New Furanone Derivatives and Alkaloids from the Co-Culture of Marine-Derived Fungi Aspergillus sclerotiorum and Penicillium citrinum

Article abstract of DOI:10.1002/cbdv.201600327

Six new compounds including two furanone derivatives sclerotiorumins A and B (1 and 2), one novel oxadiazin derivative sclerotiorumin C (3), one pyrrole derivative 1-(4-benzyl-1H-pyrrol-3-yl)ethanone (4), and two complexes of neoaspergillic acid aluminiumneohydroxyaspergillin (5) and ferrineohydroxyaspergillin (6) were isolated from the co-culture of marine-derived fungi Aspergillus sclerotiorum and Penicillium citrinum. Compound 3 was the first natural 1,2,4-oxadiazin-6-one. Compound 5 showed significant and selective cytotoxicity against human histiocytic lymphoma U937 cell line (IC₅₀?=?4.2?μm) and strong toxicity towards brine shrimp (LC₅₀?=?6.1?μm), and oppositely increased the growth and biofilm formation of Staphylococcus aureus.

Full text of DOI:10.1002/cbdv.201600327

Received Date: 09-Sep-2016
Revised Date: 14-Nov-2016
Accepted Date: 29-Nov-2016
Article Type: Full Paper
New furanone derivatives and alkaloids from the co-culture of
marine-derived fungi Aspergillus sclerotiorum and Penicillium
citrinum
Jie Bao1, Jie Wang1, Xiao-Yong Zhang1, Xu-Hua Nong1, Shu-Hua Qi1
1 Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory
of Marine Material Medica, RNAM Center for Marine Microbiology, South China Sea
Institute of Oceanology, Chinese Academy of Sciences, 164 West Xingang Road,
Guangzhou 510301, China
Correspondence
Prof. Dr. Shu-Hua Qi
Key Laboratory of Tropical Marine Bio-resources and Ecology
South China Sea Institute of Oceanology
Chinese Academy of Sciences
164 West Xingang Road
Guangzhou 510301 Guangdong
China
Phone: + 862089022112
Fax: + 862084458964
shuhuaqi@scsio.ac.cn
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been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/cbdv.201600327
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Abstract
Six new compounds including two furanone derivatives sclerotiorumins A-B (1-2), one
novel oxadiazin derivative sclerotiorumin (3), one pyrrole derivative
C
1-(4-benzyl-1H-pyrrol-3-yl)ethanone (4), and two complexes of neoaspergillic acid
aluminiumneohydroxyaspergillin (5) and ferrineohydroxyaspergillin (6) were isolated from
the co-culture of marine-derived fungi Aspergillus sclerotiorum and Penicillium citrinum.
Compound 3 was the first natural 1,2,4-oxadiazin-6-one. Compound 5 showed significant and
selective cytotoxicity against human histiocytic lymphoma U937 cell line (IC50 = 4.2 μM)
and strong toxicity towards brine shrimp (LC50 = 6.1 μM), and oppositely increased the
growth and biofilm formation of Staphylococcus aureus.
Keywords: Aspergillus sclerotiorum; furanone derivative; complexes of neoaspergillic acid;
1,2,4-oxadiazin-6-one; cytotoxicity
Introduction
Mixed fermentation of two or more microbes has been used a strategy to improve
antibiotic activity of crude extracts and increase yields of metabolites [1]. It also is an
effective approach for finding new interesting natural products [2-6]. In our previous
experiment, we found that when marine gorgonian-associated fungi Aspergillus sclerotiorum
SCSGAF 0053 and Penicillium citrinum SCSGAF 0052 were co-cultured, a red pigment
could be obviously observed in their culture medium while no red pigment could be observed
when either of the two strains was cultured alone. The special phenomenon guided us to
explore the secondary metabolites of the co-culture of A. sclerotiorum and P. citrinum, which
led to the isolation of six new compounds including two furanone derivatives sclerotiorumins
A-B (1-2), one novel oxadiazin derivative sclerotiorumin C (3), one pyrrole
derivative1-(4-benzyl-1H-pyrrol-3-yl)ethanone (4), and two complexes of neoaspergillic acid
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aluminiumneohydroxyaspergillin (5) and ferrineohydroxyaspergillin (6) (Fig. 1), along with
the known ferrineoaspergillin (7) [7-8], neohydroxyaspergillic acid [9], penicillic acid [10],
and 5,6-dihydropenicillic acid [10]. The toxicities of 1-7 were tested by brine shrimp lethality
bioassay, and 1-3 and 5-7 were also evaluated for cytotoxicity toward five human carcinoma
cell lines and antibacterial and anti-biofilm activities against five bacterial strains. In this
paper we report the isolation, structure elucidation, and bioactivity of these compounds.
