Perylenequione Derivatives with Anticancer Activities Isolated from the Marine Sponge-Derived Fungus, Alternaria sp. SCSIO41014

Seven new secondary metabolites classified as two perylenequinone derivatives (1 and 2), an altenusin derivative (3), two phthalide racemates (4 and 5), and two phenol derivatives (6 and 7), along with twenty-one known compounds (8–28) were isolated from cultures of the sponge-derived fungus, Alternaria sp. SCSIO41014. The structures and absolute configurations of these new compounds (1–7) were determined by spectroscopic analysis, X-ray single crystal diffraction, chiral-phase HPLC separation, and comparison of ECD spectra to calculations. Altertoxin VII (1) is the first example possessing a novel 4,8-dihydroxy-substituted perylenequinone derivative, while the phenolic hydroxy groups have commonly always substituted at C-4 and C-9. Compound 1 exhibited cytotoxic activities against human erythroleukemia (K562), human gastric carcinoma cells (SGC-7901), and hepatocellular carcinoma cells (BEL-7402) with IC50 values of 26.58 ± 0.80, 8.75 ± 0.13, and 13.11 ± 0.95 μg/mL, respectively. Compound 11 showed selectively cytotoxic activity against K562, with an IC50 value of 19.67 ± 0.19 μg/mL. Compound 25 displayed moderate inhibitory activity against Staphylococcus aureus with an MIC value of 31.25 μg/mL.


