3.1 General experimental procedures

Optical rotations were measured on a Rudolph Research Analytical Autopol IV automatic polarimeter. UV spectra were recorded using a Cary 300 spectrometer (Agilent Technologies, Santa Clara, CA, USA). IR spectra were recorded on a Shimadzu Fourier Transform Infrared Spectrometer using KBr pellets. Circular Dichroism (CD) spectra were obtained on a Chirascan™-plus Circular Dichroism spectrometer. High-resolution electrospray ionization mass spectrometry (HRESIMS) spectra were acquired with an Agilent 6500 series Q-TOF mass spectrometer (Agilent Technologies, Singapore). NMR spectra were recorded on Bruker Avance III HD 600 MHz and 500 MHz spectrometer with tetramethylsilane (TMS) serving as the internal standard. Column chromatography (CC) was performed using macro-porous adsorbent resin D101 (Tianjin Haoju Resin Technology Co., Ltd., Tianjin, China), silica gel (200–300 mesh, Qingdao Marine Chemical, Qingdao, China), and Sephadex LH-20 (GE Healthcare, Uppsala, Sweden). MCI GEL™ (Mitsubishi Chemical Corporation, Japan). Semipreparative ZORBAX SB-C18 (Agilent, 5 μm, 9.4 mm × 150 mm), Supersil ODS-B (Elite, 10 μm, 20.0 mm × 250 mm), Supersil ODS2 (Elite, 10 μm, 20.0 mm × 250 mm), Ultimate XB-CN (Welch, 5 μm, 10.0 mm × 250 mm) and preparative SinoPark C18 (Elite, 10 μm, 20.0 mm × 250 mm) were analyzed by an EClassical P3500 prep-HPLC system and a DAD detector (EClassical P3500, Dalian Elite Analytical Instruments Co., Ltd., Dalian, China). The progress of the experiments was monitored by pre-coated silica gel GF254 plates (Qingdao Marine Chemical). Spots were visualized under UV light (254 or 365 nm) or by spraying with 10% H2SO4 in EtOH followed by heating. All other chemicals used in this study were of analytical grade.

3.2 Fungal material

The fungal strain Trichothecium sp. DWS815 was isolated from a forest soil sample collected from Daweishan National Forest Park, Hunan Province. The strain was identified as Trichothecium sp. (GenBank Accession No. ON386019.1) based on their ITS sequences and the phylogenetic analysis (Tables S24 and S25). A voucher specimen of this strain has been deposited at the Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, China.

3.3 Fermentation, extraction and isolation

This fungus was initially cultivated on Sabouraud Dextrose Agar (SDA) medium (containing 20 g/L dextrose, 10 g/L peptone, 1 g/L yeast extract powder, 15 g/L agar and water) for 8 days at 25 °C. Subsequently, the mycelia on agar plugs were cut and transferred to the sterile rice fermentation medium. The medium was prepared with 100 g of rice and 80 mL of water in 500 mL Erlenmeyer flasks. For large-scale fermentation, a total of 14 kg of rice was distributed into 139 flasks and inoculated with this fungus. The fermentation proceeded at 25 °C in the dark for 21 days.

The rice was extracted with ethyl acetate at room temperature 10 times, and the solvent was evaporated under vacuum to yield an extract (170 g). The extract was absorbed onto 500 g macro-porous adsorbent resin D101 and eluted stepwise with water, 80% aqueous MeOH, and 100% MeOH. The 80% aqueous MeOH fraction was collected and condensed to yield 26 g residue. The 80% aqueous MeOH fraction was then fractionated using silica gel CC eluted with a step gradient of Petroleum ether–EtOAc (v/v, 100:0, 20:1, 15:1, 10:1, 5:1, 2:1, 1:1) and EtOAc–MeOH (v/v, 100:0, 10:1, 5:1, 1:1, 0:100) to provide nine fractions (A∼I).

