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Chetracins E and F, cytotoxic epipolythiodioxopiperazines from the marine-derived fungus Acrostalagmus luteoalbus HDN13-530

Two new epipolythiodioxopiperazines, named chetracins E and F (1 and 2), along with the known chetracin C (3), were isolated from the fungus Acrostalagmus luteoalbus HDN13-530. Their structures were elucidated based on the NMR, MS and CD data, as well as chemical conversion. All of the compounds exhibited cytotoxicity against the tested five cancer lines in low-micromolar or nanomolar IC50 values. The computational docking indicated that compounds 1–3 could bind to the C-terminal of heat shock protein 90 (Hsp90), which was in line with the experimental observation of decreases in levels and active forms of Hsp90 client proteins.


Introduction
The epidithiodioxopiperazine alkaloids (ETPs) characterized by a bridged polysulde piperazine ring, represent a large family of secondary metabolites with various structure types and interesting biological activities. 1,2 Among them, the representative chaetocin displayed a wide spectrum of antitumor activities in vivo in nanomolar IC 50 level. [1][2][3][4][5][6] Furthermore, recent research showed that chaetocin and the analogue chetracin B (discovered by our group), could function as heat shock protein 90 (Hsp90) inhibitors binding to the C-terminal. 7 Hsp90 has proved to be an important target for cancer treatment, and is known as a crucial facilitator of oncogene addiction and cancer cell survival. [7][8][9] Although most of the Hsp90 inhibitors undergoing clinic evaluations bind to the N-terminal (1-275 aa), those binding to the C-terminal (444-677 aa) are believed to have more potential without the tendency to induce expression of the undesired cytoprotective Hsp70 proteins. 7 Encouraged by the discovery of chetracin B as a novel Cterminal Hsp90 inhibitor from the fungus Oidiodendron truncatum GW3-13, 1,7 the ETP alkaloids attracted our particular attention. Due to the difficulty and complexity in synthesizing this kind of alkaloid, 10 we looked for more analogues from more producing fungi. As the characteristic of containing multisulfur atoms, the ETPs will show special isotope peaks in the LC-MS analysis. During the screening of our marine-derived microorganisms' library using LC-MS, the fungus, Acrostalagmus luteoalbus HDN13-530 isolated from soil of Liaodong Bay, was selected. The LC-MS prole of its fermentation extract showed signicant M + 2 isotope peak which suggests the presentation of sulfur-containing metabolites (Fig. S1 †).  Further investigation showed that the EtOAc extract has potent cytotoxicity against P388 cells (66% inhibition of P388 cells at 100 mg mL À1 ). The LC-MS-UV guided fractionation of the fermentation extract led to the discovery of two new ETPs, named chetracins E (1) and F (2), together with the known chetracin C (3) (Fig. 1). In this report, we describe the isolation, structure elucidation, and activity evaluation of these compounds.

