection. Herein, we
describe the isolation, structural elucidation and biological evaluations of these
compounds.
3
The fungal strain A. niger SCSIO Jcsw6F30 was cultured on Medium B agar
4
plates and then incubated for 7 days. The crude extract (10.5 g) was subjected to a
silica gel column chromatography (CC) and the fractions were purified by
5
semi-preparative HPLC and repeated silica gel CC, to obtain compounds 1-8 (Fig. 1).
Compounds 1 and 2 were identified as aspernigrins A and B by comparison of
3

6
,7
their NMR, MS and optical rotation (OR) data with those available in the literature.
The structures of aspernigrins A and B were identified as 2-pyridone substructure
6
7,8
20
initially, and then revised to be 4-pyridone. The OR data of 2 ([α] +38.5, c 0.1,
D
DMSO) suggests it has the same S-configuration (C-7') with aspernigrin B in the
2
0
6
reference ([α] +37.8, c 0.5, DMSO).
D
Figure 1. Chemical structures of compounds 1-8.
9
Aspernigrin C (
3
) was suggested to be an N-acylcarboxyamide derivate,
1
0
11
like the structures of pestalamide B and nygerone A , connected by
-methylsuccinic acid moiety to the carboxamide of aspernigrin B ( ), by
HR-ESI-MS and NMR analysis. The planer structure of aspernigrin C ( ),
together with some key HMBC correlations, was showed in Fig. 2 as a new
compound. The structure of aspernigrin C ( ) could be considered as the
2
2
3
3
establishment of three partial structures (fragments A-C) showed in Fig. 2.
4

Fragments A + B consist the structure of aspernigrin B (
2) we obtained, while
B + C make up the structure of pestalamide B, which is a common product of
1
2
black aspergilli strains. Aspernigrin B (2a) and 2-methylsuccinate (9) were
obtained after hydrolysis of 3 (Fig. 2). The configurations of the products 2a and
were determined to be 7'S and ''S, by comparisons of their OR data with
'S-aspernigrin B ( ) we obtained and (S)-2-methylsuccinate reported in the
literature, respectively. So, the the configuration of 3 was suggested to be 7'S, ''S.
9
3
7
2
1
1
3
The detailed structural elucidation (HR-ESI-MS, NMR and OR analyses) of
3
was showed in the Supplementary Material.
Figure 2. Key HMBC correlations of 3, and its hydrolysis products.
1
3
Aspernigrin D (4) was suggested to be the methyl ester of 3, formed during the
isolation or purification processes. The detailed structural elucidation was showed
in the Supplementary Material.
Compound
5 was identified as malformin C with the same absolute
1
4
1
13
configuration reported in the reference , by its H, C NMR and OR data
5

20
(
[α] –24.2, c 0.1, CHCl3). Compounds 6-8 were identified as rubrofusarin (6),
D
1
5
1
13
rubrofusarin B (7), and fonsecin (8) by comparison with their H and C
NMR with those reported.
Figure 3. Proposed biogenetical relationships of 1-4 and pestalamide B.
A variety of natural products containing 2-benzyl-4H-pyran-4-one and
-benzylpyridin-4(1H)-one substructures have been detected among several
2
Aspergillus stains, and those have been encountered in relatively few fungi
1
2
outside of the black aspergilli clade. By structurally fragment analysis, it is
proposed that the 2-benzylpyridin-4-one-containing metabolites
3 (fragments
6

A+B+C) was derived from pestalamide B (fragments B+C), common product
1
2
of black aspergilli strains, or
biogenetically related to aspernigrin A (
phenethyl-2-pyrone compounds, such as aspergillusol and pyrophen
2
(fragments A+B), both of which were
1
, fragment B). Some
1
6
14,16
also
found in A. niger, should be act as important precursor compounds to produce
aspernigrins (Fig. 3).
A transmitted/founder virus predominantly uses CCR5 as co-receptor to infect T
1
7
cells. Isolated compounds were evaluated for their inhibitory effects on infection by
CCR5-tropic HIV-1 SF162 in TZM-bl cells (HeLa human cervical carcinoma
1
8,19
cells),
which is a reported cell line that is commonly used to measure HIV-1
2
0
infection with advantages of being fast and cost-effective. The colorimetric XTT
2
1,22
assay was also conducted for the cytotoxicity of these compounds on TZM-bl cells.
New compound aspernigrin C (3) and malformin C (5) effectively inhibited infection
by HIV-1 SF162 with the effective concentration for 50% inhibition (IC ) being 4.7 ±
5
0
0
.4 μM and 1.4 ± 0.06 μM, respectively. Malformin C (5) showed potent antiviral
activity, which was comparable to that of abacavir (IC 0.8 ± 0.1 μM), a nucleoside
5
0
reverse transcriptase inhibitors, and ADS-J1 (IC 1.8 ± 0.3 μM), an effective HIV-1
5
0
2
3
entry inhibitor. While aspernigrin A (1) and rubrofusarin (6) also exhibited weak
antiviral activities with IC of 83.2 ± 21.4 μM and 56.2 ± 3.3 μM, respectively. All
50
these four compounds showed little or no cytotoxicity to TZM-bl cells at the
concentrations tested in the viral infection assay (Table 1 and Fig. 4). Other compounds
showed no antiviral activities at the concentration of 50 μM (data not shown).
7