Results and Discussion
Sclerotiorumin A (1) was obtained as pale yellow oil and assigned a molecular formula of
13
C14H14O5 by HRESIMS (m/z 263.0916 [M+H]+) and NMR data. Its 1H and C NMR data
revealed four methyl groups including one O-methyl (δH/δC 1.63/23.4, 1.68/5.4, 2.15/13.1,
3.75/56.3), two methines (δH/δC 6.02/107.3, 8.21/170.0), and eight quaternary carbons (δC
85.3, 112.7, 140.2, 143.8, 157.8, 181.7, 184.7, 205.1). In the HMBC spectrum (Fig.2),
correlations of H-5 with C-2/C-3/C-4, H-14 with C-3/C-4/C-5, and H-15 with C-2/C-3
suggested the presence of a 2,4-dimethylfuran-4-en-3-one moiety (fragment A) [11]. HMBC
correlations of H-8 with C-2/C-6/C-9/C-10, H-12 with C-15/C-2/C-6/C-10/C-11, H-13 with
C-8/C-9, and H-15 with C-6 revealed the presence of fragment B and the connection of
fragments A and B by the C2-C6 bond. Therefore, the planar structure of 1 was determined as
shown. Else, compound 1 was inferred as a racemic mixture by its specific rotation recorded
as zero and HPLC analysis on a chiral column (see Supporting information Fig. S30).
Although many compounds contain fragment A or B [12-15], sclerotiorumin A was obtained
as a complex of fragments A and B for the first time.
Sclerotiorumin B (2) was isolated as pale yellow oil with the molecular formula of
C14H19O6 as determined by HRESIMS (m/z 305.1000 [M+Na]+) and NMR data. Although it
1
13
was isolated as a pure compound by HPLC, its H and C NMR signals appeared in pairs
with a ratio of about1:1. This indicated that 2 wasn’t stable, and would turn into one of its
epimers immediately in air. For the two epimers, their structures were further determined by
analysis of their 2D-NMR data, respectively.
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The 1H and 13C NMR data of 2a showed four methyl groups (δH/δC 0.75/15.2, 1.26/22.9,
1.60/5.1, 3.83/59.7), one methylene (δH/δC 1.45, 2.26/35.7), three methines (δH/δC 1.72/34.1,
5.36/89.8, 8.44/173.8), and six quaternary carbons (δC 206.0, 179.3, 169.8, 113.3, 104.3,
88.3). In the HMBC spectrum (Fig.2), correlations of H-5 with C-2/C-3/C-4/C-12, H-12 with
C-3/C-4/C-5,
2,4-dimethylfuran-4-en-3-one moiety (fragment A) as that of 1. HMBC correlations of H-10
with C-8/C-9/C-11, and H-14 with C-9, suggested the presence of
and
H-13
with
C-2/C-3,
suggested
the
presence
of
a
a
4,5-dihydroxyfuran-3-en-4-one moiety (fragment B) as that of 5,6-dihydropenicillic acid [11].
Furthermore, HMBC correlations from H-6 to C-2/C-7/C-13/C-15, from H-7 to
C-2/C-6/C-8/C-15, from H-13 to C-2/C-3/C-6, and from H-15 to C-6/C-7/C-8, indicated that
fragments A and B connected together by the C2-C6-C7-C8 chain as shown. The planar
structure of 2b was inferred to be same as that of 2a by analysis of its 2D NMR spectra.
