Polyketides and Meroterpenes from the Marine-Derived Fungi Aspergillus unguis 158SC-067 and A. flocculosus 01NT-1.1.5 and Their Cytotoxic and Antioxidant Activities

Ten secondary metabolites, including a new grifolin analog, grifolin B (1); a new homovalencic acid derivative, 12-hydroxyhomovalencic acid (7); and a compound isolated from a natural source for the first time (9), along with seven known compounds, grifolin (2), averantin (3), 7-chloroaverantin (4), 1′-O-methylaverantin (5), 7-hydroxy-2-(2-hydroxypropyl)-5-pentylchromone (6), homovalencic acid (8), and bekeleylactone E (10), were isolated from two fungal strains. The structures of 1–10 were identified by detailed analysis and comparison of their spectroscopic data with literature values. Compounds 9 and 10 showed moderate cytotoxic activity against a panel of cancer cell lines (PC-3, HCT-15, MDA-MB-231, ACHN, NCI-H23, NUGC-3), with the GI50 values ranging from 1.1 µM to 3.6 µM, whereas 1 displayed a weak 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity without cytotoxicity against all tested cell lines.


Introduction
Marine habitats have been acknowledged as prolific sources of new chemical entities with various worthwhile pharmacological activities [1]. Over the past decade, more than 1000 new marine natural products have been reported annually [2]. Whereas the discovery of new compounds from tunicates, cnidarians, and sponges is diminishing, there is a remarkable increase in the number of new substances isolated from marine-derived bacteria and fungi [2]. According to the latest statistics, new natural products (NPs) reported from marine-derived fungi accounted for almost half (47%) of the total new marine NPs reported in 2019 [2].
The genus Aspergillus is one of the most ubiquitous genera of filamentous fungi, and they are the major contributor to marine-derived fungal natural products [2,3]. A great number of secondary metabolites with structural diversity, such as polyketides, alkaloids, terpenes, steroids, and peptides, have been isolated from this genus, and many of them display potent biological activities [2].
As part of our ongoing program to investigate marine-derived fungi as an underexplored source of new natural products, we focused our attention on Aspergillus unguis 158SC-067 and A. flocculosus 01NT-1.1.5 strains, which showed good antimicrobial activity in the preliminary screening. Our previous studies on the EtOAc extract of A. flocculosus 01NT-1.1.5 grown on rice medium led to the isolation of fungal metabolites having antimicrobial properties and the suppression of RANKL-induced osteoclastogenesis activities [4,5]. To further study the secondary metabolites from marine-derived fungi, the 01NT-1.1.5 grown on rice medium led to the isolation of fungal metabolites having antimicrobial properties and the suppression of RANKL-induced osteoclastogenesis activities [4,5]. To further study the secondary metabolites from marine-derived fungi, the "one strain many compounds" (OSMAC) strategy was applied by changing the culture medium from rice medium to Bennett's broth medium. Interestingly, the 1 H NMR spectra of the crude extracts from A. unguis 158SC-067 and A. flocculosus 01NT-1.1.5 grown in Bennett's broth medium showed some unique peaks in aromatic and olefinic regions, which did not appear or were much smaller when cultured in the rice medium. Therefore, the extracts from two strains were chemically investigated. As a result, two new phenolic compounds (1 and 7), together with eight known compounds (2-6 and 8-10), were isolated ( Figure 1). Herein, we report the isolation, structure determination, and bioactivities of these compounds.
The structure of a symmetrical 1,4-disubstituted benzene ring was determined by the COSY correlations from H-4 to H-5 and from H-7 to H-8, and the HMBC correlations from H-4 to C-6 and C-8, from H-5 to C-3 and C-7, from H-7 to C-3 and C-5, and from H-8 to C-4 and C-6 ( Figure 2). A carboxy methyl group attached to the benzene ring at C-3 was supported by the HMBC correlations from H 2 -2 to C-1, C-3, C-4, and C-8. The HMBC correlation from H 2 -9 to C-6 supported that a prenyl unit was attached to C-4 via an ether linkage. The fact that CH 2 -12 bears a hydroxy group was evidenced by the chemical shift values of H 2 -12 (δ H 3.98) and C-12 (δ C 67.8) as well as the molecular formula. The geometry of the double bond between C-10 and C-11 was determined as 10E by the strong NOESY correlation from H-10 to H 2 -12 ( Figure S14). Thus, 7 is a new derivative of the co-isolated compound, homovalencic acid (8) [7], and named 12-hydroxyhomovalencic acid.
Since some of the previously reported compounds isolated in this work have been shown to possess cytotoxic activity [8,12], 1, 7, 9, and 10 were evaluated for their cytotoxicity against six cancer cell lines, HCT-15 (colon), NUGC-3 (stomach), NCI-H23 (lung) ACHN (renal), PC-3 (prostate), and MDA-MB-231 (breast), which are the most common cancer types in Korea. However, only 9 and 10 showed moderate cytotoxic activity against all of the tested cell lines, with GI 50 values ranging from 1.1 µM to 3.6 µM (Table 2). Additionally, 1 and 7 were screened for their DPPH radical scavenging activity. Compound 1 showed a weak DPPH radical scavenging activity with an IC 50 value of 86.4 µM, whereas 7 showed neither cytotoxic nor DPPH radical scavenging activity.