Table 1. Inhibitory activities of compounds 1, 3, 5 and 6 on infection by HIV-1 SF162
a
and their cytotoxicities to TZM-bl cells
b
c
d
Compounds
Abacavir
IC50 (μM)
CC50 (μM)
SI
e
0.8 ± 0.1
1.8 ± 0.3
ND
ND
ND
7.0
ADS-J1
ND
Aspernigrin A (1)
Aspernigrin C (3)
Malformin C (5)
Rubrofusarin (6)
83.2 ± 21.4
4.7 ± 0.4
582.6 ± 72.3
35.0 ± 2.1
7.5
1.4 ± 0.06
56.2 ± 3.3
16.4 ± 0.3
11.4
18.1
1019.6 ± 199.1
a
b
Two independent experiments were performed in triplicate. Data are presented as mean ± standard deviations.
c
d
IC , the effective concentration for 50% inhibition. CC , 50% cytotoxicity concentration. SI, selectivity index
5
0
50
e
(
CC /IC ). ND, not done.
50 50
Figure 4. Inhibitory activities of compounds on infection by SF162 and their
cytotoxicities to TZM-bl cells. A) 1; B) 3; C) 5; D) 6.
Structurally unusual 2-benzylpyridin-4-one-containing metabolites, such
as aspernigrins A and B, were found to display cytotoxic activities and also
8

6
strong neuroprotective effect. However, antiviral activities of aspernigrins had
never been found before. Now, in our study, the structurally new compound
aspernigrin C (
3
) and aspernigrin A (
1
) showed potent and weak efficacy in
) and D (
inhibiting HIV-1 infection, respectively, while aspernigrins B (
2
4)
showed no antiviral activities. It is suggested that 2-methylsuccinic acid moiety
could remarkably improve the anti-HIV-1 activities of aspernigrins, however,
esterification of it resulted in an extremely loss in activity. The completely
structure-activity relationship will be determined in the future study.
In addition, a mycotoxin compound malformin C (
5) displayed potent
anti-HIV activity at low μM level, which was comparable to that of abacavir
2
3
and ADS-J1. Malformin C (
5
) has been found to exert antimicrobial activities,
2
4
25
including antibacterial, antimalarial and antitrypanosomal proterties. It is
also rediscovered to be promising anti-cancer agents as G2 checkpoint
1
4
inhibitor. Now, it is the first report of malformin C with its anti-HIV activity.
In summary, four 2-benzylpyridin-4-one-containing metabolites (
including two structurally new aspernigrins C ( ) and D ( ), were isolated from
the marine alga-derived A. niger SCSIO Jcsw6F30, together with other four
known compounds ( ). Aspernigrins C ( ) and malformin C ( ) exhibited
1-4),
3
4
5
-
8
3
5
significant HIV-1 inhibitory activities by SF162 infection in TZM-bl cells. The
mechanism of action is on-going and our results suggest these two compounds
are potential lead product for the development of anti-HIV therapeutics.
Acknowledgments
9

This work was supported by grants from the National Natural Science
Foundation of China (41376162, 81102482, 41406187, 31270402, and
4
1476135), 863 Program of MOST (2012AA092104), Guangdong Natural
Science Foundation (S2013010012474), the Strategic Leading Science and
Technology Project of CAS (XDA11030403), and Guangdong special support
project for outstanding young scholars. We are grateful to Dr. Z. Xiao, A. Sun,
C. Li, and Y. Zhang in the analytical facility at SCSIO for recording
spectroscopic data.
Supplementary Information
Electronic supplementary information available: structural elucidation of
compounds
and
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1
0

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and 0:1) to yield eight fractions (fr.I–fr.VIII). Fr.V (petroleum ether/EtOAc
5:1) was dissolved in methanol and chromatographed over semi-preparative
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1
8
gradient solvent system from 20% to 60% CH CN over 30 min to yield
3
1
1

compounds
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(12 mg, t 15.59 min),
3
(6.2 mg, t 17.71 min) and
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(4.6 mg,
R
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tR20.12 min). Compound
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(15 mg, t 16.73 min) was purified and obtained by
R
same semi-preparative HPLC method in fr.VI. Fr.VII (petroleum
ether/EtOAc, 1:1) was purified by silica gel CC (CHCl /MeOH, gradient
3
elution 100:1~10:1) to afford compounds
and (11.5 mg).
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13
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0
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5

Figure 1. Chemical structures of compounds 1-8.
Figure 2. Key HMBC correlations and fragments of 3. (Black and White in print)
Figure 3. Proposed biogenetical relationships of 1-4 and pestalamide B. (Black and
White in print)
Figure 4. Inhibitory activities of compounds on infection by SF162 and their
cytotoxicities to TZM-bl cells. A) 1; B) 3; C) 5; D) 6.
1
6

Graphical abstract
1
7