Phenol Derivatives From the Sponge-Derived Fungus Didymellaceae sp. SCSIO F46

Seven new phenol derivatives named coleophomones E and F (1, 2), diorcinols L and M (3, 4), 1-hydroxy-6-methyl-11-methoxy-8-hydroxymethylxanthone (5), porric acid E (6), and 7-(2-hydroxyphenyl) butane-7,8,9-triol (7), were isolated from the EtOAc extract of the marine sponge-derived fungus Didymellaceae sp. SCSIO F46, together with 10 known compounds. Their structures were determined by spectroscopic analyses, including NMR, MS, X-ray diffraction, and theoretical calculations. Each of 1 and 2 contains an unusual spiro [cyclohexane-1,2′-inden] moiety, which is relatively seldom in nature products. Cytotoxic and COX-2 inhibitory activities of all purified compounds were tested and evaluated. Compound 3 displayed obvious cytotoxicities against Huh-7, HeLa, DU145 and HL60 cells (IC50 values 5.7–9.6 μM) and weak activities against other five cell lines, while 8 showed weak cytotoxicities against HeLa and HL7702 cells. Compound 6 displayed COX-2 inhibitory activity with IC50 value of 3.3 μM.


INTRODUCTION
Natural products are still irreplaceable and continuing sources of novel drug leads, especially in the anti-infective area (Newman and Cragg, 2016). The marine ecosystem is one of the most complex and largest aquatic systems on earth, and host a huge microbial biodiversity (Agrawal et al., 2017;Corinaldesi et al., 2017). The unique and extreme characteristics of marine systems have driven a variety of biological adaptations, leading to the production of a large number of novel molecules for the treatment of many diseases (Gerwick and Fenner, 2013;Blunt et al., 2017). Marine sponges, a kind of precious marine organisms for new drug discovery, are hosts for a large community of microbes (up to 50-60% of the biomass of the sponge host) (Bergmann and Burke, 1955;Wang, 2006;Zhang et al., 2017). It was indicated that the symbiotic microbes of marine sponges might be the true producers of bioactive chemical defense substance of the sponge ecosystem (Richelle-Maurer et al., 2003;Thomas et al., 2010).

General Experimental Procedures
The NMR spectra were recorded on a Bruker AC 500 NMR (Bruker, Fällanden, Switzerland) spectrometer with TMS as an internal standard. HRESIMS data were measured on a Bruker micro TOF-QII mass spectrometer (Bruker, Fällanden, Switzerland). UV spectra were recorded on a Shimadzu UV-2600 UV-Vis spectrophotometer (Shimadzu, Kyoto, Japan). ECD spectra were performed on a Chirascan circular dichroism spectrometer (Applied Photophysics). X-ray diffraction intensity data were collected on a CrysAlis PRO charge-coupled device (CCD) area detector diffractometer with graphite monochromated Cu Kα radiation (λ = 1.54178 Å). Semipreparative reversed-phase HPLC (RP-HPLC) was performed on a YMC-Pack Pro C 18 RS column (5 µm, 250 × 10 mm id; YMC, Kyoto, Japan) with a Agilent 1260 separation module equipped with a Photodiode Array (PDA) detector. Silica gel GF254 used for TLC were supplied by the Qingdao Marine Chemical Factory, Qingdao, China. Sephadex LH-20 gel (GE Healthcare, Uppsala, Sweden) was used. Spots were detected on TLC under UV light or by heating by spraying with 12% H 2 SO 4 in H 2 O.

Fungal Material
The fungal strain SCSIO F46 was isolated from a sponge Callyspongia sp., collected from the sea area near Xuwen County, Guangdong Province, China, during August 2013. The isolate was stored on MB agar (malt extract 15 g, sea salt 10 g, agar 15 g) slants at 4 • C and then deposited at CAS Key Laboratory of Tropical Marine Bio-resources and Ecology. The fungus was identified using a molecular biological protocol by DNA amplification and sequencing of the ITS region. The nucleotide sequence of the ITS region reported in this article was assigned the GenBank accession number KU361223.

ECD Calculation
The eight possible stereoisomers (a-h) of 1 were initially performed using Confab (O'Boyle et al., 2011) with systematic search at MMFF94 force field. Room-temperature equilibrium

NMR Calculation
The two stereoisomers 1e and 1f were delivered to geometry optimization at B3LYP/6-31+G(d,p) in gas phase. The theoretical calculation of NMR was conducted using the

