Cytotoxic Polyketide Metabolites from a Marine Mesophotic Zone Chalinidae Sponge-Associated Fungus Pleosporales sp. NBUF144

Two new polyketide natural products, globosuxanthone F (1), and 2′-hydroxy bisdechlorogeodin (2), were isolated from the fungus Pleosporales sp. NBUF144, which was derived from a 62 m deep Chalinidae family sponge together with four known metabolites, 3,4-dihydroglobosuxanthone A (3), 8-hydroxy-3-methylxanthone-1-carboxylate (4), crosphaeropsone C (5), and 4-megastigmen-3,9-dione (6). The structures of these compounds were elucidated on the basis of extensive spectroscopic analysis, including 1D and 2D NMR and high-resolution electrospray ionization mass spectra (HRESIMS) data. The absolute configuration of 1 was further established by single-crystal X-ray diffraction studies. Compounds 1–5 were evaluated for cytotoxicity towards CCRF-CEM human acute lymphatic leukemia cells, and it was found that 1 had an IC50 value of 0.46 µM.

Mesophotic coral ecosystems (MCEs or "Twilight zone" coral reefs) range from 30 to 150 m deep and represent approximately 80% of potential coral reef habitats worldwide, but little is known about them compared with shallow reefs [20,21]. In an ongoing investigation covered.
Mesophotic coral ecosystems (MCEs or "Twilight zone" coral reefs) range from 30 to 150 m deep and represent approximately 80% of potential coral reef habitats worldwide, but little is known about them compared with shallow reefs [20,21]. In an ongoing investigation of fungal metabolites [22,23], rare or unexplored sponge-derived fungi from MCEs have attracted the attention of these authors and others. Several fungi with novel metabolite-producing potential were prioritized for further study after applying the combined strategy of LC-MS/MS molecular networking [24] with the OSMAC (One Strain MAny Compounds) approach [25] of crude extracts from 80 sponge-associated fungi (40 from shallow water and 40 from MCEs;Supplementary Material Figure S1). For example, in the previous work, the organism first prioritized was a Cymostachys fungus that yielded a series of new polyketide compounds, cymopolyphenols A-F [26]. Among them, cymopolyphenols D-F are low-order polymers of cymopolyphenols A that have novel chemical scaffolds. As a continuation of this work of mining new or novel fungal metabolites from MCEs, a second organism was prioritized from the same strain library after the previously reported molecular network [26] was adapted to highlight the secondary metabolite-producing activity of a Pleosporales sp. NBUF144 fungus, which was derived from a 62 m deep Chalinidae family sponge ( Figure S1). The chemical investigation of this organism yielded compounds 16 (Figure 1), and the details of these experiments and associated biological testing results are reported herein.

Bioactivity Assay
Globosuxanthone A has been reported to yield significant cytotoxicity towards eight human solid tumor cell lines, including NCI-H460, MCF-7, SF-268, PC-3, PC-3M, LNCaP, DU-145, and HCT-15, as well as T-cell leukemia Jurkat cells [27,29]. These data inspired in vitro cytotoxicity testing of the compounds isolated here. Compound 1 exhibited potent cytotoxicity in vitro against CCRF-CEM T-cell leukemia cells with a IC 50 value of 0.46 µM. Compounds 3-5 each showed no pronounced cytotoxicity at 20 µM (Figure 4), despite sharing almost identical scaffolds with globosuxanthone A and 1, suggesting the importance of the ∆3,4 unsaturation (comparing 1 to 3) and the presence of hydroxy groups at C-1 and C-2 in this scaffold (comparing 1 to 4) for yielding cancer cell cytotoxicity. Compound 6 was not obtained in sufficient quantity for testing in this study.

Bioactivity Assay
Globosuxanthone A has been reported to yield significant cytotoxicity towards eight human solid tumor cell lines, including NCI-H460, MCF-7, SF-268, PC-3, PC-3M, LNCaP, DU-145, and HCT-15, as well as T-cell leukemia Jurkat cells [27,29]. These data inspired in vitro cytotoxicity testing of the compounds isolated here. Compound 1 exhibited potent cytotoxicity in vitro against CCRF-CEM T-cell leukemia cells with a IC50 value of 0.46 μM. Compounds 3-5 each showed no pronounced cytotoxicity at 20 μM (Figure 4), despite sharing almost identical scaffolds with globosuxanthone A and 1, suggesting the importance of the Δ3,4 unsaturation (comparing 1 to3) and the presence of hydroxy groups at C-1 and C-2 in this scaffold (comparing 1 to 4) for yielding cancer cell cytotoxicity. Compound 6 was not obtained in sufficient quantity for testing in this study.

General
Optical rotations were measured with a JASCO P-2000 automatic polarimeter in MeOH at 20 °C. The CD spectra were recorded on a JASCO P-1500 spectropolarimeter. NMR spectra were recorded in CDCl3 using residual solvents as internal standards with a Bruker AVANCE NEO 600 spectrometer with a 5 mm inverse detection triple resonance (H-C/N/D) cryoprobe having z-gradients. The chemical shift values are given in parts per million (ppm) relative to TMS at 0.0 ppm, and the coupling constants are in Hertz. High-resolution electrospray ionization mass spectra (HRESIMS) were measured on an Agilent (Santa Clara, CA, USA) 6545 Q-TOF instrument. Reversed-phase HPLC purification was carried out on a Waters HPLC equipped with a 1525 binary pump, and a Thermo Scientific (Waltham, MA, USA) ODS-2 Hypersil column (5 μm, 250 × 10 mm). Normal phase column chromatography and thin-layer chromatography were performed using silica gel (200-300 mesh) and GF254 (10-20 mm) (Qingdao Marine Chemical Company, Qingdao, China). YMC*GEL ODS-A (AA12S50; YMC Co., Ltd, Japan) was used for reverse phase column chromatography. Sephadex LH-20 was a product from GE Biotechnology, USA.