General Experimental Procedures
High-resolution ESIMS data were measured with a hybrid ion-trap time-of-flight mass spectrometer (Shimadzu LC/MS-IT-TOF, Kyoto, Japan). IR spectra were obtained on a JASCO FT/IR-4100 spectrophotometer (JASCO Corporation, Tokyo, Japan). The 1D and 2D NMR spectra were recorded by a Bruker 600 MHz spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany). HPLC was performed using a semi-preparative ODS column (YMC-Triart C18, 250 × 10 mm i. The seed and mass cultures were conducted in Bennett's medium (1% glucose, 0.2% tryptone, 0.1% yeast extract, 0.1% beef extract, 0.5% glycerol, natural sea salts 3.2%, and agar 1.7% for agar medium). At first, the fungus was grown on Bennett's agar medium in a Petri dish under static condition for 7 days. Agar plugs were cut into small pieces and aseptically transferred into a 500 mL conical flask containing 300 mL of Bennett's broth medium and placed on a rotary shaker (140 rpm) at 28 • C for 7 days for the seed culture. An aliquot (0.1% v/v) from the seed culture was inoculated into 2.0 L flasks, each containing 1.0 L of the medium, and cultured under the same conditions as described for the seed culture for 14 days. In total, 20 flasks were prepared for the mass production.
The mycelium was extracted with EtOAc (3.0 L, three times) and the EtOAc solution was evaporated under reduced pressure to yield a mycelium extract (2.0 g). The extract was fractionated into 10 fractions (fraction 1m-10m) by the same procedure described for the broth extract. Aspergillus flocculosus 01NT-1.1.5 was isolated from a Stylissa sp. sponge as previously described [4]. Based on NMR-guided isolation, the 1 H NMR spectrum of the crude extract from the culture broth of A. flocculosus 01NT-1.1.5 showed some interesting peaks in olefinic and aromatic regions. Therefore, the broth extract was selected for further study. The culture broth was extracted with EtOAc, and the organic extract was fractionated into 15 fractions as described previously [13].

Cytotoxicity Test by SRB Assay
Cytotoxicity Test by SRB Assay has been described previously [14].

DPPH Radical Scavenging Assay
DPPH radical scavenging assay was performed according to the previously described method with minor modification [3,15]. The samples and a positive control, ascorbic acid, were dissolved in DMSO with final concentrations of 6.25, 12.5, 25, 50, 100, and 200 µg/mL. DPPH was dissolved in anhydrous ethanol (EtOH) with a concentration of 0.04 mg/mL. Tested samples (50 µL) were added to 50 µL of fresh DPPH, then kept in room temperature in the dark for 30 min. The optical density (OD) was measured by an AMR-100 microplate reader (Hangzhou Allsheng Instruments, Hangzhou, China) at 517 nm. The EtOH and DMSO were used as a blank and negative control, respectively. The IC 50 values were determined by the software of GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA) [3].

Conclusions
In summary, on the basis of the OSMAC strategy, ten secondary metabolites, including two new phenolic derivatives (1 and 7), and a substance isolated from a natural source for the first time (9), together with seven known compounds (2-6, 8, and 10), were isolated from two fungal strains of the genus Aspergillus. Compounds 9 and 10 showed moderate cytotoxic activity, while 1 exhibited a weak DPPH radical scavenging activity without cytotoxicity. To the best of our knowledge, the known compounds (2-6) were isolated from A. unguis for the first time. Moreover, we also found that A. flocculosus 01NT-1.1.5 produces various chemical constituents in different culture media [4,13]. This study expanded the chemical and biological diversity of natural products isolated from marine-derived fungi.
The results indicate that marine-derived fungi, particularly the Aspergillus genus, could be a promising source to search for bioactive natural products with unique structures for discovery of new anti-cancer drugs.