However, in the NOESY spectrum (Fig.3), there were some different NOE correlation
signals between 2a and 2b. For 2a, the NOE correlations of OH-8 with H-7/H-6a, H-6a with
H-13, and H-15 with H-6b were observed; while for 2b, NOE correlations of OH-8 with
H-15/H-6a, and H-7 with H-13 were observed. Although the single bonds in the open chain
could rotate freely in the room temperature, and the NOE correlation signals of open chain
couldn’t be used to determine the stereoscopic relationship, the above data indicated that 2a
and 2b were a pair of epimers at C-8. Actually, we obtained the pure compounds of 2a and
2b from the extracts by chiral column, respectively, but they would turn into each other
immediately after collection (see Fig. S30).
Sclerotiorumin C (3) had a molecular formula of C12H20N2O5 as determined by its
HRESIMS (m/z 295.1265 ([M+ Na]+) and NMR data. The 1H and 13C NMR spectra revealed
two exchangeable protons (δH 10.36, 9.07), four methyl groups (δH/δC 0.84/15.8, 0.86/18.5,
0.94/22.9, 0.99/23.8), one methylene (δH/δC 1.96, 1.86/46.2), four methines (δH/δC 1.76/23.4,
2.37/29.4, 4.29/77.1, 8.04/162.0), and three quaternary carbons (δC 168.7, 166.4, 83.7). The
HMBC spectrum (Fig. 4) showed correlations of H-3 with C-5, NH-4 with C-3/C-5/C-6, H-7
with C-5/C-6/C-9/C-10, H-8 with C-5/C-9/C-10, H-9/H-10 with C-7/C-8, H-11 with
C-5/C-12/C-13, and H-14/H-15 with C-13/C-11. Combining with the molecular formula,
these data established the planar structure of 3 as shown. However, the absolute configuration
of 3 remained un-determined. Although there were several reports about the synthesis of
1,2,4-oxadiazin-6-ones [16-17], 3 was the first natural 1,2,4-oxadiazin-6-one derivative.
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1-(4-Benzyl-1H-pyrrol-3-yl)ethanone (4) was isolated as a white powder with a molecular
formula of C13H13NO as determined by HRESIMS (m/z 200.1077 [M+H]+) and NMR data.
1
The H NMR spectrum showed one exchangeable proton (δH 11.72) and five aromatic
protons (δH 7.17-7.29), and the 13C and DEPT135 NMR spectra displayed one methyl (δC
25.1), one methylene (δC 33.0), seven methines (δC 128.4, 128.4, 128.2, 128.2, 126.0, 117.7)
and four quaternary carbons (δC 186.1, 139.5, 139.4, 131.0). These data revealed the presence
of mono-substituted phenyl group in 4. In the HMBC spectrum, correlations from H-3 to
C-2/C-4/C-5, and from H-4 to C-2/C-3/C-5, established a pyrrole unit. Furthermore, the
HMBC spectrum showing correlations of H-8 with C-4/C-5/C-9/C-10/C-14, and H-7 with
C-2/C-6, and the coupling constant of J3,4 = J1,3 = J1,4 = 3.0 Hz [18-21], suggested a
2,5-disubstitutedpyrrole. So, the structure of 4 was determined as shown.
Aluminiumneohydroxyaspergillin (5) was isolated as a yellowish solid, and established to
have the molecular formula C36H57AlN6O9 on the basis of HRESIMS (m/z 745.4087
1
[M+H]+). The H NMR spectrum showed signals with irregular broad peaks including four
methines (δH 7.82, 4.53, 2.30, 2.16), one methylene (δH 2.76), and four methyl groups (δH
13
1.04, 0.83-0.92). The C and DEPT135 NMR showed 12 carbon signals in cluster. These
NMR data showed similarity to those of aluminiumneoaspergillic [10], except the additional
presence of one oxymethine (δH/δC 4.53/74.6) in 5 instead of one methylene (δH/δC
2.56–2.84/36.36) in aluminiumneoaspergillic and the lower-field shift of C-8 [22]. Alkali
hydrolysis of 5 gave neohydroxyaspergillic acid (5A) [9] and Al(OH)3 sediment, which
indicated that 5 was an aluminium complex of neohydroxyaspergillic acid. The zero value for
the specific rotation of 5 suggested that 5 was a racemic mixture, which was supported by the
HPLC analysis of 5 and 5A on a chiral column (see Fig. S30).