General
Optical rotations were measured with a JASCO P-2000 automatic polarimeter in MeOH at 20 • C. The CD spectra were recorded on a JASCO P-1500 spectropolarimeter. NMR spectra were recorded in CDCl 3 using residual solvents as internal standards with a Bruker AVANCE NEO 600 spectrometer with a 5 mm inverse detection triple resonance (H-C/N/D) cryoprobe having z-gradients. The chemical shift values are given in parts per million (ppm) relative to TMS at 0.0 ppm, and the coupling constants are in Hertz. High-resolution electrospray ionization mass spectra (HRESIMS) were measured on an Agilent (Santa Clara, CA, USA) 6545 Q-TOF instrument. Reversed-phase HPLC purification was carried out on a Waters HPLC equipped with a 1525 binary pump, and a Thermo Scientific (Waltham, MA, USA) ODS-2 Hypersil column (5 µm, 250 × 10 mm). Normal phase column chromatography and thin-layer chromatography were performed using silica gel (200-300 mesh) and GF254 (10-20 mm) (Qingdao Marine Chemical Company, Qingdao, China). YMC*GEL ODS-A (AA12S50; YMC Co., Ltd., Japan) was used for reverse phase column chromatography. Sephadex LH-20 was a product from GE Biotechnology, USA.

Fungal Strains
The fungal strain was isolated from a Chalinidae family sponge collected in 2018 by scientific-technical SCUBA diving at the depth of 62 m near Apo Island, Negros Oriental, Philippines (9 • 04 40.6" N 123 • 15 57.3" E and 9 • 04 33.0" N 123 • 15 59.1" E). The inner tissue of the sponge was sliced into squares (0.5 × 0.5 × 0.5 cm 3 ) and incubated on plates Mar. Drugs 2021, 19, 186 6 of 9 containing modified fungal mediums described elsewhere [22]. The sponge sample loaded petri-dishes were then incubated at 28 • C for two weeks. The fungal colonies were picked and sub-cultivated on PDA (Potato Dextrose Agar; potato 200.0 g, glucose 20.0 g, sea salt 35.0 g, agar powder 20.0 g, and H 2 O up to a total volume of 1 L) media.
The fungal strain further grown and evaluated in this study was identified as a Pleosporales sp. based on morphological traits and sequence analysis of the ITS region (GenBank accession no. MW386403).

Cultivation of the Fungi
For chemical investigation, the Pleosporales sp. was cultured in 250 × 1 L Erlenmeyer flasks, each containing 400 mL PDB medium (80 g potato, 8 g glucose, 14.0 g sea salt, 400 mL H 2 O), for 15 days at 28 • C with agitation (120 rpm). The culture broth was filtered through cheesecloth to separate it into filtrate and mycelia (retained in the cheese), and both of them were extracted with EtOAc (v/v, 1:1) for 5 times and MeOH and DCM (v/v, 1:1) for 3 times, respectively. The separated extracts of the filtrate and mycelia were combined for their similar spots in TLC analysis to afford a total crude organic extract (280 g).

Extraction and Isolation
The crude extract was subjected to column chromatography (CC) over silica gel

Single-Crystal X-ray Diffraction Analysis
The crystals obtained for 1 and 2 were tested on a Bruker APEX-II CCD diffractometer through Ga Kα (λ = 1.34139 Å). The structures were solved by direct methods (SHELXT-2014) and refined via full-matrix least-squares difference Fourier techniques using SHELXL-2018/3. Crystallographic data for the structures have been deposited with the Cambridge Crystallographic Data Centre. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-(0)1223-336033 or e-mail: deposit@ccdc.cam.ac.uk).
Crystallographic data for 1:

Cytotoxicity Assay
CCRF-CEM human T lymphoblast cells were obtained from the National Collection of Authenticated Cell Cultures, Shanghai. Compounds 1-5 were diluted serially from 10 uM mother solution in DMSO to make the final concentration from 0 to 20 µM and the final DMSO concentration was ≤0.1% in the reaction mixture. CCRF-CEM cells (5 × 10 4 in 100 µL) and the tested compounds were co-incubated in a 96-well plate and the absorbance at 490 nm were recorded after published protocols [38,39]. The IC 50 values were calculated using GraphPad Prism 7 software.

Conclusions
The chemical investigation of the MCE sponge-associated fungus Pleosporales sp. NBUF144, after prioritization by a combination of the OSMAC approach and LC-MS/MS molecular networking, led to the isolation of two new polyketide natural products (1-2). Compounds 1-5 were screened for in vitro cytotoxicity towards CCRF-CEM cells. Compound 1 showed strong cytotoxic activity with an IC 50 value of 0.46 µM, while 3 was not active at concentrations up to 20 µM, indicating the importance of the ∆3,4 olefin moiety in the scaffold of these compounds for this biological activity. Since compound 4 was also not active, in comparison with 1, it is suggested that the C-1 and C-2 hydroxy groups are also important for cytotoxicity. Together, this extends the known potential of this chemical class, as drugs leading to the development of new anticancer agents, as well as restricting the flexibility for modification according to the preliminary natural structure-activity relationship (SAR). Further natural product research of sponge-derived fungi, especially those obtained from understudied MCEs, is expected to continue to yield new chemistry and associated biological activity for evaluation in drug development and ecological research.

Data Availability Statement:
The datasets generated for this study can be found in the Cambridge Structural Database https://www.ccdc.cam.ac.uk/structures.