COX-2 Inhibitory Activity Assay
According to the manufacturer's instructions. The test compounds were dissolved in DMSO and the final concentration was set as 10 µM. The percentage inhibition has been calculated
The COSY cross-peaks of H-13 (δ H 2.61, m) /H-14 (δ H 4.39, dd, J = 9.2, 5.9 Hz), and H-12 (δ H 6.33, d, J = 5.9 Hz) delineated the spin system C 12 -C 13 -C 14 . Moreover, the HMBC correlations of H-12/C-10 and C-14, H-13/C-9, C-11, and C-12, and H-14/C-1, C-8 and C-9 indicated the presence of a α,β-unsaturated ketone (ring C) and rings B/C are connected by C-9. Finally, a 2-methyl-2 butene group was attached to C-11 (δ C 138.4) by the evidence of the HMBC correlations of H 3 -18 (δ H 1.54, s)/C-16 (δ C 121.3), C-17 (δ C 132.2), and C-19 and H 2 -15 (δ H 2.72, s)/C-10, C-11, C-12, C-13, C-16, and C-17. NOESY correlations and Mosher method were failure to determine the configurations of 1, so theoretical calculations were used to solve it. There are eight possible stereoisomers (a-h) of 1, as shown in Figure 3A. Computational studies of electron circular dichroism (ECD) were carried out. All stereoisomers (a-h) were selected for theoretical calculations using time dependent density functional theory (TDDFT) B3LYP/6-311G (d,p) level with the IEFPCM model in MeOH (Tables S1, 2, 4). A comparison of the experimental spectrum of 1 with the calculated ECD spectra of eight possible stereoisomers (a-h) were presented in Figure 3B. The measured ECD curve exhibits two negative Cotton effects (CEs) at 219 and 315 nm, and two positive cotton effects at 238 and 267 nm, matching well with the calculated ECD curve of 1e (1S, 9R, 14S) and 1f (1S, 9R, 14R). Then, computed 13 C-NMR chemical shifts was carried out to define the stereochemistry of C-14. Computed 13 C-NMR chemical shifts of each conformer were first Boltzmann-weighted averaged, and then fitted to experimental values by Ordinary Least Squares (OLS) Linear Regression method in order to remove systematic error that results from the conformational search and random error from experimental conditions (Tables S3, 5). As a result, the computed chemical shift for C-14 of 1e is δ C = 68.7 ppm, with only a deviation of 1.3 ppm from the experimental value (δ C = 67.4 ppm) ( Table S8). All in all, the computed chemical shifts of 1e showed good agreement with the experimental values and has the higher R 2 and R 2 adj values than that of 1f, which suggesting that 1e (1S, 9R, 14S) be the true isomer of 1 ( Figure 3C, Table S7).
The molecular formula of 2 was determined as C 21 H 24 O 6 by its HRESIMS (m/z 373.1638 [M + H] + ), corresponding to 10 units of unsaturation. Its UV and NMR date were similar to those of 1, except for the presence of a methoxy (δ H 3.72, δ C 60.6) in 2 ( Table 1). The extra methoxy (C-21) was located at C-3 by HMBC correlations from H 3 -21 to C-3 (δ C 148.5) (Figure 2). The absolute configuration of 2 was suggested as (1S,9R,14S), as its chemical shift, coupling constant, optical FIGURE 4 | X-ray crystallographic structure of 3. rotation and CD effect (Table 1, Figure S20) almost the same as those of 1.
The molecular formula of 3 was assigned as C 16 H 16 O 5 by its HRESIMS ion peak at m/z 289.1070 [M + H] + and NMR date. The 1 H NMR spectrum of 3 exhibited two hydroxyl proton at δ H 9.95 and 9.50, five aromatic signals at δ H 6.41, 6.35, 6.22, and 6.14×2, one O-methyl at δ H 3.67, and two single methyls at δ H 2.18 and 2.20 ( Table 2). Analysis of the 13 C and DEPT-135 NMR spectra of 3 indicated the presence of 16 carbons, including one carbonyl carbon (δ C 167.7), 12 aromatic carbons (four oxygenated ones at δ C 159.7, 158.9, 158.0 and 155.8), one methoxy and two methyls. These spectra of 3 were similar to those of diorcinol (Tian et al., 2015b), except for the presence of a metheyl formate group (one carbonyl carbon at δ C 167.7 and one methoxy at δ H 3.67/δ C 52.2). However, relying solely on the NMR date, the location of metheyl formate was more difficult to determine. In order to determine location of metheyl formate of 3, a single-crystal X-ray diffraction pattern was obtained using the anomalous scattering of Cu Kα radiation shows an ORTEP drawing (Figure 4, Table S9) and unambiguously determined metheyl formate at C-2 ′ . Thus, the structure of 3 was determined, and named as diorcinol L.
Compound 4 was obtained as brown powder. The molecular formula of was determined as C 17 H 17 NO 6 by its HRESIMS (m/z 332.1131 [M + H] + ), which corresponded to ten units of unsaturation. The 1 H and 13 C NMR data of 4 were similar to those of 3, except for the presence of one amide [δ C 169.2 (CONH 2 )/ δ H 7.45, 7.35 (CONH 2 )] ( Table 1). The extra amide was determined at C-2 by the HMBC correlations of H-4, H-6/C-2, as well as H 3 -7/C-2, C-3, C-4, and C-10 ( Figure 5). Hence, the structure of was elucidated and the trivial name diorcinol M was assigned.

CONCLUSION
In conclusion, 17 phenol derivatives were isolated from the EtOAc extract of a marine sponge-derived fungus Didymellaceae sp. SCSIO F46. Coleophomones E and F (1 and 2) possess unprecedented spiro [cyclohexane-1,2 ′ -inden] moiety, which is relatively seldom in natural products. Other new compounds 3-7 represent common types of phenol derivatives, which are widely found in fungal metabolites. Amongst, compounds 3 and 8 displayed a wide range of cytotoxicities against several tumor cell lines. In addition, 6 displayed significant COX-2 inhibitory activity with the prominent IC 50 value of 3.3 µM.

AUTHOR CONTRIBUTIONS
YT designed the experiments and performed the isolation and characterization of all the compounds and wrote the manuscript. XL performed the isolation and purification of the fungal strain. XZ designed the research work and revised the manuscript. YL contributed in project design and manuscript preparation. All authors reviewed the manuscript and approved for submission.