The Fr.A was subjected to MCI to give five subfractions (Fr.A1∼Fr.A5). Fr.A2 was separated using semipreparative Supersil ODS-B (CH3CN: H2O, 55:45) to afford compound 24 (tR = 20.9 min, 22.5 mg) and Fr.A2-1. Fr.A2-1 was fractionated using Supersil ODS-B (MeOH: H2O, 57:43) to afford compounds 7 (tR = 36.9 min, 3.5 mg) and 21 (tR = 40.1 min, 1.9 mg). Fr.A4 was separated by semipreparative ZORBAX SB-C18 (CH3CN: H2O, 49:51) to obtain compound 20 (tR = 23.9 min, 50.2 mg). Fr.B was subjected to Sephadex LH-20 (MeOH) to give five subfractions (Fr.B1∼Fr.B5). Fr.B3 was isolated on the preparative SinoPark C18 (MeOH: H2O, 62:38) to obtain four fractions (Fr.B3-1∼Fr.B3-4). Fr.B3-1 was purified using semipreparative Supersil ODS2 (CH3CN: H2O, 28:72) to afford compounds 1 (tR = 21.1 min, 0.3 mg) and 2 (tR = 29.2 min, 0.6 mg). Fr.B3-4 was purified using semipreparative Supersil ODS-B (MeOH: H2O, 53:47) to yield compound 17 (tR = 47.0 min, 24.5 mg). Fr.C was applied to semipreparative ZORBAX SB-C18 with a step gradient of CH3CN-H2O (v/v, from 20:100 to 50:50 in 35 min, flow speed: 3 mL/min) to afford compounds 22 (tR = 19.8 min, 1.6 g), 16 (tR = 17.7 min, 4.9 mg), 8 (tR = 19.4 min, 6.8 mg), and 5 (tR = 32.8 min, 6.58 mg). Fr.D was subjected to Sephadex LH-20 (MeOH) and then was further fractionated using semipreparative Ultimate XB-CN (CH3CN: H2O, 19:81) to give 19 (tR = 14.6 min, 1.1 mg), 18 (tR = 9.5 min, 1.9 mg), 9 (tR = 12.3 min, 1.5 mg), 13 (tR = 28.9 min, 8.4 mg), 26 (tR = 20.0 min, 3.5 mg), 25 (tR = 24.9 min, 9.9 mg), and 12 (tR = 17.0 min, 2.3 mg). Fr.E was subjected to Sephadex LH-20 (MeOH) and then was purified using semipreparative Supersil ODS-B (MeOH: H2O, 26:74) to afford 14 (tR = 15.9 min, 1.4 mg), 15 (tR = 12.2 min, 41.9 mg), 4 (tR = 10.7 min, 3.09 mg), 23 (tR = 23.5 min, 3.3 mg), and 11 (tR = 8.7 min, 1.6 mg). Fr.F was subjected to Sephadex LH-20 (MeOH) and then was purified using semipreparative ZORBAX SB-C18 with a step gradient of CH3CN–H2O (v/v, from 5:95 to 21:79 in 100 min, flow speed: 3 mL/min) to yield 3 (tR = 81.6 min, 1.3 mg). Fr.I was also chromatographed on Sephadex LH-20 (MeOH) and purified using semipreparative Ultimate XB-CN with a step gradient of CH3CN–H2O (v/v, from 25:75 to 45:55 in 40 min, flow speed: 3 mL/min) to give compounds 6 (tR = 29.8 min, 0.5 mg) and 10 (tR = 35.1 min, 1.9 mg).

Trichotheciumone A (1). White powder; \([\alpha]^_\)+ 37.5 (c 0.04, MeOH); UV (MeOH) λmax (log ε): 210 (3.48) nm; IR (KBr) νmax: 3442, 2979, 2950, 2923, 1714, 1647, 1294, 1178, 1115, 1028, 817 cm−1; CD (c 1.19 mM, MeOH): 205 nm (Δε –4.84), 227 nm (Δε + 4.38), 269 nm (Δε –1.91); for 13C NMR (CDCl3, 150 MHz) and 1H NMR (CDCl3, 600 MHz) data, see Tables 1 and 4; HRESIMS (positive) m/z 367.1758 [M + H]+ (calcd for C19H27O7, 367.1751, Δ + 1.9064 ppm).

