Cytotoxic trichothecene-type sesquiterpenes from the sponge-derived fungus Stachybotrys chartarum with tyrosine kinase inhibition

Bioassay-guided fractionation of the sponge (Niphates recondite)-associated fungus Stachybotrys chartarum revealed that the EtOAc fraction shows inhibitory effects against tumor cell lines. Chromatographic separation of the active fraction resulted in the isolation of 15 trichothecene-based sesquiterpenes, including four new compounds, namely chartarenes A–D (1–4). The structures of the new compounds were determined on the basis of extensive spectroscopic analysis and chemical conversion. Compounds 1–15 exerted potent or selective inhibition against a panel of tumor cell lines including HCT-116, HepG2, BGC-823, NCI-H1650, and A2780, with IC50 values ranging from 10 2 to 10 mM. In addition, these compounds showed potent inhibition against tumor-related kinases FGFR3, IGF1R, PDGFRb, and TRKB.


Introduction
Trichothecenes are a group of sesquiterpene-based mycotoxins, [1][2][3][4][5][6] which are markedly cytotoxic toward eukaryotic organisms, 7 including high phytotoxicity. [8][9][10] The mechanism of toxicity is attributed to the inhibition of protein and DNA synthesis, and the mitochondrial electron transport system. 11,12 Biogenetically, the mevalonate pathway is involved in the generation of this typical scaffold and chemically related derivatives such as apotrichothecene-type and sambucinol-type analogues. 13,14 Apart from the phytotoxicity and mammalian intoxications, trichothecene-related compounds exhibited potent inhibitory effects for tumors, 4 and diacetoxyscirpenol (anguidine) has even been developed for clinical trials as an anticancer agent. 15 The structure-activity relationships revealed that the structural variety directly affects the cytotoxic activity, while destruction of the epoxide moiety and rearrangement of the ring system resulted in the virtual elimination of cytotoxicity. 16 It was noted that a slight change in trichothecenes could dramatically reduce the toxicity but still retain the activity of apoptosis induction in human cancer cells, as exemplied by 2 0 ,3 0 -epoxymyrothecine A and myrothecine A inducing the apoptosis of tumor cell lines through the induction of PARP cleavage and JNK phosphorylation with low cytotoxicity. 3 The derivatives of roridins A and H and verrucarins A and J exhibited extraordinary activities in vivo against P388 mouse leukemia with high therapeutic index, 17,18 and trichothecinol A strongly inhibited TPA-induced tumor promotion for cancer preventive activity. 19 These ndings suggested that trichothecenes are promising leads which are applicable for cancer chemotherapy. During our study on antitumor-active metabolites from marinederived fungi, we found that the EtOAc extract of a rice solid cultured fungus, Stachybotrys chartarum, isolated from a sponge (Niphates recondite), showed signicant cytotoxicity against a panel of tumor cell lines including HCT-116 and HepG2 with IC 50 values <10 mg mL À1 . This fungus was then subjected to large-scale fermentation, while chromatographic separation of the EtOAc fraction resulted in the isolation of 15 trichothecenebased sesquiterpenes (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) (Fig. 1).

Microorganism
The fungal strain WGC-25C-6 was isolated from the sponge Niphates recondite, which was collected from the inner coral reef at a depth of 10 m in Beibuwan Bay, Guangxi Province of P. R. China, in August 2010. The fungal strain was identied by comparing the morphological character and 18S rDNA sequence with those of standard records. Specically, the morphological examination was performed by scrutinizing the fungal culture, the mechanism of spore production, and the characteristics of the spores. For inducing sporulation, the fungal strain was separately inoculated on PDA. All experiments and observations were repeated at least twice leading to the identication of the strain as Stachybotrys chartarum. The fungal strain WGC-25C-6 was deposited at the State Key Laboratory of Natural and Biomimetic Drugs, Peking University, China.

Fermentation
Large-scale fermentation was carried out in 30 Fernbach asks (500 mL) each containing 80 g of rice. Distilled H 2 O (120 mL) was added to each ask, and the contents were soaked overnight before autoclaving at 15 psi for 30 min. Aer cooling to room temperature, each ask was inoculated with 5.0 mL of the spore inoculum and incubated at 25 C for 40 days.

