Sorbicillasins A–B and Scirpyrone K from a Deep-Sea-Derived Fungus, Phialocephala sp. FL30r

Two new nitrogen-containing sorbicillinoids named sorbicillasins A and B (1 and 2) and a new 3,4,6-trisubstituted α-pyrone derivative, scirpyrone K (3), together with two known biosynthetically related polyketides (4–5), were isolated from the deep-sea-derived fungus Phialocephala sp. FL30r by using the OSMAC (one strain-many compounds) method. The structures of 1–3, including absolute configurations, were deduced based on MS, NMR, and time-dependent density functional theory (TD-DFT) calculations of specific ECD (electronic circular dichroism) spectra. Compounds 1 and 2 possessed a novel hexahydropyrimido[2,1-a] isoindole moiety, and compound 3 exhibited weak radical scavenging activity against DPPH (2,2-diphenyl-1-picrylhydrazyl) with an IC50 value of 27.9 μM.


Introduction
Filamentous fungi are known as prolific microbial factories for the production of a wide range of metabolites having extensive biological activity [1]. However, previous genomic analysis of fungi revealed a large number of biosynthetic genes that were unexpressed under common laboratory culture conditions, which offers a great opportunity for natural product discovery research [2]. To induce the expression of silent biogenetic clusters and increase the chemical diversity of the secondary metabolome, the approach "one strain-many compounds (OSMAC)" has been widely and successfully practiced by altering media constituents and manipulating culture conditions [3][4][5].
During our ongoing search for bioactive secondary metabolites from deep-sea-derived fungi [6], the fungal strain Phialocephala sp. FL30r was found to be an extensive producer of diverse polyketides, including monomeric sorbicillinol derivatives, bisorbicillinoids, and trisorbicillinoids [7][8][9][10]. Based upon the biosynthetic capability of this strain, the OSMAC approach was employed to further enhance the structural diversity of secondary metabolites. When the fungal strain was cultured on a mannitol-based medium, the HPLC-UV profile ( Figure S1) of the fungal extract differed from those generated previously from culture in a potato-based medium [7][8][9][10]. Further chemical assessment of the organic extract led to the isolation of two new nitrogen-containing sorbicillinoids named sorbicillasins A and B (1 and 2) and a new 3,4,6-trisubstituted α-pyrone derivative, scirpyrone K (3), together with two known biosynthetically related polyketides (4)(5) [11,12]. Among them, a] isoindole tricyclic skeleton. The radical scavenging activities of the new compounds against DPPH (2,2-diphenyl-1-picrylhydrazyl) were tested, and 3 showed weak activity with an IC50 value of 27.9 μM. Herein, we report the details of the isolation, structure elucidation, and biological activities of compounds 1-5.

