Five New Cytotoxic Metabolites from the Marine Fungus Neosartorya pseudofischeri

The marine fungus Neosartorya pseudofischeri was isolated from Acanthaster planci from the South China Sea. In a preliminary bioactivity screening, the crude methanol extract of the fungal mycelia showed significant inhibitory activity against the Sf9 cell line from the fall armyworm Spodoptera frugiperda. Five novel compounds, including 5-olefin phenylpyropene A (1), 13-dehydroxylpyripyropene A (4), deacetylsesquiterpene (7), 5-formyl-6-hydroxy-8-isopropyl-2- naphthoic acid (9) and 6,8-dihydroxy-3-((1E,3E)-penta-1,3-dien-1-yl)isochroman-1-one (10), together with eleven known compounds, phenylpyropene A (2) and C (3), pyripyropene A (5), 7-deacetylpyripyropene A (6), (1S,2R,4aR,5R,8R,8aR)-1,8a-dihydroxy-2-acetoxy-3,8-dimethyl-5- (prop-1-en-2-yl)-1,2,4a, 5,6,7,8,8a-octahydronaphthalene (8), isochaetominine C (11), trichodermamide A (12), indolyl-3-acetic acid methyl ester (13), 1-acetyl-β-carboline (14), 1,2,3,4-tetrahydro-6-hydroxyl-2-methyl-l,3,4-trioxopyrazino[l,2-a]-indole (15) and fumiquinazoline F (16), were obtained. The structures of these compounds were determined mainly by MS and NMR data. The absolute configuration of 9 was assigned by the single-crystal X-ray diffraction studies. Compounds 1–11 and 15 showed significant cytotoxicity against the Sf9 cells from S. frugiperda.


Introduction
The fall armyworm Spodoptera frugiperda is a pest feeding on the gramineous plants occurring worldwide and causing serious damage to several economically-important crops, such as the maize, sorghum, sugarcane, as well as the cotton, cruciferous and Cucurbitaceae plants.Although other alternative methods have been investigated on the biological control, as well as the development of resistant transgenic plants, the control of S. frugiperda still relies mainly on the use of chemical pesticides, easily inducing resistance and causing severe damage to the environment and consumers.However, some natural products with higher selectivity and safety become an important potential source of new biopesticides for the control of S. frugiperda [1][2][3][4][5].Sf9 cells are clonal isolates of S. frugiperda Sf21 cells (IPLB-SF21-AE) [6].A cultured Sf9 cell line is commonly used for primary screening to determine insecticidal activity [7] and in biomedical research for the purpose of recombinant protein expression using insect-specific viruses called baculoviruses [8].

Structural Elucidation
5-Olefin phenylpyropene A (1) was isolated as a yellowish oil.The molecular formula was established as C 32 H 36 O 9 based on the analysis of 13 C-NMR data and HR(+)ESIMS m/z 565.2448 [M + H] + (calcd.for C 32 H 36 O 9 , 565.2432), implying fifteen degrees of unsaturation (Supplementary Figure S1).The IR spectrum indicated the presence of a carbonyl group (1680 cm ´1) and a benzene ring (3073, 1574 and 1507 cm ´1).UV maxima at 232, 278 and 322 nm supported the long conjugated system containing a benzene ring.The 13 C-NMR and DEPT spectra displayed six methyls, four methylenes, ten methines and twelve quaternary carbons.The diagnostic aryl protons at δ H 7.81 (m, 2H) and 7.44 (m, 3H) revealed a monosubstituted benzene ring.Two alkenyl protons at δ H 6.46 (s) and 6.36 (s) indicated there were two trisubstituted double bonds in the molecule.Three methyl group singlets at δ H 2.16, 2.10 and 2.04 showed HMBC correlations with the carbonyl groups at δ C 169.9, 171.1 and 170.4,respectively, confirming there are three acyloxy groups.The other three methyl group singlets at δ H 1.57, 1.25 and 0.87 are connected with the quaternary carbons at δ C 21.2, 24.2 and 13.3, respectively.In the 1 H-    1 and Supplementary Figures S2-S7).Compounds 2 and 3 were identified as phenylpyripyropene A and C [15,16], by comparing their NMR data to the reference values.The double bond between the C-5 and C-13 positions in 1 was replaced by the saturated carbon-carbon bond in 2. Compounds 1 and 2 have triacyloxy groups at the C-1, C-7 and C-11 positions; however, Compound 3 has only one acyloxy group at the C-1 position (Supplementary Figures S8-S11).

