Secondary metabolites from a deep-sea derived fungal strain of Phomopsis lithocarpus FS508

Abstract One new isoindolinone lactam lithocarlactam A (1) and one new triterpene lithocarin D (2), along with nine known natural products (3–11) were isolated from the broth extract of marine fungus Phomopsis lithocarpus FS508, which was isolated from a deep-sea sediment sample. Their structures were elucidated by interpretation of spectroscopic analysis and comparison with previously published literatures for the known compounds. Moreover, compounds 1 and 2 were evaluated for cytotoxic activity against SF-268, MCF-7, HepG-2, and A549 cell lines. However, both of them showed no activity against the tumor cell lines. Graphical Abstract


Introduction
Marine-derived fungi are demonstrated to be one of the most rich and reliable sources of biologically meaningful natural products and has attracted increasing attention in recent decades. Considerably growing number of secondary metabolites with attractive skeletons and promising bioactivities have been isolated and characterized from marine fungi in recent years (Aly et al. 2011;Rateb and Ebel 2011;Deshmukh et al. 2017;Carroll et al. 2021). On our continuous searching for novel metabolites from the marine-derived fungi Chen et al. 2020), Phomopsis lithocarpus FS508 has successfully attracted our attention. The chemical composition analysis of its solid culture showed that this strain was fruitful in structurally special polyketides including lithocarpins (Xu et al. , 2021, lithocarols (Xu et al. 2019), lithocaldehydes , and tenellones .
The complete structure of 1 was determined by the analysis of its 2 D NMR data. The HMBC correlations from H-4 to C-2 (d C 117.3) and C-6 (d C 115.8), H-5 to C-3 (d C 156.4) and C-7 (d C 151.6) coupling with H-6 to C-2 and C-4 (d C 115.4) indicated the presence of an aryl ring A in the molecule. Moreover, the HMBC correlations from the C-methyl resonating at d H 2.17 to both aromatic methine carbons at d C 114.4 (C-12) and 119.6 (C-14), enabled the methyl to be positioned at position C-13. The HMBC correlations of H-12 to C-10 (d C 143.8) and C-14, H-14 to C-10 and C-12 further confirmed the existence of a 1,2,3,4-treta-sustituted phenyl ring B in 1. Furthermore, the remaining two methyls and three oxygenated carbons resonated at d C 71.6 (C-1 0 ), 77.7 (C-2 0 ), 72.6 (C-3 0 ), 26.9 (C-4 0 ), 25.1 (C-5 0 ), as well as the HMBC correlations of H 2 -1 0 to C-3 0 , H-2 0 to C-4 0 and C-5 0 , H 3 -4 0 and H 3 -5 0 to C-2 0 and C-3 0 , suggested the presence of an isopentenyl unit. The isopentenyl moiety was concluded to be located at the C-11 position by the critical HMBC correlation from H-1 0 to C-11. Then, the remaining unassigned atoms of 1 consisted of a C 2 H 2 NO unit including a carbonyl group at d C 173.9. With all atoms and nine of the 10 degrees for 1 had to incorporate a third ring, and the only plausibility was the formation of a five-membered lactam ring, which was further verified by the HMBC correlations of H-8 to C-1 (173.9) and C-2. Therefore, the planar structure of 1 was established as shown in Figure S18.
Finally, the X-ray diffraction experiment was carried out on the CuKa with a Flack parameter of 0.56(13), clarifying the relative configuration of 1 ( Figure S19). Compound 1 might be existed as racemic enantiomers due to the lack of specific optical rotation and CD Cotton effects. Moreover, it is worth mentioning that other structurally similar isoindolinone alkaloids have also been isolated as racemic mixtures, such as pestalachloride A (Li et al. 2008) and entonalactam A (Vanida et al. 2015). In light of the aforementioned evidence, the structure of 1 was established as shown in Figure 1, and assigned as 3-(3-(2,3-dihydroxy-3-methylbutoxy)-2-hydroxy-5-methylphenyl)-7-hydroxyisoindolin-1-one with the trivial name of lithocarlactam A. Compound 2 was obtained as a colorless oil. Its molecular formula was established as C 30 H 52 O 3 with five indices of hydrogen deficiency, based on the molecular ion peak [M þ H] þ determined at m/z 461.3986 with the aid of HRESIMS spectrum. The 13 C NMR data (Table S2) of 2 displayed 30 carbon signals, which can be sorted into eight methyls, ten methylenes, six methines, and six quaternary carbons. Moreover, the 1 H NMR (Table  S2) spectrum showed resonances for eight methyl groups including three vinyl ones [d H 1.62 (3H, s, H-25), 1.60 (6H, s, H-26, 27)]. The 1 H NMR data showed characteristic signals for three down-shielded multiplets at d H 5.14 (2H) and 5.19, which were assigned to vinyl moiety of H-10, H-13, and H-17. From the above evidence, compound 2 was concluded to be a squalene derivative with the structure featuring a bicyclic system.

In vitro cytotoxicity assay
Compounds 1 and 2 were evaluated for their in vitro cytotoxicities against SF-268, MCF-7, HepG-2, and A549 cell lines with adriamycin as the positive control. However, compounds 1 and 2 exhibited no growth inhibitory activities against the four tumor cell lines even at the concentration of 100 mM.

Fungal material and identification
The strain FS508 used in this work was isolated from a sediment sample, which was collected at the depth of 3606 m in the Indian Ocean (111 53.335' E, 16 50.508' N) in May, 2016. The sequence data for this strain have been submitted to the GenBank under accession No. MG686131. By using BLAST (nucleotide sequence comparison program) to search the GenBank database, FS508 has 99% similarity to Phomopsis lithocarpus CZ105B (Accession No. FJ755236). The strain was preserved at the Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Institute of Microbiology, Guangdong Academy of Sciences.

Fermentation, extraction, and isolation
The marine fungus Phomopsis lithocarpus FS508 was cultured on potato dextrose agar (PDA) at 28 C for 5 days to prepare the seed culture, and then inoculated into flasks (3 L) each containing 30 g of glucose, 9 g of peptone, 45 g of yeast extract, and 1.5 L natural filtered seawater. After that, all flasks (50 L) were incubated on a rotary shaker (120 rpm) at room temperature for 12 days. All fermentation products were extracted three times with EtOAc and then concentrated under reduced pressure to obtain a dark brown crude extract (37.1 g).

In vitro cytotoxicity assay
Cytotoxicities of compounds 1-4 were assayed against four tumor cell lines including SF-268 (human glioma cell line), MCF-7 (human breast adenocarcinoma cell line), HepG-2 (human liver cell line), and A549 (human lung cancer cell line). Assays were performed by the SRB method (Skehan et al. 1990).

Conclusion
In conclusion, a liquid culture fermentation of Phomopsis lithocarpus FS508 was carried out and the chemical constituents of the fermentation broth of this strain was investigated. As a result, two new secondary metabolites lithocarlactam A (1) and lithocarin D (2) together with nine known natural products were isolated. The new compounds 1 and 2 were evaluated for in vitro cytotoxicity by SRB method. Unfortunately, compounds 1 and 2 showed no activity against the tumor cell lines.