Structural Elucidation
Compound 1 was obtained as a dark red powder. Its molecular formula was established as C20H16O4 by 13 C NMR data and the high resolution electrospray ionization mass spectroscopy (HRESIMS) [M + Na] + peak at m/z 343.0939 (calculated for C20H15O4Na, 343.0941), indicating thirteen degrees of unsaturation. Its UV spectrum showed maxima at 323 and 368 nm, which suggested that 1 featured a polycyclic aromatic hydrocarbons system. Its 1 H NMR data (Table 1)
Compound 3 possessed a molecular formula of C14H16O6 on the basis of its NMR data and the HRESIMS ion peak at m/z 303.0844 [M + Na] + , indicating seven indices of hydrogen deficiency. The 1 H NMR spectrum ( Table 2)  Because one benzene ring and one carbonyl accounted for five degrees of unsaturation, double ring fragment was required for the structure of 3. Its NMR data were similar to those of dihydroaltenuenes A (19) [15], the obvious differences being the disappearance of a methylene signal (δC 28.1) and the downfield shift of the oxygenated methine in 3, which indicated that a fragment of the six-membered Compound 2 was obtained as a dark red powder. Its molecular formula was assigned as C 24 H 20 O 7 based on its [M + Na] + ion at m/z 443.1110 and [M + H] + ion at m/z 421.1284 in the HRESIMS spectrum, indicating fifteen degrees of unsaturation. Analysis of its 1 H NMR and 13 C NMR data revealed that the structural features of 2 were similar to those of xanalteric acid II [5], except for the presence of an oxygenated n-butyl group (δ C/H 65.1/4.09, CH 2 -1 ; 30.0/1.46, CH 2 -2 ; 18.3/1.14, CH 2 -3 ; 13.4/0.71, CH 3 -4 ), which was confirmed by its COSY cross-peaks of The location of the oxygenated n-butyl group was ascertained by the HMBC correlation from H 2 -1 to C-13. Based on the HMBC correlations ( Figure 2), the planar structure of 2 was determined as butyl 2,4,9-trihydroxy-10-oxo-2,10-dihydro-1H-phenaleno[1,2,3-de] chromene-2-carboxylate and named butyl xanalterate. Due to the existence of the cyclic hemi-ketal, compound 2 was not stable under the protic solvent condition, meaning that the absolute configuration was uncertain.
Compound 3 possessed a molecular formula of C 14 H 16 O 6 on the basis of its NMR data and the HRESIMS ion peak at m/z 303.0844 [M + Na] + , indicating seven indices of hydrogen deficiency. The 1 H NMR spectrum (Table 2)  one sp 3 methylene, three sp 3 methines (two oxygenated at δ C 80.1, C-7; 71.2, C-8), two sp 2 methines (δ C 101.0, C-4; 108.9, C-6), four sp 2 non-protonated carbon, and an ester carbonyl (δ C 170.0, C-2). Because one benzene ring and one carbonyl accounted for five degrees of unsaturation, double ring fragment was required for the structure of 3. Its NMR data were similar to those of dihydroaltenuenes A (19) [15], the obvious differences being the disappearance of a methylene signal (δ C 28.1) and the downfield shift of the oxygenated methine in 3, which indicated that a fragment of the six-membered ring in 19 was replaced by a five-membered ring in 3. The deduction was confirmed by following COSY correlations of H-7a/H-7/H-8/H 2 -9, as well as HMBC correlations, from H-7 and H-7a to C-6a; from H-6, H 2 -9 and H 3 -10 to C-7a; and from H-8, H 2 -9 and H 3 -10 to C-9a ( Figure 2). The NOESY spectrum in DMSO-d 6 ( Figure 4) exhibited the cross-peaks of H-7a to H-9α, H 3 -10, OH-7, and OH-8, and also of H-9β to H-7, which suggested that CH 3 -10, H-7a, OH-7, and OH-8 were on the same side. The Cu Kα radiation for the X-ray diffraction experiment with the refined Flack parameter of −0.02(7) allowed the assignment of the absolute configuration of all the stereogenic centers in 3 as 7R, 7aR, 8S, and 9aS. Thus, the absolute structure of 3 was unambiguously elucidated and named nordihydroaltenuenes A. and 9aS. Thus, the absolute structure of 3 was unambiguously elucidated and named nordihydroaltenuenes A.  Compounds 4 and 5 were initially believed to be a single compound, which was obtained as a yellow oil. The molecular formula was established as C11H15O5 based on the HRESIMS and 13 C NMR data. A literature survey suggested that the 1 H NMR and 13 C NMR data ( Table 2) closely resembled those of isoochracinic acid [16], except for the presence of a methoxy group (δC/H 52.5/3.69, C-10) and an upfield shift of 1.3 ppm of the carbonyl (δC 171.6, C-9), which indicated the carboxyl in the isoochracinic acid was methylated. The deduction was confirmed by the HMBC correlation of H3-10 to C-9. The planar structure of 4/5 was further established by the COSY and HMBC correlations ( Figure 2). The optical rotation was small ([α] 25 D + 1.6) and the value of the ECD spectrum close to zero, suggesting that compounds 4 and 5 belonged to a racemate. Using a chiral-phase column (Daicel Chiraloak IC-3, 250 × 4.6 mm, 5 μm), the racemate was resolved to two enantiomers, 4 and 5, whose scale was nearly 1:1 ( Figure S30  Compounds 4 and 5 were initially believed to be a single compound, which was obtained as a yellow oil. The molecular formula was established as C 11 H 15 O 5 based on the HRESIMS and 13 C NMR data. A literature survey suggested that the 1 H NMR and 13 C NMR data ( Table 2) closely resembled those of isoochracinic acid [16], except for the presence of a methoxy group (δ C/H 52.5/3.69, C-10) and an upfield shift of 1.3 ppm of the carbonyl (δ C 171.6, C-9), which indicated the carboxyl in the isoochracinic acid was methylated. The deduction was confirmed by the HMBC correlation of H 3 -10 to C-9. The planar structure of 4/5 was further established by the COSY and HMBC correlations ( Figure 2). The optical rotation was small ([α] 25 D +1.6) and the value of the ECD spectrum close to zero, suggesting that compounds 4 and 5 belonged to a racemate. Using a chiral-phase column (Daicel Chiraloak IC-3, 250 × 4.6 mm, 5 µm), the racemate was resolved to two enantiomers, 4 and 5, whose scale was nearly 1:1 ( Figure S30). The optical rotation of 4 ([α] 25 D −21, c 0.1, MeOH) and 5 ([α] 25 D +26, c 0.1, MeOH), as well as the ECD spectrum ( Figure 5), were opposite to each other. In general, the 3R-phthalides displayed a positive optical rotation, while 3S-phthalides had a negative one [17][18][19][20]. Therefore, the absolute configuration of compound 4 was proposed as 3S and named (S)-isoochracinate A1, while compound 5 was proposed as 3R, and named (R)-isoochracinate A2. Compounds 6 and 7, obtained as a yellow powder, were also believed to be a single compound at first. The HRESIMS spectrum supported a molecular formula of C13H14O4, requiring 7 degrees of unsaturation. The 1 H NMR and 13 C NMR data ( Table 2) of 6/7 were almost identical to those of 4′-(S)-(3,5-dihydroxyphenyl)-4'-hydroxy-6'-methylcyclopent-1′-en-5′-one isolated from Penicillium sp. HN29-3B1 [21], indicating that 6/7 were a structural analog, except for the presence of a methoxy group (δC/H 55.7, C-7). The deduction was confirmed by the HMBC correlation (Table 2) from H3-7 to C-3. Analyzed by a chiral-phase column (Phenomenex Lux Cellulose-2, 250 × 4.6 mm, 5 μm), compounds 6/7 were found to be stereoisomeric mixture, whose mass ratio was about 1:3.5 ( Figure  S39). The single compound 6 ([α]  MeOH) were isolated, and they had opposite ECD spectra ( Figure 5). The optical rotation of 6 was consistent with that of 4′-(S)-(3,5-dihydroxyphenyl)-4′-hydroxy-6′-methylcyclopent-1′-en-5′-one [21], indicating that they had the same S configuration at C-4′, opposite to that of 7. Thus, the absolute structure of 6 was determined as 4′-(S)-(3-Methoxy-5-hydroxyphenyl)-4′-hydroxy-6′-methylcyclopent-1′-en-5′-one and named (S)-alternariphent A1, while the absolute structure of 7 was determined as 4′-(R)-(3-Methoxy-5-hydroxyphenyl)-4′-hydroxy-6′-methylcyclopent-1′-en-5′-one and named (R)-alternariphent A2.