Ferrineohydroxyaspergillin (6) was obtained as a red pigment and had the molecular
formula of C36H57FeN6O9 as determined by HRESIMS (m/z 774.3708 [M+H]+). No NMR
signals appeared when it was tested, revealing the existence of Iron ion. Alkali hydrolysis of
6 gave neohydroxyaspergillic acid (5A) [9] and Fe(OH)3 sediment. These data displayed that
6 was a ferricomplex with three molecules of neohydroxyaspergillic acid. It was also inferred
as a racemic mixture by testing its specific rotation ([α]20 0 (c 0.2, MeOH) and HPLC
D
analysis on a chiral column (see Fig. S30). Compounds 5 and 6 were reported as complexes
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of metal ion and neohydroxyaspergillic acid for the first time, while previous reports were
about the complexes of metal ion and neoaspergillic acid/aspergillic acid [7, 22-23].
Ferrineoaspergillin (7) was obtained as a red pigment, and no NMR signals appeared
when it was tested, revealing the existence of Iron ion. Alkali hydrolysis of 7 gave
neoaspergillic acid (7A) [7-8] as reported and Fe(OH)3 sediment. These data displayed that 7
was a ferricomplex with three molecules of neoaspergillic acid.
Compounds 1-7 were deduced to be produced by the strain SCSGAF 0053 by comparison
of the HPLC analysis data of the extracts of the mono-culture and co-culture of the strains
SCSGAF 0052 and SCSGAF 0053 (see Fig. S31-S32 ).
The bioassay of toxicity of 1-7 towards brine shrimp showed that 5 had strong toxicity
with LC50 value of 6.1 μM by comparison with the positive control toosendanin (LC50 = 1.73
μM), and 4, 6 and 7 had medium toxicity with LC50 values of 46.2, 11.5, 27.8 μM,
respectively, while 1-3 showed weak toxicity with LC50 >100 μM. In addition, 1-3 and 5-7
were tested for cytotoxicity against human helacytongartleri (Hela), breast cancer MCF-7,
liver hepatocellular carcinoma HepG2 and Huh7, and histiocytic lymphoma U937 cell lines.
The results displayed that 5 exhibited significant selective cytotoxicity against U937 cell line
with IC50 value of 4.2 μM, and mild cytotoxicity against Hela and MCF-7 cell lines with IC50
values of 29.3 and 24.8 μM, respectively, and 6 and 7 showed mild cytotoxicity against U937,
Hela and MCF-7 cell lines with IC50 values of 42.0 and 48.0 μM, 39.3 and 57.1 μM, 42.5 and
89.1 μM, respectively, while all the tested compounds showed weak cytotoxicity towards
HepG2 and Huh7 cell lines with IC50 >100 μM. The IC50 values of positive control
doxorubicin towards U937, Hela, MCF-7, HepG2, and Huh7 cell lines were 0.06, 0.8, 23.1,
3.3, and 1.2 μM, respectively. Moreover, 1-3 and 5-7 were also tested for antibacterial and
anti-biofilm activities against two common bacteria Staphylococcus aureus and Pseudomonas
aeruginosaand three marine-derived bacteria Pseudoalteromonasnigrifaciens, Bacillus
amyloliquefaciens, and Bacillus stearothermophilus. However, none of these compounds
showed obvious antibacterial and anti-biofilm activity, and oppositely 4 and 5 could increase
the growth of S. aureus by 1 fold at 100 μg/ml, 5 could increase the biofilm formation of S.
