Sorbicillinoid Derivatives From Sponge-Derived Fungus Trichoderma reesei (HN-2016-018)

Six new sorbicillinoids, trichoreeseione A (1) and B (2), trichodermolide B (3), 13-hydroxy-trichodermolide (4), 24-hydroxy-trichodimerol (5), 15-hydroxy-bisvertinol (7), together with three known analogs, trichodimerol (6), 24-hydroxy-bisvertinol (8), and bisvertinol (9), were isolated from the sponge-derived fungus Trichoderma reesei (HN-2016-018). Their structures including absolute configurations were elucidated by analysis of NMR, MS data, and calculated ECD spectra. Compounds 1 and 2 with a characteristic naphthalene-trione ring were firstly reported in sorbicillinoid family. Compounds 3 and 4 were two rare sorbicillinoids containing a unique bicycle [3.2.1] lactone skeleton, while 3 with a propan-2-one moiety was also recorded first time in this family. Compound 5 displayed cytotoxic activity against A549, MCF-7, and HCT116 cell lines with the IC50 values of 5.1, 9.5, and 13.7 μM, respectively.


General Experimental Procedure
The optical rotations were measured on a JASCO P-1020 digital polarimeter. IR spectra were recorded on a Nicolet-Nexus-470 spectrometer using KBr pellets (Thermo Electron, Waltham, MA, United States). UV spectra were recorded using a Milton Roy UV-Vis spectrophotometer. NMR spectra were acquired using a JEOL JEMECP NMR spectrometer (JEOL, Tokyo, Japan) (600 MHz for 1 H, 150 MHz for 13 C) and BRUKER AVANCE NEO NMR spectrometer (BRUKER, United States) (400 MHz for 1 H, and 100 MHz for 13 C), using TMS as an internal standard. ECD spectra were recorded on a JASCO J-815 circular dichroism spectrometer (JASCO Electric Co., Ltd., Tokyo, Japan). ESIMS spectra were measured on a Micromass Q-TOF spectrometer (Waters Corp., Manchester, United Kingdom). HPLC separation was performed using a Hitachi L-2000 HPLC system (Hitachi High Technologies, Tokyo, Japan) coupled with a Hitachi L-2455 photodiode array detector. A Kromasil C 18 semi-preparative HPLC column (250 × 10 mm, 5 µm) (Eka Nobel, Bohus, Sweden) was used. Silica gel (Qingdao Marine Chemical Group Co., Qingdao, China) and Sephadex LH-20 (Amersham Biosciences Inc., Piscataway, NJ, United States) were used for column chromatography. Precoated silica gel GF254 plates (Yantai Zifu Chemical Group Co., Yantai, China) were used for analytical TLC.

Fungal Material
The fungal strain T. reesei (HN-2016-018) was separated from the internal fresh tissue of an unidentified sponge collected from the South China Sea in October 2016. The fungal strain was recognized on the basis of its morphological traits and by amplification and sequencing of the DNA sequences of the ITS region. The fungus was identified as T. reesei whose 500 base pair ITS sequence had 99% sequence identity to that of T. reesei. The phylogenetic tree (Supplementary Figure S50) was constructed using the neighbor joining method (Saitou and Nei, 1987). The distance calculations, tree construction, and bootstrap analysis were performed with the software MEGA 7 (Felsenstein, 1985). The strain was deposited in the Key Laboratory of Marine Drugs, the Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao, China, with the Gene Bank (NCBI) accession number MT367415.