Trichotheciumone B (2). White powder; \([\alpha]^_\) + 264.86 (c 0.037, MeOH); UV (MeOH) λmax (log ε): 210 (2.88) nm; IR (KBr) νmax: 3434, 2924, 2855, 2788, 1718, 1578, 1435, 1384, 1180 cm−1; CD (c 1.05 mM, MeOH): 200 nm (Δε + 5.29), 226 nm (Δε –2.83), 270 nm (Δε –0.51); for 13C NMR (CDCl3, 150 MHz) and 1H NMR (CDCl3, 600 MHz) data, see Tables 1 and 4; HRESIMS (positive) m/z 353.1599 [M + H]+ (calcd for C18H25O7, 353.1595, Δ + 1.1326 ppm).

Trichothecinoside A (3). White powder; \([\alpha]^_\) –2.0 (c 0.05, MeOH); UV (MeOH) λmax (log ε): 200 (3.17) nm; IR (KBr) νmax: 3420, 2962, 2935, 2876, 1715, 1649, 1383, 1291, 1184, 1083, 1027, 959 cm−1; CD (c 1.00 mM, MeOH): 217 nm (Δε –7.13); for 13C NMR (CDCl3, 150 MHz) and 1H NMR (CDCl3, 600 MHz) data, see Tables 1 and 4; HRESIMS (positive) m/z 499.2519 [M + H]+ (calcd for C25H39O10, 499.2538 Δ –3.8057 ppm).

Trichothecrotocin T (4). White powder; \([\alpha]^_\) –12.5 (c 0.037, MeOH); UV (MeOH) λmax (log ε): 200 (2.82) nm; IR (KBr) νmax: 3459, 3371, 2981, 2932, 2859, 1628, 1457, 1135, 1125, 1098, 1051 cm−1; CD (c 1.35 mM, MeOH): 200 nm (Δε –28.13); for 13C NMR (CDCl3, 150 MHz) and 1H NMR (CDCl3, 600 MHz) data, see Tables 1 and 4; HRESIMS (positive) m/z 297.1700 [M + H]+ (calcd for C16H25O5, 297.1697, Δ + 1.0009 ppm).

Trichothecrotocin U (5). White powder; \([\alpha]^_\) –10.9 (c 0.064, MeOH); UV (MeOH) λmax (log ε): 200 (3.21) nm; IR (KBr) νmax: 3434, 2978, 2946, 2922, 2859, 1737, 1714, 1647, 1436, 1248, 1179, 1133, 1049 cm−1; CD (c 1.37 mM, MeOH): 200 nm (Δε –30.89), 210 nm (Δε + 0.19), 225 nm (Δε –8.52); for 13C NMR (CDCl3, 150 MHz) and 1H NMR (CDCl3, 600 MHz) data, see Tables 2 and 4; HRESIMS (positive) m/z 469.2211 [M + H]+ (calcd for C 27H33O7, 469.2221, Δ –2.1312 ppm).

Trichothecrotocin V (6). White powder; \([\alpha]^_\)+ 12.5 (c 0.008, MeOH); UV (MeOH) λmax (log ε): 210 (2.94) nm; IR (KBr) νmax: 2971, 2928, 2855, 1717, 1576, 1445, 1384, 1183, 1081, 971 cm−1; CD (c 1.50 mM, MeOH): 208 nm (Δε –11.61), 291 nm (Δε + 0.92); for 13C NMR (CDCl3, 150 MHz) and 1H NMR (CDCl3, 600 MHz) data, see Tables 2 and 4; HRESIMS (positive) m/z 335.1851 [M + H]+ (calcd for C19H27O5, 335.1853, Δ –0.5967 ppm).

Trichothecrotocin W (7). white powder; \([\alpha]^_\) –6.98 (c 0.043, MeOH); UV (MeOH) λmax (log ε): 210 (3.08) nm; IR (KBr) νmax: 2959, 2928, 1716, 1647, 1466, 1366, 1287, 1183, 1081, 966, 816 cm−1; CD (c 1.29 mM, MeOH): 213 nm (Δε –4.32), 293 nm (Δε + 1.46); for 13C NMR (CDCl3, 150 MHz) and 1H NMR (CDCl3, 600 MHz) data, see Tables 2 and 4; HRESIMS (positive) m/z 335.1863 [M + H]+ (calcd for C19H27O5, 335.1853, Δ + 2.9834 ppm).