Results and discussion
The fungal strain A. luteoalbus HDN13-530 was fermented (30 L) under static conditions at 28 C for 4 weeks. Guided by LC-MS-UV data, the EtOAc extract (40 g) of the fermentation was fractionated by silica gel vacuum liquid chromatography, C-18 ODS column chromatography, Sephadex LH-20 column chromatography, ODS MPLC, and nally HPLC to yield compounds 1 (20.0 mg), 2 (15.0 mg) and 3 (15.0 mg). Chetracin E (1) was isolated as a pale yellow, amorphous powder. Based on the HRESIMS adduct ion detected at m/z 777.0432 [M + H] + , its molecular formula was established as C 30 H 28 N 6 O 7 S 6 , requiring 20 degrees of unsaturation. The major 1D NMR resonances was categorized into three methyls with two nitrogenized ones (d C 27.5 and 28.2), one oxygenated methylene (d C 60.1), twelve methines (including eight aromatic ones), fourteen non-protonated carbons including four carbonyls (d C 163.1, 164.1, 167.5, and 167.8) ( Table 1). The 1D NMR data of 1 were nearly superimposable to those of chetracin C (3) ( Table 1). 1 The only difference was that one oxygenated methylene in 3 was replaced by a methyl in 1, which was also conrmed by the HMBC correlations from H 3 -13 0 to C-3 0 , C-4 0 (Fig. 2).
The relative conguration of 1 was established based on the NOESY experiments (Fig. 3). The NOESY correlations from H-10 to H-11 and H-10 0 to H-11 0 indicated that H-11 and H-11 0 faced to the same orientation to C-10a-C-10b bond and C-10a 0 -C-10b 0 bond, respectively. Since the H-5a/H-5a 0 and the C-10b-C-10b 0 bond must be on the same side of disubstituted indole fragment because of structural rigidity, H-5a and the C-10b-C10b 0 bond should be trans to H-11, H-5a 0 and the C-10b-C10b 0 bond   Table. also should be trans to H-11 0 . 1 In order to determine the relative conguration of sulfur-bridged section, the tetrakis(methylsulfanyl) derivative (1a) ( Fig. 1 and S2, Table S1 †), was produced by treatment of 1 with NaBH 4 and MeI. The NOESY correlations from 11a-SCH 3 to H-11 and 3a-SCH 3 , as well as 11a 0 -SCH 3 to H-11 0 and 3a 0 -SCH 3 indicated that H-11 and H-11 0 were cis to sulfur-bridged (Fig. 3). The absolute conguration of 1 was determined to be the same as 3, evidenced by the similar CD spectra of 1 and 3, as well as the almost identical CD curves between 1a and chetracin D 1 which was the tetrakis(methylsulfanyl) derivative of 3 (Fig. 4). Chetracin F (2) was isolated as a pale yellow, amorphous powder. The molecular formula was assigned as C 30 H 28 N 6 O 8 S 7 by HRESIMS adduct ion detected at m/z 825.0105 [M + H] + , indicating the presence of one additional sulfur atom in the molecule compared to 3. Unlike compound 3 which was composed by two symmetric monomers, the asymmetric NMR signals of 2 indicated the existence of a tetrasulde bridge containing the additional sulfur atom. The tetrasulde bridge was assigned in the second monomeric subunit (between C-1 0 and C-13 0 ) according to the chemical shis (Fig. 1, Table 1), and the planar structure of compound 2 was also conrmed by the COSY and HMBC correlations (Fig. 2). The stereochemistry of compound 2 was deduced by chemical conversion. The tetrakis(methylsulfanyl) derivative of 2 showed identical spectroscopic data to those of chetracin D, which indicated the same absolute conguration of them. In addition, when kept at room temperature for two weeks, compound 2 could convert to 3 partially in DMSO induced by free radical reaction (Fig. S3 †), 1,11 suggesting that they share the same absolute conguration.
Biological evaluation using an MTT method showed that 1-3 exhibited extensive cytotoxicity against all the ve tested cancer cell lines (Table 2). Among them, compound 1 showed the strongest cytotoxicity on H1975 cells with IC 50 value 0.2 mM.
In light of discovery of the novel C-terminal Hsp90 inhibitors chaetocin and chetracin B, the interactions between compounds 1-3 and Hsp90 were investigated primarily in silico. The docking results displayed that 1-3 could bind to the 526-570 region (C-terminal) of Hsp90a by forming hydrogen bonds and hydrophobic interactions, with the average binding energy of À9.58 kcal, À6.21 kcal and À9.59 kcal, respectively. Distinguished from the phenotypic cytotoxicity, compound 2 showed a high binding energy, which possibly because of ignoring a potential cation-pi interaction between side chain K(570) of Hsp90 and the aromatic ring of 2 (Fig. 5). The cationpi interaction is about À4 kcal that was not taken into account by the docking soware. Anyway, the docking data suggest that all the compounds will be potential C-terminal Hsp90 inhibitors. To conrm the docking result, we estimated the levels of expression and phosphorylation of Hsp90 client oncoproteins induced by compounds 1-3 (with chetracin B used as reference drug). Similar to chetracin B, the treatment of 1-3 at the concentration of 0.5 mM reduced the expressions of EGFR, Akt, and the active forms of EGFR, Stat3, Akt and Erk in H1975 cells (Fig. 6). These results suggested that compounds 1-3 also could inhibit Hsp90 by binding to the Cterminal, which may subsequently induce the degradation of a serious of client oncoproteins. In addition, as chetracin B and compounds 1-3 show the effect of similar levels (Fig. 6), the number of sulfur atoms in the bridge and the hydroxyl