Biological Assay
Human tumor cells, including colonic (HCT116 and SW480), lung carcinoma (A-549), hepatocellular carcinoma (HepG2), cervical carcinoma (HeLa), breast cancer (MCF-7), and human normal cells, including human umbilical vein endothelial cells (HUVEC) and hepatocytes CLiver cells were cultured in RPMI 1640 medium supplemented with 10% heat inactivated FBS (fetal bovine serum), 2 mM L-glutamine and combination of antibiotics penicillin 100 units/ml and streptomycin 100 g/ml were used to avoid contamination in culture medium. All samples were dissolved in DMSO. The adriamycin was used as a positive control and DMSO was used as a negative control (Wu et al., 2020).
The cytotoxicity of the isolated compounds was determined by sulphorhodamine B assay (Skehan et al., 1990). Cells in logarithmic growth stage were inoculated into 96-well tissue culture plates with 5000 cells/well (180 µL/well) for 24 h before treatment with the tested compounds to allow attachment of the cells to the plate. Cells were exposed to the six different concentrations (1.25, 2.5, 5, 10, 20, and 40 µM) in four parallel. After 72 h of drug action, cold trichloroacetic acid (TCA, 50% w/v) was added into each well to fix the cell. After several washings, cells were stained by 0.4% (w/v) SRB solution for 10 min in dark place. Excess stain was washed with 1% (v/v) glacial acetic acid. After drying overnight, the SRB-stained cells were dissolved with 150 µL/pore Tris solution and the color

Statistical Analysis
The bioassay results were expressed as mean values ± SD. The IC 50 values, i.e., the concentrations necessary for 50% inhibition, were calculated from the dose response curves using non-linear regression.  that two rings should be present in 1. A substituted 2,6,8,10tetra-methyl-8-hydroxy naphthalene 1,4,7-trione skeleton was deduced based on its spectroscopic features, combining with the key HMBC correlations from H-3 to C-1 and C-5, from H 3 -18 to C-1, C-2, and C-3, from H 3 -19 to C-4, C-5, C-6, and C-7, from H 3 -20 to C-7, C-8, and C-9, and from H 3 -21 to C-1, C-5, C-9, and C-10 (Figure 2). A typical sorbyl side chain was deduced from the 1 H-1 H COSY correlations H-13/H-14/H-15/H-16/H 3 -17 and HMBC correlation from H-14 to C-12 (Figure 2). Further, the 1 H-1 H COSY signal H-9/H 2 -11 and the HMBC correlations from H 2 -11 to C-8, C-9, C-10, and C-12 (Figure 2) demonstrated that the sorbyl side chain was connected to C-9 bridged by CH 2 -11. The relative configuration of 1 was determined by coupling constants and NOESY spectrum (Figure 3). The E-configurations of double bonds in the sorbyl side chain were deduced on the basis of the large coupling constant (J H−13/H−14 = 15.5 Hz) and the NOESY correlation between H-14 and H-16. The NOESY correlation between H 3 -20 and H 3 -21 indicated the same orientation of these two methyls, whereas the protons H 3 -20 and H 3 -21 were simultaneously correlated with H 2 -11, reflecting that proton H-9 should locate at the other orientation. Therefore, the relative configuration of 1 was assumed as 8R * ,9S * ,10S * . The absolute stereochemistry of 1 was resolved by quantum chemical time-dependent density functional theory (TDDFT) calculation of its electronic circular dichroism (ECD) spectra of (8R,9S,10S)-1 and (8S,9R10R)-1. The experimental ECD spectrum of 1 displayed the intense positive cotton effect at 252 nm and negative cotton effects at 227 and 288 nm, respectively, which were consistent with the theoretical ECD spectrum for (8R,9S,10S)-1 (Figure 4). Therefore, the absolute configuration of 1 was determined as 8R,9S,10S. Trichoreeseione B (2) was assigned a molecular formula of C 21 H 24 O 6 by HR-ESIMS, showing one additional oxygen atom compared to that of 1. The NMR data of 2 (Table 1) suggested the same skeleton as 1, apart from a hydroxy-methylene resonance (δ H 4.31, δ C 62.9) in 2 instead of the methyl signals (δ H 1.87, δ C 18.9) in 1. The 1 H-1 H COSY and HMBC correlations confirmed the planar structure of 2, in which a terminal hydroxymethylene in the sorbyl side chain replaced the methyl group in 1 (Figure 2). The coupling constants, NOESY correlations (Figure 3), and ECD spectrum of 2 (Figure 4) demonstrated that its stereochemistry was the same as 1. It is worth mentioning that compounds 1 and 2 represented the first example of sorbicillinoids with a characteristic naphthalene-trione ring.

