*3.3. Fermentation, Extraction, and Isolation*

The fungal strain *Eutypella* sp. D-1 was cultivated in seed medium (PDB 100 mL) in 250 mL Erlenmeyer flasks on a rotatory shaker (180 rpm) at 20 ◦C for 3 days. Subsequently, seed medium (10 mL) was transferred into 60 × 250 mL Erlenmeyer flasks (40 g of rice and 60 mL of water) and 60 plates of about 20 cm diameter (sucrose 51.4 g, NaNO3 3.3 g, K2HPO4·3H2O 0.07 g, MgSO4·7H2O 0.4 g, KCl 0.625 g, yeast extract 0.7 g, CoCl2·6H2O 0.003125 g, FeSO4 0.01875 g, CaCl2 0.0065 g, and L-ornithine hydrochloride 15 g, and agar

20.0 g, dissolved in 1 L of water), respectively, and then cultured under static conditions at 20 ◦C for 45 days.

The rice fermentation was combined and then extracted with CH2Cl2−MeOH (1:1, 1 L) three times. The organic solvent was concentrated under reduced pressure and partitioned with EtOAc and H2O to yield the EtOAc extract (24.5 g). The EtOAc extract was subjected to vacuum liquid chromatography (VLC) on silica gel via gradient elution using CH2Cl2/MeOH (80:1, 60:1, 40:1, 20:1, 15:1, 10:1, 0:1, *v*/*v*) as the solvent to give seven fractions (A–G). Fraction B (1.23 g) was chromatographed on a Sephadex LH-20 column using CH2Cl2−MeOH (1:1) as mobile phase to afford three subfractions (Fr. B1−B3), and subfraction B1 was further purified by reversed-phase HPLC eluting 43% MeCN/H2O at a flow rate of 2 mL/min to afford **1** (3.2 mg, tR = 16.6 min). Compounds **2** (7.3 mg, tR = 23.4 min) and **3** (1.4 mg, tR = 46.6 min) were isolated using reversed-phase HPLC (63% MeOH/H2O) from subfraction B2. Fraction F (3.75 g) was separated using MPLC on an ODS (50 μm) column to give seven fractions (Fr. F1–F7). Fr. F3 was subjected to reversed-phase HPLC (65% MeOH/H2O, 2 mL/min) to afford **11** (15.3 mg, tR = 15.3 min). Fr. F4 was separated with reversed-phase HPLC (40% CH3CN/H2O, 2 mL/min) to give **12** (40.1 mg, tR = 11.3 min).

The defined medium fermentation was combined and then extracted with CH2Cl2−MeOH (1:1, 1 L) three times. The organic solvent was concentrated under reduced pressure to yield the extract (6.86 g). The extract was subjected to silica gel VLC, eluting with a gradient of petroleum ether/EtOAc (100:1, 80:1, 50:1, 30:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, *v*/*v*) to obtain 20 fractions (Fr.A−Fr.T). Fraction O (0.4 g) was subjected to an ODS (50 μm) column via MPLC (MeOH/H2O, 50–100%) to give eight fractions, Fr. O1−Fr.O8. Fr. O6 (17.1 mg) was then purified with semipreparative HPLC (MeOH/H2O, 63:37, *v*/*v*; 2.0 mL/min) at 250 nm to afford **5** (6.2 mg, tR = 32.1 min). Fr. P (0.42 g) was separated with MPLC (MeOH/H2O, 60–100%) to afford five fractions, Fr. P1−Fr. P5. Fr. P4 (27.2 mg) and Fr. P5 (19.5 mg) were purified with HPLC on an RP C18 column to give **4** (7.4 mg, MeCN/H2O 40:60, 2.0 mL/min, tR = 24.9 min) and **6** (6.3 mg, MeCN/H2O 50:50, 2.0 mL/min, tR = 30.1 min), respectively. Fr. Q (0.15 g) was separated with reversed-phase ODS (50 μm) MPLC eluting with a MeOH/H2O gradient (from 60% to 100%) to afford six subfractions, Fr.Q1−Fr.Q6. Fr. Q5 (17.2 mg) was purified on an RP C18 column with HPLC (80% MeOH/H2O, 2.0 mL/min), yielding **2** (2.4 mg, tR = 28.8 min). Fr. R (1.04 g) was chromatographed over ODS via MPLC using a gradient elution of MeOH−H2O (from 50% to 100%) to get five fractions (Fr. R1−R5). Fr. R3 (475.1 mg) was then subjected to a silica gel CC (petroleum ether/EtOAc, 3:1, *v*:*v*) to give five fractions, Fr. R3a−Fr. R3e. Fr. R3c (168.0 mg) was then purified with semipreparative HPLC on an RP C18 ODS (CH3CN/H2O, 30:70, *v*/*v*; 2.0 mL/min) to afford **8** (3.5 mg, tR = 52.2 min) and **11** (116.8 mg, tR = 60.5 min). Fr. R3d (275.7 mg) was further purified with 37% CH3CN via HPLC (2.0 mL/min) to afford **7** (6.8 mg, tR = 33.0 min) and **9** (174.0 mg, tR = 39.8 min). Fr. S (0.55 g) was chromatographed over ODS using a gradient elution of MeOH/H2O (from 50% to 100%) to obtain three fractions (Fr. S1−S3). Fr. S3 (322.0 mg) was further purified with 35% CH3CN via HPLC to afford compound **10** (243.8 mg, tR = 18.6 min).