Results and Discussion
The fungus Phialocephala sp. FL30r was cultured in mannitol-based medium (45.0 L) with agitation. The EtOAc extract (15.0 g) of fermentation was fractionated by silica gel column chromatography, Sephadex LH-20 column chromatography, medium-pressure preparation liquid chromatography (MPLC; ODS), and semi-preparative HPLC to afford compounds 1 (4.5 mg), 2 (2.7 mg), 3 (7.0 mg), 4 (4.4 mg), and 5 (2.0 mg) ( Figure 1). Sorbicillasins A and B (1 and 2) were both obtained as yellow oils with the molecular formula C19H22N2O6 according to the protonated peak at m/z 375.1550 ( Figure S9, calcd. for C19H23N2O6, 375.1151) and the sodinated peak at m/z 397.1376 (Figure S17, calcd. for C19H22N2O6Na, 397.1370) as analyzed by HRESIMS, respectively. The 1D NMR data (Table 1) of 1 and 2 were highly similar. Each set of data suggested the presence of 19 carbons, assigned as 3 methyls, 3 methylenes, 3 methines with 2 vinyl moieties, and 10 non-protonated carbons, including 3 carbonyls. Among the three carbonyls, one was proven to be a carboxylic group based on the exchangeable proton signal at δH 13.12, together with the IR absorptions at 3524 and 1670 cm −1 . The planar structures of 1 and 2 were determined to be the same by interpretation of 1D and 2D NMR spectroscopic data ( Figure 2, Table  1, Figure S2-S7, and Figure S11-S15). The methylated olefinic hydrocarbon chain (from C-16 to C-20) was established by the sequential COSY correlations of H2-16/H2-17/H-18/H-19/H3-20. The presence of a fully substituted benzene ring was indicated by the aromatic non-protonated carbon signals (C-1/C-6) in the 13 C-NMR spectrum. Consistent with this analysis, diagnostic HMBC correlations were observed from H3-14 to C-1, C-2, and C-3; from H3-15 to C-3, C-4, and C-5; from 3-OH to C-2, C-3, and C-4; and from 5-OH to C-6. The presence of a tetrahydro-pyrimidinone ring was postulated based on the COSY correlation (H-10/H-11) and the HMBC correlations from 8-NH to C-7, C-9, and C-10; from H2-10 to C-9 and C-11; and from H-11 to C-7, C-9, and C-10. The HMBC correlations from 8-NH and H2-16 to C-6 confirmed the connection of the tetrahydro-pyrimidinone structure and the benzene ring. The HMBC correlation from H2-16 to C-7 positioned the olefinic chain at C-7. Based on the key  (Table 1) of 1 and 2 were highly similar. Each set of data suggested the presence of 19 carbons, assigned as 3 methyls, 3 methylenes, 3 methines with 2 vinyl moieties, and 10 non-protonated carbons, including 3 carbonyls. Among the three carbonyls, one was proven to be a carboxylic group based on the exchangeable proton signal at δ H 13.12, together with the IR absorptions at 3524 and 1670 cm −1 . The planar structures of 1 and 2 were determined to be the same by interpretation of 1D and 2D NMR spectroscopic data ( Figure 2, Table 1, Figures S2-S7, and Figures S11-S15). The methylated olefinic hydrocarbon chain (from C-16 to C-20) was established by the sequential COSY correlations of H 2 -16/H 2 -17/H-18/H-19/H 3 -20. The presence of a fully substituted benzene ring was indicated by the aromatic non-protonated carbon signals (C-1/C-6) in the 13 C-NMR spectrum. Consistent with this analysis, diagnostic HMBC correlations were observed from H 3 -14 to C-1, C-2, and C-3; from H 3 -15 to C-3, C-4, and C-5; from 3-OH to C-2, C-3, and C-4; and from 5-OH to C-6. The presence of a tetrahydro-pyrimidinone ring was postulated based on the COSY correlation (H-10/H-11) and the HMBC correlations from 8-NH to C-7, C-9, and C-10; from H 2 -10 to C-9 and C-11; and from H-11 to C-7, C-9, and C-10. The HMBC correlations from 8-NH and H 2 -16 to C-6 confirmed the connection of the tetrahydro-pyrimidinone structure and the benzene ring. The HMBC correlation from H 2 -16 to C-7 positioned the olefinic chain at C-7. Based on the key HMBC correlations from H-11 to C-13 and C-21, together with the chemical shift of C-11 (δ C 48.2) and C-13 (δ C 169.0), two carbonyls (C-13 and C-21) were connected to N-12 and C-11, respectively. Finally, when accounting for the molecular formula and the degree of unsaturation, C-1 was linked to C-13, and one hydroxyl group was attached to C-21, thus completing the planar structure of 1.
Mar. Drugs 2018, 16, x FOR PEER REVIEW 3 of 9 HMBC correlations from H-11 to C-13 and C-21, together with the chemical shift of C-11 (δC 48.2) and C-13 (δC 169.0), two carbonyls (C-13 and C-21) were connected to N-12 and C-11, respectively. Finally, when accounting for the molecular formula and the degree of unsaturation, C-1 was linked to C-13, and one hydroxyl group was attached to C-21, thus completing the planar structure of 1. The relative configuration of 1 was deduced based on the NOESY correlations ( Figure 3 and Figure S8). The E geometries of double bonds in the olefinic chain were deduced by the correlations between H-17 and H-19 and between H-18 and H-20. The NOEs of H-10a/H-11 and H-10b/H-16 indicated that the carboxylic group and methylated olefinic hydrocarbon chain were to the same face of the pyrimidinone ring. Thus, the relative configuration of 1 was suggested to be 7R*, 11S*. The absolute configuration of 1 was determined by comparing the experimental ECD curve with the one calculated from the truncated model (7R,11S)-1a using time-dependent density functional theory (TD-DFT). The DFT re-optimization of the initial MMFF (Merck molecular force field) minima was performed at the B3LYP/6-31+g(d) level with a polarizable continuum model (PCM) solvent for MeOH. The strong agreement between the calculated ECD spectra of (7R,11S)-1a with experimental results suggested the absolute configuration of 1 as 7R, 11S ( Figure 4 and Figure S27).