Biological Activity
The cytotoxic activities of compounds 1, 2, and 8-12 against human erythroleukemia (K562), human gastric carcinoma cells (SGC-7901), and hepatocellular carcinoma cells (BEL-7402) were evaluated using the CCK-8 method, and paclitaxel was used as a positive control with IC 50 values of 0.18 ± 0.20, 0.89 ± 0.15, and 0.54 ± 0.20 µg/mL, respectively. Among the tested compounds, compound 1 exhibited cytotoxic activity against the K562, SGC-7901, and BEL-7402 cell lines with IC 50 values of 26.58 ± 0.80, 8.75 ± 0.13, and 13.11±0.95 µg/mL, respectively. Compound 11 showed selectively cytotoxic activity against K562 with an IC 50 value of 19.67 ± 0.19 µg/mL. All compounds were tested for their antibacterial activities against Staphylococcus aureus. Compounds 10 and 25 with 50 µg/disc displayed an inhibition zone with a diameter of about 21 and 15 mm, respectively ( Figure S1). Furthermore, their minimum inhibitory concentrations (MIC) were tested, and the MIC value of compound 25 was 31.25 µg/mL, while compound 10 showed more than 500 µg/mL-perhaps due to its poor solubility. Ampicillin was used as a positive control with an MIC value of 6.25 µg/mL.

Fungal Material
The fungal strain SCSIO41014 was obtained from a Callyspongia sp. sponge, which was collected from the sea area near Xuwen County, Guangdong Province, China. The producing strain was stored on MB agar (malt extract 15 g, agar 16 g, sea salt 10 g, water 1 L, pH 7.4-7.8) slants at 4 • C and deposited at the Key Laboratory of Tropical Marine Bio-resources and Ecology, Chinese Academy of Science. The ITS1-5.8S-ITS2 sequence region (508 base pairs, GenBank accession No. MH444654) of strain SCSIO41014 was amplified by PCR, and DNA sequencing showed it shared a significant homology to several species of Setosphaeria. The 508 base pairs of the ITS sequence had a 99% sequence identity to that of the Alternaria alternata strain SCAU091 (GenBank accession No. MF061753.1). It was thus designated as a member of Alternaria sp. and named Alternaria sp. SCSIO41014.