Trichothecrotocin X (8). White powder; \([\alpha]^_\) –16.67 (c 0.024, MeOH); UV (MeOH) λmax (log ε): 208 (3.29) nm; IR (KBr) νmax: 2965, 2925, 2857, 1716, 1602, 1384, 1185, 1083, 969 cm−1; CD (c 0.72 mM, MeOH): 203 nm (Δε –13.10), 230 nm (Δε + 0.50); for 13C NMR (CDCl3, 125 MHz) and 1H NMR (CDCl3, 500 MHz) data, see Tables 2 and 4; HRESIMS (positive) m/z 333.1708 [M + H]+ (calcd for C19H25O5, 333.1697, Δ + 3.3016 ppm).

Trichothecrotocin Y (9). White powder; \([\alpha]^_\) –51.35 (c 0.037, MeOH); UV (MeOH) λmax (log ε): 200 (2.60) nm; IR (KBr) νmax: 3452, 2965, 2923, 2852, 1664, 1623, 1593, 1447, 1422, 1281, 1185, 1074, 955 cm−1; CD (c 1.49 mM, MeOH): 256 nm (Δε –43.19); for 13C NMR (CDCl3, 125 MHz) and 1H NMR (CDCl3, 500 MHz) data, see Tables 3 and 4; HRESIMS (positive) m/z 249.1482 [M + H]+ (calcd for C15H21O3, 249.1485, Δ –1.2041 ppm).

Trichothecrotocin Z (10). White powder; \([\alpha]^_\) –63.16 (c 0.019, MeOH); UV (MeOH) λmax (log ε): 210 (3.15) nm; IR (KBr) νmax: 2917, 2852, 1732, 1717, 1606, 1590, 1384, 1189, 1101, 1082, 1064, 1028, 957 cm−1; CD (c 0.60 mM, MeOH): 226 nm (Δε –13.12); for 13C NMR (dimethyl sulfoxide-d6, 150 MHz) and 1H NMR (dimethyl sulfoxide-d6, 600 MHz) data, see Tables 3 and 4; HRESIMS (positive) m/z 339.1563 [M + H]+ (calcd for C19H24O5Na, 339.1567, Δ –1.1793 ppm).

Isocrotocol B (11). White powder; \([\alpha]^_\) –18.0 (c 0.05, MeOH); UV (MeOH) λmax (log ε): 200 (2.71) nm; IR (KBr) νmax: 3507, 3435, 3332, 2978, 2939, 2876, 1637, 1452, 1321, 1135, 1100, 1055, 973, 898, 817 cm−1; CD (c 1.77 mM, MeOH): 221 nm (Δε + 1.42); for 13C NMR (CDCl3, 150 MHz) and 1H NMR (CDCl3, 600 MHz) data, see Tables 3 and 4; HRESIMS (positive) m/z 283.1542 [M + H]+ (calcd for C15H23O4, 283.1540, Δ + 0.7063 ppm).

Trichothecolone (12). White powder; \([\alpha]^_\) –5.22 (c 0.23, MeOH); UV (MeOH) λmax (log ε): 200 (2.02) nm; IR (KBr) νmax: 3502, 2982, 2966, 2931, 1715, 1458, 1280, 1081, 957 cm−1; CD (c 8.64 mM, MeOH): 200 nm (Δε –71.20), 292 nm (Δε + 15.78); for 13C NMR (methanol-d4, 150 MHz) and 1H NMR (methanol-d4, 600 MHz) data, see Tables 3 and 4; HRESIMS (positive) m/z 267.1585 [M + H]+ (calcd for C15H23O4, 267.1591 Δ –2.2458 ppm).