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aureus by 1.3 folds at 25 μg/ml, and 7 could increase the biofilm formation of P. aeruginosa
by 1 fold at 100 μg/ml. As reported, neoaspergillic acid derivatives were a member of
mycotoxins with antibacterial, antifungal or toxic activity, such as hydroxyaspergillic acid,
aspergillic acid, etc[7, 9, 23].Our results further revealed the toxicity and potent cytotoxicity
of neoaspergillic acid derivatives, however, the three complexes of neoaspergillic acid didn’t
show antibacterial activity in here. According to the toxicities and cytotoxicities of 5-7 and
our previous report about the toxicities of aluminiumneoaspergillin and
zirconiumneoaspergillin [23], it seems that the toxicity and cytotoxicity of the aluminium
complex derivatives of neoaspergillic acid are stronger than that of the corresponding
ferricomplex.
Acknowledgments
We are grateful for the financial support provided by the National Natural Science
Foundation of China (41376160, 81673326), National Marine Public Welfare Research
Project of China (201305017), Marine Economic Development Regional Innovation
Demonstration Project of Guangdong Province (GD2012-D01-002), National Natural
Science Foundation of China (41506148), and Shandong Excellent Young Scientist Award
Fund (BS2015HZ005).
Materials and methods
General experimental procedures.
NMR spectra were recorded on a Bruker AV- 500 MHz NMR spectrometer with TMS as
an internal standard. UV spectra were measured on a Shimadzu UV-VIS spectrophotometer.
IR spectra were obtained using a Shimadzu IR Affinity-1 Fourier Transform Infrared
spectrophotometer. HRESIMS spectra were got on a Bruker microTOF-QII mass
spectrometer. The optical rotations were recorded on an Anton Paar MCP 500 polarimeter.
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Semi-preparative HPLC was performed on a Shimadzu LC-20AT pump with a Shimadzu
SPD-M20A Photodiode Array Detector using an Phenomenex Gemini C18 5 µ 110A column
(250×10 mm, 5 μm). CD spectra were got on a Chirascan Circular Dichroism Spectrometer
(Applied Photophysics). Column chromatography (CC) was performed on Silica gel
(200-300 mesh, Qingdao Marine Chemical), sephadex LH-20 or Rp-18 (Pharmacia Co. ODS).
Precoated silica gel plates (Yan Tai Zi Fu Chemical Group Co.) were used for thin layer
chromatography.
Fungal material.
The two strains SCSGAF 0052 and SCSGAF 0053 were both isolated from gorgonian
Muricella flexuosa collected from the South China Sea, Sanya (18°11’N, 109°25’E), Hainan
Province, China. They were identified as Penicillium citrinum and Aspergillus sclerotiorum
respectively, with a molecular biological protocol calling for DNA amplification and ITS
region sequence comparison with the GenBank database [24]. The BLAST sequenced data
have been deposited at GenBank (accession no.JN851003.1 for SCSGAF 0052 and
JN851004.1 for SCSGAF 0053). The two strains were deposited in the RNAM center, South
China Sea Institute of Oceanology, Chinese Academy of Sciences.
Fermentation and extraction.
Strains SCSGAF 0052 and SCSGAF 0053 were both grown on PDA agar medium. The
spores (1ml, about 108 CFU/ml) of SCSGAF 0053 and a very small amount of spores (1ml,
about 104 CFU/ml) of SCSGAF 0052 were inoculated into 1L flasks containing 300 ml liquid
medium (containing glucose 1.0%, soluble starch 1.0%, MgSO4 0.1%, KH2PO4 0.1%,
peptone 0.1%, sea salt 3.0% and pH 6.5-7.0) followed by static incubation for 30 days at 28
oC.
After fermentation, the total 40 L broth was harvested and centrifuged to yield the
supernatant and mycelia. The supernatant was extracted with ethyl acetate, the extracts were
combined and solvent was removed under reduced pressure. The mycelia were extracted with
80% acetone. The acetone extract was evaporated under reduced pressure to afford an
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aqueous solution, and then extracted with ethyl acetate. The two organic extracts were
combined to give a crude gum.
Isolation and purification.