The slight discrepancies of 1 and 2 in the NMR data suggested they might be isomers. Further NOESY correlation ( Figure 3 and Figure S16) of H-11/H2-16 indicated that the relative configuration of 2 was 7S*, 11S*. The absolute configuration of 2 was then further determined as 7S, 11S by the agreement between the calculated ECD spectra of 2 and the experimental results according to the truncated model (7S,11S)-1b ( Figure 5 and Figure S28).  The relative configuration of 1 was deduced based on the NOESY correlations (Figures 3 and S8). The E geometries of double bonds in the olefinic chain were deduced by the correlations between H-17 and H-19 and between H-18 and H-20. The NOEs of H-10a/H-11 and H-10b/H-16 indicated that the carboxylic group and methylated olefinic hydrocarbon chain were to the same face of the pyrimidinone ring. Thus, the relative configuration of 1 was suggested to be 7R*, 11S*. The absolute configuration of 1 was determined by comparing the experimental ECD curve with the one calculated from the truncated model (7R,11S)-1a using time-dependent density functional theory (TD-DFT). The DFT re-optimization of the initial MMFF (Merck molecular force field) minima was performed at the B3LYP/6-31+g(d) level with a polarizable continuum model (PCM) solvent for MeOH. The strong agreement between the calculated ECD spectra of (7R,11S)-1a with experimental results suggested the absolute configuration of 1 as 7R, 11S (Figures 4 and S27   The slight discrepancies of 1 and 2 in the NMR data suggested they might be isomers. Further NOESY correlation (Figures 3 and S16) of H-11/H 2 -16 indicated that the relative configuration of 2 was 7S*, 11S*. The absolute configuration of 2 was then further determined as 7S, 11S by the agreement        Compound 3 was obtained as a white amorphous powder, and the molecular formula was determined to be C 10 H 12 O 5 by HRESIMS peaks at m/z 213.0764 ( Figure S25, calcd. for C 10 H 13 O 5 , 213.0757). The 1D NMR data ( Table 2) of 3 suggested the presence of two methyls, including one methoxy (δ C 52.0 and δ H 3.58), two methylenes, one methine, and five non-protonated carbons. Comparison of the 1 H and 13 C NMR spectra (Figures S19-S21) of 3 with those of scirpyrone H revealed the presence of an extra methyl group (δ C 8.8 and δ H 1.72) and the replacement of the 4-methoxyl group by a 4-hydroxyl group in 3 [13]. Further 2D NMR ( Figures S22-S24) data and key HMBC correlations from H 3 -11 to C-2, C-3, and C-4, from H-5 to C-3, C-4, and C-6, and from 4-OH to C-3, C-4, and C-5 supported the locations of the 4-hydroxyl group and the 11-methyl group. Compounds 4 and 5 were identified as trichopyrone [11] and peniginseng A [12] based on the comparison of their spectroscopic data (NMR and MS) with those reported in the literature.
The cytotoxicity against K562 and MGC-803 cell lines [14,15] and the radical scavenging activity [16] against DPPH of the new compounds 1-3 were evaluated. All of them were non-cytotoxic. Compound 3 exhibited weak activity against DPPH with an IC 50 value of 27.9 µM (ascorbic acid was used as a positive control with an IC 50 value of 14.2 µM), whereas compounds 1 and 2 were not active (IC 50 > 500 µM). According to the literature [11,12], the known compound 4 showed weak radical scavenging activity, but the radical scavenging activity of 5 has not been reported.
Sorbicillinoids belong to a large family of polyketides with highly diverse carbon skeletons and bioactivities [17]. Since first reported in 1948, about 90 sorbicillinoids have been isolated from terrestrial-and marine-derived fungi [17]. Among them, the nitrogen-containing analogues are rare, with only eight related cases reported, including sorbicillactones A and B [18], sorbicillinoid urea [19], and sorbicillamines A-E [20]. The sources of nitrogen atoms in the reported nitrogenous sorbicillinoids were deduced to be L-alanine, urea, and an aminotransferase enzyme [18][19][20]. In this report, sorbicillasins A and B (1 and 2) were probably formed by adding a whole molecule of L-asparagine to 2 ,3 -dihydrosorbicillin [21] (Figure 6) via sequential intermolecular/intramolecular nucleophilic reactions. The hexahydropyrimido[2,1-a] isoindole ring system in compounds 1 and 2, which compose a 6/5/6 tricyclic ring system, have not been found in nature, with only related synthetic structures reported [22][23][24]. The above result shows that the OSMAC approach is a useful method to discover structurally diversified metabolites from a deep-sea-derived fungal strain.