Epi-trichothecinol B (13). White powder; \([\alpha]^_\)+ 87.5 (c 0.04, MeOH); UV (MeOH) λmax (log ε): 200 (2.71) nm; IR (KBr) νmax: 3468, 2985, 2962, 2938, 1712, 1643, 1434, 1376, 1313, 1245, 1175, 1078, 1034, 996, 972, 962 cm−1; CD (c 1.77 mM, MeOH): 221 nm (Δε + 1.42); for 13C NMR (CDCl3, 150 MHz) and 1H NMR (CDCl3, 600 MHz) data, see Tables 3 and 4; HRESIMS (positive) m/z 335.1856 [M + H]+ (calcd for C19H27O5, 335.1853, Δ + 0.8950 ppm).

3.4 Quantum chemical calculations

Conformer–rotamer ensemble sampling tool (CREST) was used to search the conformational space of compounds at the GFN0 level of theory in the gas phase [29, 30], followed by optimization at the GFN2-xTB level to avoid the errors caused by poor initial geometry [31], with a 4 kcal/mol energy window to remove high-energy conformers. Optimization and frequency calculation of each conformer were performed at the r2-SCAN-3c level of theory in the conductor-like polarizable continuum model (CPCM) to ensure they were true local minima on the potential energy surface [32, 33]. Under STS protocol [34], DFT GIAO 13C NMR calculation was calculated at the ωB97x-D/6-31G* (IEFPCM, chloroform) level or ωB97x-D/6-31G* (IEFPCM, methanol) level of theory. Time-dependent density-functional theory (TDDFT) ECD calculations were performed at the B3LYP/TZVP (IEFPCM, methanol) level of theory with the consideration of solvent effects. The calculated shielding tensors and ECD curves of conformers were Boltzmann averaged based on Gibbs free energy. In the TDDFT-ECD calculations, 40 excited states were calculated for each conformer. The ECD curves of all conformers were Boltzmann averaged based on Gibbs free energy calculated at the r2SCAN-3c level of theory and generated by Multiwfn [35, 36]. All DFT calculations were performed by Gaussian 16 or ORCA 6.0.0 software packages [37,38,39]. The DFT optimized geometry data, relative energies, and conformational population of all calculated structures were provided in the supplemental material. The 3D molecular structures were rendered using VMD (1.9.3) [40]. Details are provided in the Supplementary Information.

3.5 Cell lines and culture conditions

The cells of GES-1, HCT116, 4T1 and MHCC97H were cultivated in DMEM medium enriched with 10% fetal bovine serum and 1% penicillin–streptomycin. Cultures were maintained at 37 °C in a humidified environment with 5% CO2.

3.6 Cell viability analysis

Cell viability was determined with CCK-8 method [18]. All cell lines were plated into 96-well plates at a concentration of 10,000 cells per well and incubated for an additional 24 h. Then, the cells were treated with concentrations 5 μM and 50 μM of the test compounds and positive control (doxorubicin) for a duration of 48 h. In addition to the experimental groups, blank controls (wells with CCK-8 solution but no cells) and negative controls (wells with vehicle) were included. After treatment, 10 μL of CCK-8 reagent was added to each well and incubated for 1 h. The optical density at 450 nm was then measured on a microplate reader (Feyond-A300, allsheng, Hangzhou, China). The IC50 value represents the half of maximal inhibitory concentration. The cells of HCT116, 4T1, and MHCC97H were treated with a concentration gradient of selected compounds (7, 8, 10, 13, 17, 18, 20, and 24) to obtain IC50 values, using either the range of (0.04 μM–10 μM) or (0.5 μM–50 μM).

3.7 Cell cycle assays

The cell cycle distribution was appraised, using the Propidium Iodide (PI) Flow Cytometry Kit, followed by the flow Cytometry analysis [12]. Briefly, HCT116 cells were planted on 6-well plates and incubated overnight. Next, the cells were treated with 7 and 8 at concentrations of 0, 1 and 2 μM, and incubated for 24 h in triplicate manner. Cells were harvested and washed with cold phosphate buffered saline (PBS), and fixed overnight with ice-cold 70% ethanol. Then, the ethanol was removed, and the cells were rinsed with PBS and stained with the DNA fluorochrome PI at room temperature in the dark for 20 min. CYTEK™ NL-3000 flow cytometer was then used to test samples.