The 60 g extract was applied to silica gel CC eluting with step gradient CHCl3/MeOH to
give nine fractions (Fr.1 – Fr.9). Fr.2 was subjected to MPLC with an ODS column, eluting
with gradient MeOH/H2O to give six subfractions (Fr.2-1–Fr.2-6). Fr.2-2 was washed with
methanol repeatedly to give neohydroxyaspergillic acid (12.2 mg), Fr.2-3 was purified by
semipreparative HPLC, eluting with MeOH/H2O (v/v 46:54, 3 ml/min) to obtain 1 (tR = 16.3
min, 7.0 mg), and Fr.2-6 was purified by semipreparative HPLC, eluting with MeOH/H2O
(v/v 51:49, 3 ml/min) to obtain 4 (tR = 38.4 min, 5.0 mg). Fr.5 was repeatedly subjected to
silica gel CC, eluting with step gradient CHCl3/Acetone to give seven fractions
(Fr.5-1–Fr.5-7). Fr.5-2 was purified by MPLC with an ODS column and preparation TLC
plate to yield7 (40.0mg), and Fr.5-3 was isolated by MPLC with an ODS column to give four
subfractions (Fr.5-3-1–Fr.5-3-4). Fr.5-3-1 was subjected to MPLC with an ODS column, and
then purified by HPLC eluting with ACN/H2O (v/v 20:80, 3.0 ml/min) to obtain penicillic
acid (tR = 17.7 min, 50.0 mg) and 5,6-dihydropenicillic acid (tR = 22.8 min, 30.2 mg), while
Fr.5-3-3 was applied to sephadex LH-20 column, and then purified by HPLC eluting with
MeOH/H2O (v/v 22:78, 1 ml/min) to obtain 2a (tR = 22.8min, 7.0 mg) and 2b (tR = 23.7 min,
8.0 mg). Fr.6 was applied to silica gel CC eluting with step gradient CHCl3/Acetone to give
seven fractions (Fr.6-1–Fr.6-7). Fr.6-5 was separated by preparation TLC plate to afford 5
(7.0 mg) and 6 (10.2 mg). Fr.6-6 was subject to MPLC with an ODS column to give four
fractions (Fr.6-6-1–Fr.6-6-4), and then Fr.6-6-3 was purified by preparation TLC plate to give
3 (6.0 mg).
SclerotioruminA (1). [α]20D 0 (c 0.2, MeOH); UV (MeOH) λmax (logε): 205 (4.10), 264
1
13
(3.80) nm; IR νmax3372, 1524, 1485, 1419, 1161, 1018, 984 cm-1; H and C NMR spectral
data, see Table 1; (+)-HRESIMS m/z 263.0916 [M+H]+ (calcd for 263.0914, C14H15O5).
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Sclerotiorumin B (2). [α]20D -3.1 (c 0.2, MeOH); UV (MeOH) λmax (logε): 224 (3.96), 269
(3.18) nm; IR νmax 3383, 1755, 1689, 1640, 1612, 1454, 1350, 1219, 1177, 1134, 1030, 918,
1
810 cm-1; H and 13C NMR spectral data of 2a and 2b, see Table 1; (+)-HRESIMS m/z
305.1000 [M+Na]+ (calcd for 305.0996, C14H18O6Na).
SclerotioruminC (3). [α]20D +61.8 (c 0.4, MeOH); UV (MeOH) λmax (logε): 202 (3.31), 281
(2.56), 371 (2.04) nm; CD (c 1.91 mM, MeOH) λmax (Δε) 212 (-4.61), 231 (4.76); IR νmax
3229, 2963, 1751, 1693, 1647, 1535, 1466, 1393, 1273, 1223, 1173, 1150, 1049, 961, 795
1
cm-1; H and 13C NMR spectral data, see Table 2; (+)-HRESIMS m/z 295.1265
[M+Na]+(calcd for 295.1264, C12H20N2O5Na ).
1-(4-Benzyl-1H-pyrrol-3-yl)ethanone (4). UV (MeOH) λmax (logε): 203 (4.17), 246 (3.65),
299 (4.30) nm; IR νmax3217, 1682, 1616, 1489, 1392, 1312, 1215, 1611, 1045, 995. 930, 791,
13
772 cm-1; 1H and C NMR spectral data, see Table 2; (+)-HRESIMS m/z 200.1077 [M+H]-
(calcd for 200.1070, C13H14NO ).