Structure Elucidation
Trichodermolide B (3) was given a molecular formula C 21 H 26 O 5 based on HR-ESIMS, indicating 9 degrees of unsaturation. The 1 H NMR and 13 C NMR data ( Table 2) showed four carbonyls, three pairs of olefinic carbons, five methyls, three methylenes, one methine, and two quaternary carbons. These spectroscopic features suggested that 3 should be a sorbicillinoid analog and closely resembled to trichodermolide isolated from Trichoderma longibrachiatum (strain UAMH 4159) collected from cotton duck shelter (Andrade et al., 1996). The main differences were absence of four olefinic carbons, and addition of a propan-2-one moiety and one methylene. The HMBC correlations from H 2 -7 to C-2, C-3 and C-4 suggested the propan-2-one moiety was attached to C-3. The 1 H-1 H COSY linkage between H 2 -10 and H 3 -11 indicated the presence of one ethylene group, which was located at C-4 based on the HMBC correlations from H 3 -11 to C-4, and from H 2 -10 to C-4, C-5, and C-21 (Figure 2).
The two double bonds in sorbyl side chain of 3 were also assigned as E configuration on the basis of their coupling constants (J H−14/H−15 = 15.5 Hz), and the NOESY correlation between H-14/H-16 and H-15/H-17 (Figure 3). The relative configurations of the stereocenters at C-4, C-5, and C-6 were determined by NOESY spectrum. The NOESY correlations of H-5/H 3 -20, and H-5/H 3 -11 suggested the same side of these protons. Therefore, the relative configuration of 3 was assumed as 4S * ,5R * ,6R * . The absolute configuration of 3 was determined by ECD calculation. The Boltzmann-weighted ECD curve of (4S,5R,6R)-3 agreed with the experimental one (Figure 4), and hence, the absolute configuration of 3 was assigned as 4S,5R,6R.
The  (Figure 3). The relative configurations of three stereocenters, C-4, C-5, and C-6 of 4 were determined by comparison of its 1 H NMR data with already reported compound dihydrotrichodermolide (Li et al., 2011). The similarity of chemical shifts of H-5 (δ H 3.33), H 2 -14 (δ H 3.23 and 2.46), and two methyl groups H 3 -22 (1.23) and H 3 -23 (δ H 1.46) in 4 with those of in dihydro-trichodermolide, combing the NOESY correlations between H-5/H 3 -22 and H-5/H 3 -23, established that the relative configuration of 4 was in accordance with that of  dihydro-trichodermolide. The absolute configuration of 4 was determined by comparing its ECD data (Figure 4) with those of known analogs (Andrade et al., 1996;Li et al., 2011;Cao et al., 2019). The ECD spectrum of 4 demonstrated nearly similar negative and positive cotton effects to those of reported analogs (Figure 4 and Supplementary Table S4). Consequently, the absolute configuration of 4 was determined as 4S,5R,6R.
A literature survey revealed that bicycle (3.2.1) lactone skeleton was rare in the sorbicillinoid family. So far, only three compounds with this distinctive skeleton have been reported, including trichodermolide (Andrade et al., 1996), dihydro-trichodermolide (Li et al., 2011) and 13-hydroxydihydro-trichodermolide (Cao et al., 2019). In this study, two new sorbicillinoids (3 and 4) with a bicycle [3.2.1] lactone FIGURE 4 | ECD spectra of compounds 1-8. skeleton was discovered. Among them, 3 was unique in a sense that the propan-2-one side chain was reported first time for sorbicillinoid family.
15-Hydroxy-bisvertinol (7) displayed a [M − H] − ion at m/z 513.2136 in its HR-ESIMS, in accordance with the molecular formula C 28 H 33 O 9 , which indicated 12 degrees of unsaturation. The 1D NMR and HSQC spectra displayed the presence of five methyls, two methylenes, nine methines, and twelve quaternary carbons. Careful examination of the NMR data of 7 (Table 3) disclosed that its skeleton has resemblance with the known compound bisvertinol (9), primarily isolated from fungus Verticillium intertextum (Trifonov et al., 1986). The obvious difference was that the methyl group at C-15 on one of the sorbyl side chains in 9 was replaced by a hydroxy methylene in 7.
The relative configuration of 7 was addressed by the coupling constants, NOESY spectrum and biogenetic relationship. In the NOESY spectrum, key cross peaks were observed between H-9a and H 3 -25 and H 3 -17 (Figure 3), indicating that the methyls H 3 -17 and H 3 -25 were at the same side with H-9a. The 4a-OH was deduced in the same face with H-9a, H 3 -25 and H 3 -17 by considering the higher stability of a cis 5-6 ring junction over a trans 5-6 ring junction (Andrade et al., 1992). The configuration of C-4 was presumed to be identical to that of the co-isolated 8 and 9 on the basis of biogenetic relationship. The experimental ECD spectra of 7 and 8 (Figure 4) were parallel to each other, possessing positive Cotton effect at 346 nm and negative at 403 nm, and both gave negative optical rotation values. Consequently, the absolute configuration of 7 was deduced as 4S,4aR,5aS,9aR,9bR.