Extraction and isolation
The fermented material was extracted successively with EtOAc (3 Â 500 mL). The EtOAc layer was evaporated to dryness under vacuum to afford a crude extract (30 g).

Acidic hydrolysis of 1
Compound 1 (2 mg) was dissolved in acetone (2 mL) and then was treated with 6 N HCl (1 mL) and heated at 90 C for 12 h. The which were used in all calculations. The nal R 1 was 0.0404 (I > 2s(I)) and wR 2 was 0.1049 (all data). Flack parameter ¼ 0.2 (2).
Colorless crystals of 6 were obtained from MeOH.

Cytotoxicity assay
Test samples were dissolved in dimethyl sulfoxide (DMSO) to make stock solutions and further diluted in culture medium upon assay. The cancer cell lines were cultured in RPMI 1640 medium, containing 10% fetal bovine serum. The cell lines were maintained at 37 C in a humidied environment containing 5% CO 2 . To determine the effects of the compounds on cell viability, the cell number was quantied using a standard colorimetric MTT assay. Cells were plated in a 96-well plate (5 Â 10 3 cells per well) and allowed to attach overnight. Cells were treated with 1, 2, 4, 8, and 10 mM of each compound in culture medium for 72 h. Then, new culture medium was added with 20 mL of MTT (5 mg mL À1 stock in PBS) per well and incubated for 4 h at 37 C. Finally, the culture medium was discarded, and 150 mL of DMSO was added per well to dissolve the purple formazan crystals. Absorbance of the solution was measured using a microplate reader spectrophotometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA), at a wavelength of 490 nm. The absorbance of untreated cells in the medium (negative control) was 100%. Paclitaxel was used as a positive control and showed cytotoxic activity with IC 50 values of 0.03, 0.01, 0.01, 0.04, and 0.01 mM against the HCT-116, HepG2, BGC-823, NCI-H1650, and A2780 tumor cell lines, respectively. The compound with IC 50 < 1 mM was further screened by the diluted concentrations of 0.1, 0.2, 0.4, and 0.8 mM, while the compound with IC 50 < 0.1 mM was diluted to 0.01 mM for additional assay. The human malignant cell lines (colon carcinoma HCT-116, hepatocellular carcinoma HepG2, gastric carcinoma BGC-823, non-small cell lung carcinoma NCI-H1655, and ovarian carcinoma A2780) used in this study were purchased from the China Infrastructure of Cell Line Resources.