Eutypelleudesmane A (**1**): light-brown oil; [*α*] 25 *<sup>D</sup>* –23.0 (*c* 0.10, MeOH); UV (MeOH) (log *ε*) λmax 241 (4.07) nm; CD (MeOH) (Δ*ε*) 242 (+17.1); IR (KBr) *ν*max 3357, 2956, 2929, 2873, 1741, 1650, 1455, 1438, 1376, 1232, 1153, 1116, 1068, 1024, 958, 883,850,719 cm<sup>−</sup>1; 1H and 13C NMR data, see Table 1; HRESIMS *m/z* 601.4074 [M + H]+ (calcd for C36H57O7, 601.4104).

Cytosporin Y (**2**): light brown oil; [*α*] 25 *<sup>D</sup>* +14.0 (*c* 0.10, MeOH); UV (MeOH) (log ε) λmax 241 (3.89) nm; CD (MeOH) (Δε) 238 (+9.1); IR (KBr) *ν*max 3359, 2956, 2927, 2857, 1454, 1376, 1261, 1014, 842, 725 cm<sup>−</sup>1; 1H and 13C NMR data, see Table 2; HRESIMS *m/z* 367.2125 [M + COOH]− (calcd for C20H31O6, 367.2121).

Cytosporin Z (**3**): light-brown oil; [*α*] 25 *<sup>D</sup>* +12.3 (*c* 0.10, MeOH), [*α*] 25 *<sup>D</sup>* +1.9 (*c* 0.1, CDCl3); UV (MeOH) (log ε) λmax 210 (5.37), 312 (3.13) nm; IR (KBr) *ν*max 3378, 2954, 2927, 2856, 1708, 1614, 1513, 1434, 1380, 1369, 1255, 1218, 1184, 1143, 1064, 1029, 977, 852 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS *m/z* 319.1912 [M − H]<sup>−</sup> (calcd for C19H27O4, 319.1909).

Cytosporin Y1 (**4**): light-yellow oil; [*α*] 25 *<sup>D</sup>* –15.8 (*c* 0.1, MeOH); UV (MeOH) λmax (log ε) 221 (3.71), 240 (3.77) nm; IR νmax 3367, 2955, 2926, 2857, 1743, 1601, 1378, 1072, 1023 cm<sup>−</sup>1; CD (MeOH) (Δε) 208 (–6.4); 1H NMR and 13C NMR, see Table 3; HRESIMS *m/z* 363.2139 [M + Na]+ (calcd for C19H32O5Na, 363.2142).

Cytosporin Y2 (**5**): light-yellow oil; [*α*] 25 *<sup>D</sup>* –60.1 (*c* 0.1, MeOH); UV (MeOH) λmax (log ε) 214 (3.96), 244 (4.28) nm; IR νmax 3381, 2956, 2926, 2857, 1786, 1647, 1344, 1219, 1049, 1023, 1001, 822, 760 cm<sup>−</sup>1; CD (MeOH) (Δε) 222 (–13.2); 1H NMR and 13C NMR, see Table 3; HRESIMS *m/z* 389.1928 [M + Na]+ (calcd for C20H30O6Na, 389.1935).

Cytosporin Y3 (**6**): light-yellow oil, [*α*] 25 *<sup>D</sup>* +37.8 (*c* 0.1, MeOH); UV (MeOH) λmax (log ε) 217 (3.64), 241 (3.81) nm; IR νmax 3383, 2956, 2928, 2858, 1783, 1595, 1361, 1270, 1182, 1068, 907, 770, 737 cm−1; CD (MeOH) (Δε) 241 (+4.87); 1H NMR and 13C NMR, see Table 4; HRESIMS *m/z* 389.1928 [M + Na]+ (calcd for C20H30O6Na, 389.1935).

Cytosporin E1 (**7**): light-yellow oil, [*α*] 25 *<sup>D</sup>* +12.9 (*c* 0.1, MeOH), UV (MeOH) λmax (log ε) 200 (4.10) nm; IR νmax 3393, 2926, 2856, 1783, 1464, 1361, 1184, 1158, 1086, 1058, 1023, 917, 772, 629 cm−1; CD (MeOH) (Δε) 210 (+14.2) nm; 1H NMR and 13C NMR, see Table 4; HRESIMS *m/z* 363.2145 407.2034 [M + Na]+ (calcd for C20H32O7Na, 407.2040).