Bioassays of Compounds
All of the isolated new compounds (1-5 and 7) were assessed for their cytotoxic activities against five human tumor cell lines, including A549, HepG2, HCT 116, HeLa, MCF-7 and two healthy human cell lines HUVEC and CLiver. Compound 5 displayed cytotoxic activity against A549, MCF-7 and HCT 116 cell lines (Figure 5) with the IC 50 values of 5.1, 9.5 and 13.7 µM, respectively, whereas displayed no significant cytotoxic activity against normal cell lines (HUVEC and CLiver) with the IC 50 values higher than 40 µM. The selectivity index values (SI, IC 50 normal cell line/IC 50 cancer cell line) for compound 5 were found FIGURE 5 | The dose response curve of the cytotoxicity of compound 5 toward A549, MCF-7 HCT 116, HUVEC and CLiver cell lines. Cells were exposed to compound 5 with different concentrations for 72 h. Cell inhibition was determined by SRB stain. Frontiers in Microbiology | www.frontiersin.org to be higher than 7.8, 4.2, and 2.9, respectively, indicating its selective cytotoxicity.

CONCLUSION
In summary, we report here nine sorbicillinoid derivatives (1-9), including six new compounds, isolated from the spongederived fungus T. reesei (HN-2016-018). To date, more than 130 sorbicillinoid derivatives have been reported (Meng et al., 2016(Meng et al., , 2018(Meng et al., , 2019Zhang et al., 2018Zhang et al., , 2019Cao et al., 2019;Wang et al., 2019;Yu et al., 2019). In this study, we discovered two novel sorbicillinoids (1-2) with a characteristic naphthalenetrione ring and two rare sorbicillinoids (3-4) possessing a bicycle (3.2.1) lactone skeleton (Figure 6), where only three compounds with such distinctive skeleton have been reported previously. Furthermore, compound 3 represented the first reported sorbicillinoid with a propan-2-one side chain, while a terminal hydroxylation at the side chain of compounds 2, 4, 5, and 7 was also rare in the sorbicillinoid family. Compound 5 displayed strong cytotoxic activity against A549, MCF-7, and HCT116 cell lines. This study enriched the structural diversity of sorbicillinoids and provided the chemical entities for the development of marine bioactive natural products.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, and further inquiries can be directed to the corresponding author/s.

AUTHOR CONTRIBUTIONS
C-YW and C-LS conceived of and proposed the idea. SR contributed to fermentation, extraction, and isolation. SR and L-JY contributed to the manuscript preparation. WH contributed to bioactivities test. SR, L-JY, J-SW, TS, and Y-HZ contributed to data analysis, write up, revision, and proofreading of the manuscript. All authors read and approved the final manuscript.