Proposed Biosynthetic Pathways
Tomoda et al. described the biosynthetic origin of pyripyropene A (5), which was established by feeding experiments using various [ 13 C] and [ 14 C] precursors.Pyripyropene A ( 5) is derived from three mevalonates, five acetates and one nicotinic acid.The pyridino-α-pyrone moiety is produced via condensation of a primer nicotinic acid with two acetates in a "head-to-tail" fashion; an all-trans farnesyl pyrophosphate is produced via the mevalonate pathway; the two parts are linked and cyclized to form the core skeleton, and then three acetyl residues from the acetates are introduced into the skeleton to yield 5 [28].Based on the above finding, we proposed that the phenyl-α-pyrones (1-3) shared a similar biosynthetic pathway (Figure 5).Benzoic acid, instead of nicotinic acid, is one of the precursors.It is very interesting that Compound 2 was observed slowly changing into Compound 1 when stored in DMSO-d 6 .The change was recorded by 1 H-NMR spectra.After 18 days, Compound 2 was changed into Compound 1 thoroughly.However, the pure Compounds 1 and 2 are stable at the temperature range from ´20 ˝C-90 ˝C.

Cytotoxicity
Compounds 1-11 and 15 showed significant cytotoxicity against the Sf9 cells (Table 3).After 48 h of treatment at the concentration of 50 mg/L, the cell growth inhibition rates of compounds 2, 3, 7-9, 11 and 15 were greater than 90%.Therefore, further in-depth studies to identify the insecticidal activities in vivo and the mechanism are warranted for their development as biorational pesticides.

Fungal Material
The marine fungus N. pseudofischeri was isolated from the inner tissue of the starfish A. planci collected from the Hainan Sanya National Coral Reef Reserve, China.The fungus was identified on the basis of the DNA sequences of the ITS1-5.8S-ITS2(Internal Transcribed Spacer, ITS) regions of their rRNA gene.The ITS gene sequence showed 100% homology with that of the fungus N. pseudofischeri in GenBank (Accession Number KF999816).

Bioassay
To evaluate the biological activities of these compounds, cytotoxic assays were carried out with insect cultured cell line Sf9 from S. frugiperda.Sf9 cells were maintained at 27 ˝C in TC-199-MK medium supplemented with 10% FCS (v/v), 1% L-glutamine 200 mmol/L and penicillin-streptomycin-neomycin solutions (v/v).The cell growth inhibition was measured by the MTT method.Cells were seeded in 96-well microtitration plates at the exponential growth phase.Different concentrations of compounds diluted with medium were added at their log-phase growth stage.Additionally, 0.2 µL of solvent (DMSO) only was added as the control (CK).The final concentration of solvent in the cultures assayed was 1%.Compounds were solubilized with DMSO at concentrations of 10 and 50 mg/L.The rotenone was used as the positive control.After 6 h, 12 h, 24 h and 48 h of treatment, 5 mg/mL MTT were dissolved in PBS, and 20 µL of this stock solution were added to the culture cells.After an additional 3 h of incubation, the medium was discarded, and the 96-well plates were dried in the air.Then, 100 µL of DMSO were added to dissolve the formazan crystals, and the absorbance was measured at 570 nm by a microplate reader (Spectramex, 190 Molecular Devices Inc., Sunnyvale, CA, USA).
The cytotoxic effect was expressed as the relative percentage of inhibition calculated as follows: cell growth inhibition rate (%) = [(A control ´A treatment)/A control] ˆ100 [29].

Conclusions
In summary, sixteen compounds, including five new compounds (1, 4, 7, 9 and 10), were obtained from the mycelium of marine fungus N. pseudofischeri.Their structure types include phenylpyripyropenes, pyripyropenes, sesquiterpenoids, isocoumarin and alkaloids.Our findings provide further evidence that the marine fungus N. pseudofischeri has the tremendous potential of biosynthesis.The potent cytotoxicity of these compounds against the Sf9 cells revealed the prospect to develop biorational pesticides.

Table 2 .
1H and13C-NMR data of 7, 9 and 10, δ in ppm.The formula of Compound 8 was identified as C 17 H 26 O 4 , based on the analysis of 13 C-NMR data and HREIMS peak at m/z 294.1828 [M] + (calcd.for C 17 H 26 O 4 , 294.1826).The NMR spectroscopic data of Compound 8 were very similar to those of Compound 7, except that the C-2 hydroxyl group in 7 was replaced by an acetoxy group in 8 (Supplementary Figures