Aluminiumneohydroxyaspergillin(5). [α]20D 0 ( c 0.2, MeOH); UV (MeOH) λmax (logε):
206 (4.48), 224 (4.57), 320 (4.28). 392 (3.07) nm; IR νmax 3364, 1597, 1535, 1493, 1427,
13
1161, 1022, 988, 775, 752 cm-1; 1H and C NMR spectral data, see Table 3; (+)-HRESIMS
m/z 745.4087 [M+H]+ (calcd for 745.4075, C36H58AlN6O9 ).
Ferrineohydroxyaspergillin(6). [α]20D0 ( c 0.2, MeOH); UV (MeOH) λmax (logε): 205
(3.57), 226 (4.23), 313 (4.63). 394 (4.51) nm; IR νmax 3372, 1589, 1524, 1486, 1420, 1234,
1161, 1018, 984, 772, 748 cm-1; 1H and 13C NMR spectral data couldn’t be measured, and 1H
and 13C NMR spectral data of alkali hydrolysis product neohydroxyaspergillic acid (5A), see
Table 3; (+)-HRESIMS m/z 774.3708 [M+H]+ (calcd for 774.3615, C36H58FeN6O9).
1
Ferrineoaspergillin (7). H and 13C NMR spectral data of alkali hydrolysis product
neoaspergillic acid (7A), see Table 3;
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Alkaline hydrolysis of 5-7.
Compounds 5-7 were applied to alkaline hydrolysis as reported [23].
Brine shrimp bioassay.
Brine shrimp lethality bioassay was evaluated using Brine shrimp (Artemiasalina) eggs
(Aquatic Lifeline, Inc., Utah, USA) by the method as described previously [18].
Cytotoxicity.
Compounds were tested for cytotoxicity against Hela, MCF-7, HepG2 and Huh7 human
carcinoma cell lines using a MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium
bromide)method, and against U937 human carcinoma cell line using a CCK-8
(2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium,
monosodium salt) method. The media for U937 and the other four cell lines were RPMI1640
and DMEM, respectively. Cells in the logarithmic phase were seeded into 96-well plates in a
o
100 μL volume of medium, maintained at 37 C in a 5% CO2 incubator overnight, then
treated with the corresponding compounds or vehicle control at the indicated concentration.
After 48 h of incubation, 5 μL of MTT or CCK-8 was added into the 96-well plates and
incubated for additional 2-3 h, then the absorbance was measured in a microplate reader at
450 nm and 650 nm. Cell toxicity was evaluated as the ratio of the absorbance of the sample
to that of the control. The IC50 were further calculated using Graphpad Prism 5 (Graphpad
Software, Inc.).
Antibacterial and Anti-biofilm Assays.
Bacterium and its biofilm cultivation in sterile 96-well polystyrene microliter plates was
carried out essentially as previously described [25-26].Briefly, overnight cultures of five
strains grown in LB medium were adjusted to an optical density at 600 nm of 0.05 in LB
medium. The diluted cultures with different concentration of compounds were transferred to
well of polystyrene microliter plates (150 μL per well) and incubated for the desired time.
Afterwards, for the observation of antibacterial activity, the absorbance of wells was
determined at 600 nm; while for the observation of anti-biofilm activity, the wells were
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washed with distilled water, dried in an inverted position, and stained with 170 μL crystal
violet (0.5% w/v) for 30 min. The wells were then washed three times with distilled water,
dried in an inverted position, and dissolved in 200 μL sodium acetate (33% w/v), and the
absorbance was determined at 560 nm. The experiment was run in three replicates.
Supporting Information
1H, 13C NMR, 2D NMR, HRESIMS and IR spectra of compounds 1–6 are available as
Supporting Information.
Conflicts of Interest
All the authors declare no conflict of interest.
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Table 1 1H (500 MHz) and 13C NMR (125 MHz) spectral data for 1-2(in DMSO-d6, δ ppm)
1
2a
2b
Pos.