Fermentation and Extraction
The strain SCSIO41014 was cultured in 100 mL flasks (×59) each containing 10 mL seed medium (malt extract: 15 g, sea salt: 2.5 g, distilled water: 1 L, pH 7.4-7.8) at 27 • C on a rotary shaker (172 rpm) for 48 h. The mass fermentation of this fungus was carried out at 25 • C for 32 days using a rice medium (rice: 200 g/flask, sea salt: 2.5 g/flask, tap water: 200 mL/flask) in the 1 L flasks (×59). The flasks were incubated statically at 25 • C under the normal day and night cycle. After 32 days, cultures were soaked in acetone (400 mL/flask), mashed into small pieces, and vibrated with ultrasound for 20 min. Then the acetone solution was evaporated under reduced pressure to afford an aqueous solution, which was extracted with ethyl acetate (EtOAc) three times. Concurrently, the rice residue was extracted with EtOAc in order to make another EtOAc solution. Both of the EtOAc solutions were combined and concentrated under reduced pressure to produce a crude extract. The extract was then suspended in MeOH and partitioned with equal volumes of petroleum ether (PE). Finally, the MeOH solution was concentrated under reduced pressure to obtain a reddish-brown extract (78.0 g).

X-ray Crystal Structure Analysis
Crystallographic data for compound nordihydroaltenuenes A (3) were collected on a Rigaku XtaLAB PRO single-crystal diffractometer using Cu Kα radiation. The structure of 3 was solved by direct methods (SHELXS 97), expanded using difference Fourier techniques, and refined by using the full-matrix least-squares calculation. The non-hydrogen atoms were refined anisotropically, and hydrogen atoms were fixed at calculated positions. Crystallographic data for the structure 3 has been deposited with the Cambridge Crystallographic Data Centre with the supplementary publication number CCDC-1847869. Copies of the data can be obtained free of charge from the CCDC at www. ccdc.cam.ac.uk.

ECD Calculations
The theoretical calculations of new compound 1 was performed by using the density functional theory (DFT) as carried out in the Gaussian 03 [36]. Conformational analysis was initially conducted by using SYBYL-X 2.0 software. All ground-state geometries were optimized at the B3LYP/6-31G(d) level. TDDFT at B3LYP/6-31G(d) was employed to calculate the electronic excitation energies and rotational strengths in methanol [37,38]. Solvent effects of methanol solution were evaluated at the same DFT level by using the SCRF/PCM method [39].

Antibacterial Activity Assay
Compounds 1-28 were tested for antibacterial activities against Staphylococcus aureus using the agar filter-paper diffusion method. Then, compounds which had an inhibition zone were evaluated in 96-well plates using a modified version of the broth microdilution method, and ampicillin was used as a positive control [40].

Antitumor Activity Assay
The in vitro cytotoxic activities against the three tumor cell lines (K562, SGC-7901 and BEL-7402) were assessed by the CCK-8 method, and the positive control was taxol [41].

Conclusions
Seven new structurally diverse polyketide derivatives (1-7), along with 21 known compounds , were isolated from cultures of the sponge-derived fungus, Alternaria sp. SCSIO41014. The structures and absolute configurations of these new compounds (1-7) were determined by spectroscopic analysis, X-ray single crystal diffraction, chiral separation, and comparison of ECD spectra to the calculations. Altertoxin VII (1) was the first example to possess a novel 4,8-dihydroxy substituted perylenequinone derivative, while the phenolic hydroxy groups always commonly substituted at C-4 and C-9. Compound 1 exhibited cytotoxic activities against the K562, SGC-7901, and BEL-7402 cell lines, with IC 50 values of 26.58 ± 0.80, 8.75 ± 0.13, and 13.11 ± 0.95 µg/mL, respectively. Compound 11 showed selectively cytotoxic activity against K562 with an IC 50 value of 19.67 ± 0.19 µg/mL. Compound 25 displayed moderate inhibitory activity against Staphylococcus aureus, with an MIC value of 31.25 µg/mL.
Author Contributions: X.P., J.W., and Y.L. contributed conception and design of the study. X.P. performed experiments, analyzed data and wrote the manuscript, X.L. did the isolation and identification of the fungus. All authors contributed to manuscript revision, read and approved the submitted version.