Structure elucidation of new compounds
Chartarene A (1) has a molecular formula of C 21 H 34 O 7 as determined by HRESIMS and NMR data, containing ve degrees of unsaturation. The 1 H NMR spectrum exhibited the resonances of three methyl singlets (d H 0.81, 0.99, 1.61), an olenic proton at d H 5.47 (brs, H-10), as well as a number of alkyl protons and hydroxylated methine and methylene protons. The 13 C NMR spectrum showed a total of 21 carbon resonances, including two olenic carbons at d C 134.7 (C-9) and 122.1 (C-10) for a double bond. Based on the 2D NMR data analyses, an apotrichothecene nucleus 20 structurally related to apotrichodiol 21  3), C-9 and C-10, H 3 -14 (d H 0.99) to C-5 (d C 56.1), C-6 (d C 45.2), C-7 (d C 27.5), and C-11 (d C 81.1), H 3 -13 (d H 0.81) to C-1 (d C 95.2), C-4 (d C 36.9), C-5 and C-6, and H 2 -12 (d H 3.31, 3.88) to C-1, C-2 (d C 37.0), and C-5. The HMBC interaction between H-11 (d H 4.12) and C-1 claried an ether bridge between C-1 and C-11 (Fig. 2). Thus, a tricyclic core with methyl substitution at C-5, C-6 and C-9 and an oxymethylene at C-1, as well as a double bond at C-9 and C-10 was deduced. The and H-5 0 , while H-2 0 was in equatorial orientation. Acidic hydrolysis 22 of 1 resulted in the generation of a sugar, which was identical to D-mannose based on the comparison of its HPLC retention time, specic rotation and NMR data with those of authentic sample. The linkage of sugar to C-12 of the tricyclic nucleus through an ether bond was determined by the HMBC interaction between H 2 -12 and C-1 0 (d C 101.9). The NOE interactions from H 3 -13 to H-11, H 2 -12 and H b -7, in association with the NOE relationship between H 3 -14 and H a -7 (Fig. 3) indicated the trans junction of rings A and B and cis junction of rings B and C, while H 3 -13 and H 3 -14 were in opposite faces.
The molecular formula of chartarene B (2) was determined as C 16  2), C-2 (d C 85.7) and C-5 (d C 57.6) and the HMQC correlations between H-2/C-2 and H-4/C-4 (d C 69.2) indicated the substitution of oxygen atom at C-2 and C-4. In addition, the HMBC correlation between H-2 and a methoxy carbon at d C 58.2 claried a methoxy substitution at C-2. Thus, C-4 was located with a hydroxy group, and this assignment was supported by the COSY relationship between H-4 and OH-4 (d H 4.50). The NOE interactions between H-2/H-4 and H 3 -14/H-11, and from H 2 -12 to OMe and H 3 -13 (Fig. 3), determined the cis fusion of rings A/B and B/C, while H-2 and H-4 were oriented in the same face as H 3 -14.
The HRESIMS and NMR data indicated the molecular formula of chartarene C (3) to be C 15 H 22 O 4 , requiring ve degrees of unsaturation. The 1D and 2D NMR data established a sambucinol-type analogue with the planer structure of 3 to be related to loukacinol A. 5 The distinction was attributed to C-8, where a methylene group of 3 replacing a ketone of loukacinol A was evident from H 2 -8 (d H 1.92, 2.10) correlated to the olenic carbons C-9 (d C 143.9) and C-10 (d C 117.5) in the HMBC spectrum. The similar NOE interactions between 3 and loukacinol A indicated both compounds possessing the same congurations. These assignments were conrmed by the analyses of the X-ray diffraction data using the Flack parameter (Fig. 4).
The molecular formula of chartarene D (4) was determined as C 29    and an olenic carbon C-6 00 (d C 150.5) and from H-3 00 and H 2 -2 00 to a carbonyl carbon C-1 00 . The linkage of units A and B by an ester bond across C-14 and C-1 0 was deduced by the HMBC interaction between H 2 -14 (d H 3.83, 4.28) and C-1 0 , while the conjunction of units B and C through an ester bond across C-4 and C-1 00 was indicated by the HMBC correlation between H-4 (d H 5.98) and C-1 00 (d C 170.1) (Fig. 2). The connection of units B and C with an ether bond between C-5 0 and C-6 00 was deduced by the HMBC relationship between H 2 -5 0 (d H 3.77, 4.17) and C-6 00 . Thus, 4 was established as a roridoid-type analogue, structurally related to roridin E with the migration of the olenic bonds to C-3 00 /C-4 00 and C-5 00 /C-6 00 and the substitution of a ketone at C-7 00 . The NOE interaction between H-2 0 and H 2 -4 0 in association with the chemical shi of the methyl carbon C-6 0 (d C 18.3, <20 ppm) determined 2 0 E geometry. The J H-3 00 /H-4 00 value (12.0 Hz) and the NOE interaction between H 3 -8 00 and H-5 00 conrmed 3 00 Z and 5 00 Z geometries. Acidic hydrolysis of 4 produced a product which was identical to verrucarol, con-rming the stereogenic centers in moiety A of 4 to be the same as those of verrucarol. Moreover, the known compounds were identical to mytoxin A (5), 24 satratoxin G (6), 25 roridin L-2 (7), 26 satratoxin H (8), 27 muconomycin B (9), 28 roridin E (10), 29 verrol (11), 30 trichodermadienediol B (12), 31 isotrichoverrol B (13), 32 2,4,12-trihydroxyapotrichothecene (14), and trichodermol (15), 33 based on the comparison of their NMR and MS data in association with specic rotation with those reported in the literature. 2,4,12-Trihydroxyapotrichothecene (14) was isolated from a microorganism for the rst time, while the absolute congurations of 14 and satratoxin G (6) were uncertain in the literature. In the present work, the absolute congurations of satratoxin G (6) and 2,4,12-trihydroxyapotrichothecene (14) were established by the analyses of the X-ray diffraction data using Flack parameters (Fig. 4).