Fungal Material
The fungal strain FL30r has been previously described [7][8][9][10]. The strain was deposited at the Key Laboratory of Marine Drugs, the Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao, China.

Fermentation and Extraction
Erlenmeyer flasks (500 mL) containing 150 mL fermentation medium were directly inoculated with spores. The media contained mannitol (20.

Fungal Material
The fungal strain FL30r has been previously described [7][8][9][10]. The strain was deposited at the Key Laboratory of Marine Drugs, the Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao, China.

Fermentation and Extraction
Erlenmeyer flasks (500 mL) containing 150 mL fermentation medium were directly inoculated with spores. The media contained mannitol (20. collected seawater (Huiquan Bay, Yellow sea). The flasks were cultured at 28 • C on a rotary platform shaker at 180 rpm for 9 days. The fermentation broth (45.0 L) was filtered through cheese cloth to separate the supernatant from mycelia. The supernatant was extracted with EtOAc (3 × 45.0 L) and evaporated under reduced pressure to produce the crude gum (15.0 g) [26].

Biological Assay
Cytotoxic activities of 1-3 were evaluated using an MGC-803 cell line by the SRB (Sulforhodamine B) method [14] and the K562 cell line by the MTT method [15]. The positive control was doxorubicin hydrochloride. In the DPPH scavenging assay [16], samples to be tested were dissolved in MeOH and the solution (160 µL) was dispensed into wells of a 96-well microtiter tray. Forty microliters of the DPPH solution in MeOH were added to each well. The mixture was shaken and left to stand for 30 min. After the reaction, absorbance was measured at 510 nm, and the percent inhibition was calculated. IC 50 values denoted the concentration of sample required to scavenge 50% of the DPPH free radicals [27].

Computation Section
Conformational searches were run by employing the "systematic" procedure implemented in Spartan 14 [28] using MMFF (Merck molecular force field), which were all reoptimized with DFT calculations at the B3LYP/6-31+G(d) level using the Gaussian09 program [29]. The geometry of various initial conformations was optimized. Vibrational frequency calculations confirmed the presence of minima. Time-dependent DFT calculations were performed on the three lowest-energy conformations for (7R,11S)-1a and three lowest-energy conformations for (7S,11S)-1b (>4% population) using 30 excited states and a polarizable continuum model (PCM) for MeOH. ECD spectra were generated using the program SpecDis [30] by applying a Gaussian band shape with 0.30 eV width for 1a and 0.35 eV width for 1b, from dipole length rotational strengths. The dipole velocity forms yielded negligible differences. The spectra of the conformers were combined using Boltzmann weighting, with the lowest-energy conformations accounting for 100% of the weights. The calculated spectra were shifted for 1a (4 nm) and for 1b (0 nm) to facilitate comparison to the experimental data.