δH(J in Hz)
8.21 (s)
δC
δH(J in Hz)
δC
δH(J in Hz)
δC
87.9, C
2
3
4
5
6
85.3, C
205.1, C
112.7, C
88.3, C
206.0, C
113.3, C
206.0, C
113.0, C
173.6, CH
37.0, CH2
170.0, CH 8.44 (s)
173.8, CH 8.42 (s)
2.26 (d, 14.5)
1.69 (d,14.0)
140.2, C
35.7, CH2
1.45 (dd, 14.5, 9.5)
1.48 (dd, 14.5, 8.5)
1.72 (m)
1.63 (m)
7
8
184.7, C
107.3, CH
157.8, C
181.7, C
143.8, C
34.1, CH
104.3, C
179.3, C
89.8, CH
169.8, C
5.1, CH3
33.6, CH
104.3, C
179.1, C
89.7, CH
169.8, C
5.0, CH3
22.6, CH3
59.86, CH3
15.1, CH3
6.02 (s)
9
10
11
12
13
14
15
8-OH
5.36 (s)
5.34 (s)
1.60 (s)
2.15 (s)
3.75 (s)
1.68 (s)
1.63 (s)
13.1, CH3 1.60 (s)
56.3, CH3 1.26 (s)
22.9, CH3 1.19 (s)
59.7, CH3 3.81 (s)
15.2, CH3 0.98 (d, 6.5)
7.62 (s)
5.4, CH3
3.83 (s)
23.4, CH3 0.75 (d, 6.5)
7.51 (s)
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Table 2 1H (500 MHz) and 13C NMR (125 MHz) spectral data for 3-4 (in DMSO-d6, δ ppm)
3
4
Pos.
δC
δH ( J in Hz)
δC
δH ( J in Hz)
11.72 (s)
1
2
3
4
5
6
7
131.0, C
162.0, CH
8.04 (d, 1.0)
9.07 (s)
117.7, CH 6.87 (t, 3.0)
108.3, CH 5.91 (t, 3.0)
139.4, C
83.7, C
166.4, C
46.2, CH2
186.1, C
1.96 (dd, 14.0, 5.5)
1.86 (dd, 14.0, 6.5)
25.1, CH3 2.28 (s)
8
23.4, CH
22.9, CH3
18.5, CH3
77.1, CH
168.7, C
1.76 (m)
33.0, CH2 3.91 (s)
139.5, C
9
0.94 (d, 6.5)
0.86 (d, 6.5)
4.29 (d, 2.5)
10
11
12
13
14
15
128.4, CH 7.17-7.29 (m)
128.2, CH
126.0, CH
29.4, CH
15.8, CH3
23.8, CH3
2.37 (m)
128.2, CH
0.84 (d, 6.5)
0.99 (d, 7.0)
10.36 (s)
126.0, CH
-OH
Table 3 1H (500 MHz) and 13C NMR (125 MHz) spectral data for 5, 5A and 7A (δ ppm)
Pos.
5a
5Ab
7Ab
δC
δH a ( J in Hz) δH ( J in Hz)
δH ( J in Hz)
2
153.0, C
149.7, C
3
5
129.1, CH 7.82 (m)
137.6, C
7.28 (s)
7.15 (s)
6
7
74.6, CH
32.5, CH
4.53 (m)
2.16 (m)
4.53 (s)
2.57 (d, 7.5)
2.16 (m)
8
2.05 (brs)
0.95 (d, 6.0)
9
19.2, CH3 1.04 (m)
0.89 (d, 6.5)
0.89 (d, 6.5)
2.57 (d, 7.5)
2.05 (m)
10
11
12
13/14
7-OH
18.1, CH3 0.83-0.91 (m) 0.78 (d, 6.0)
41.0, CH2 2.76 (m)
27.7, CH 2.30 (m)
2.63 (dd, 14.0,
2.17 (m)
22.2, CH3 0.83-0.91 (m) 0.90 (d, 6.5)
3.76 (m)
0.90 (d, 0.5)
a Recorded in CDCl3. b Recorded in DMSO-d6
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Fig. 1 Structures of compounds 1-7.
Fig. 2 The key HMBC correlations of compounds 1 and 2.
Fig. 3 The key NOE correlations observed in the NOESY spectrum of 2.
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Fig. 4 The key HMBC correlations of 3 and 4.
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