Fungal material
The fungal strain A. luteoalbus HDN13-530 was isolated from soil of Liaodong Bay, China. It was identied by ITS sequence and the sequence data have been submitted to GenBank (accession number KP969081). The voucher specimen was deposited in our laboratory at À80 C.

Fermentation and extraction
The fungus A. luteoalbus HDN13-530 was cultured under static conditions at 28 C in 1 L Erlenmeyer asks containing 300 mL of liquid culture medium, composed of glycerin (20.0 mL L À1 ), peptone (2.0 g L À1 ), yeast extract, power (2.0 g L À1 ), and seawater (Huiquan Bay, Yellow Sea). Aer 4 weeks of cultivation, 30 L of whole broth was ltered through cheesecloth to separate the supernatant from the mycelia. The former was extracted three times with EtOAc, while the latter was extracted three times with acetone and concentrated under reduced pressure to afford an aqueous solution, which was extracted three times with EtOAc. Both EtOAc solutions were combined and concentrated under reduced pressure to give the organic extract (40 g).

Isolation
The organic extract was subjected to vacuum liquid chromatography over a silica gel column using a gradient elution with petroleum ether-CH 2 Cl 2 -MeOH to give six fractions (fractions 1-

Formation of tetrakis(methylsulfanyl) derivative from compound 2
In the pyridine (0.1 mL) and MeOH (0.16 mL) solution of compound 2 (4 mg), MeI (0.1 mL) and NaBH 4 (2 mg) were added, aer stirring for 30 min at room temperature, the reaction mixture was then diluted with water and extracted with diethyl ether, and the residue evaporated under reduced pressure was puried by HPLC (CH 3 OH : H 2 O ¼ 50-100%, 3 mL min À1 ) to afford the known compound chetracin D (2.0 mg).

Cytotoxicity assay
Cytotoxic activities of 1-3 were evaluated by an MTT method using A549, HCT116, K562, H1975 and HL-60 cell lines. Dox (doxorubicin hydrochloride) was used as reference drug. The detailed methodology for biological testing had already been described in a previous report. 12

Computational modeling
The 3D structures of Hsp90 (PDB: 2CGE) 13 was taken from the Protein Data Bank (http://www.rcsb.org/pdb). The initial structures of compounds 1-3 were sketched in Sybyl 2.0 and their 3D structures were minimized using 3000 steps of conjugated minimization method in Sybyl 2.0. Ligand docking of compounds 1-3 to Hsp90 was performed using AutoDock 4.2. 14 Gasteiger charges were used and non-polar hydrogens of the macromolecule and the ligand were merged. A grid box with dimensions of 60 Â 60 Â 60Å and a grid spacing of 0.375Å was set up and centered on the binding pocket of Hsp90. Docking was performed using a Lamarckian Genetic Algorithm (LGA), with the receptor treated as rigid. For each of the above compounds, 50 complexes were generated and the best-ranked score from the largest cluster (with the RMSD threshold set at 2.0Å) was selected as the nal pose.
Western blotting assay for expression and activation levels of multiple oncoproteins The detailed methodology has already been described in a previous report. 7 Chetracin B (C. B) was used as reference drug.

Conclusions
In summary, three ETPs including two new ones were isolated from the fungus A. luteoalbus HDN13-530 by UPLC-MS-UV guided fractionation. Compounds 1-3 exhibited extensive cytotoxicity in low-micromolar or nanomolar IC 50 values and could function as Hsp90 C-terminal inhibitors.

Conflicts of interest
There are no conicts to declare.