Bioassay
Compounds 1-15 were tested against a panel of human tumor cell lines, including human colon carcinoma (HCT-116), human hepatocellular carcinoma (HepG2), gastric cancer (BGC-823), non-small-cell lung adenocarcinoma (NCI-H1655), and human ovarian cancer (A2780), by the MTT method. 34 As shown in Table 3, all compounds exhibited selective inhibition against the tumor cell lines, while compounds 5, 6, 8, and 10 showed potent cytotoxic activities against the panel of tumor cell lines with IC 50 values less than 10 nM. Preliminary analyses of the structure-activity relationship revealed that the cytotoxic effects are related to the scaffolds and the substitution. Apotrichothecene-based analogues with a sugar moiety at C-12 and without hydroxy group at C-2 and C-4 (1) or with a methoxy group at C-2 (2) showed weaker cytotoxicity in comparison with the 2,4,12-trihydroxy analogue (14). For the trichodermol-type analogues, those with a macrocyclic ring along with a tetrahydropyran ring (5,6,8) exerted remarkable effects, whereas the analogues without a tetrahydropyran ring in the macrocyclic moiety (4,9) showed less cytotoxicity. However, compound 10 is an exception which showed IC 50 within 10 nM, while the structural distinction of 4 and 10 was attributed to the olenic rearrangement and the substitution at C-7 00 of the macrocyclic ring. Compounds 7 and 11-13 with a linear moiety at C-4 or C-14 had decreased inhibitory effects in comparison with 5.
In order to gain an insight into the mechanism of the antitumor-active compounds, a panel of tumor growth-related tyrosine kinases including FGFR3 (broblast growth factor receptor 3), IGF1R (insulin-like growth factor 1 receptor), PDGFRb (b-type platelet-derived growth factor receptor), and TRKB (tropomyosin receptor kinase B) were selected for testing. The bioassay results revealed that most compounds exerted potent inhibitory activities (Table 4), except for 1 and 13 showing weak activity with IC 50 values of more than 20 mM.
FGF signaling plays a role in various aspects of cancer biology, involving proliferation, anti-apoptosis, drug resistance, 139.2 C 10 119.6 CH 5.37, d (J ¼ 3.  angiogenesis, EMT, and invasion; thus FGFR-targeted therapeutics is an active topic in the eld of clinical oncology. 35,36 IGFIR regulates the tumor microenvironment to promote cancer progression and metastasis, 37,38 while PDGFR inhibition is an antiangiogenic approach potentially affecting all solid tumors due to tumor vasculature having a pericyte coverage. 39,40 Meanwhile, high levels of TrkB are correlated with poor patient prognosis in neuroblastomas and ovarian, pancreatic, colon, prostate, and gastric cancers, while TrkB is overexpressed in ovarian adenocarcinoma metastases compared with primary lesions. 41,42 The potent inhibition toward the spectrum of tyrosine kinases suggested that the antitumor effects of the trichothecene-type derivatives were mediated by multiple targets.

Conclusion
Trichothecene-type sesquiterpenoids are frequently found in Stachybotrys species in different terrestrial localities, while the unique scaffold of trichothecenes may be considered as chemotaxonomic markers. This work rstly reported the sponge-derived Stachybotrys chartarum to be a new fungal strain of functionalized trichothecenes. Trichothecene with a sugar linkage such as 1 was found from nature for the rst time. The potent cytotoxic effects of the isolated compounds against a panel of tumor cell lines are induced by the inhibition of the multiple kinase targets FGFR3, IGF1R, PDGFRb, and TRKB. These ndings provided additional data indicating that trichothecenes are promising kinase inhibitors for the development of antitumor leads.