*3.3. Cultivation and Isolation of Metabolites*

A culture of *Teratosphaeria* sp. AK1128 grown on PDA for two weeks was used for extraction. The PDA cultures from 20 T-flasks were combined and extracted with MeOH (5.0 L) in an ultrasonic bath for 1.0 h. After filtration, the MeOH solution was concentrated to around one-third of its volume in vacuo, and the resulting solution was extracted with EtOAc (3 × 700.0 mL). The EtOAc solution was concentrated in vacuo to afford the crude extract (1.223 g). The crude extract, which showed activity in cytotoxicity assay, was fractionated by solvent–solvent partitioning using 80% aq. MeOH (100.0 mL) and hexanes (3 × 100.0 mL), followed by 50% aq. MeOH (obtained from 80% aq. MeOH layer by adding calculated volume of water) and CHCl3 (3 × 100.0 mL) to afford hexanes, CHCl3, and 50% aq. MeOH fractions of which only the CHCl3 fraction was found to be cytotoxic. Therefore, the CHCl3 fraction (1.094 g) was subjected to gel-permeation chromatography on Sephadex LH-20 (50.0 g). The column was eluted with 250.0 mL each of 1:4 hexanes-CH2Cl2, 3:2 CH2Cl2-acetone, 1:4 CH2Cl2-acetone, and MeOH to give four fractions. The cytotoxic 3:2 CH2Cl2-acetone fraction (778.6 mg) was further fractionated by SiO2 gel (100.0 g) column chromatography using 95:5 CHCl3-MeOH as eluting solvent. Eight combined fractions (fractions 1–8) were obtained by combining the fractions based on their TLC profiles. Fraction 1 (12.5 mg) was separated by reversed-phase HPLC (70% aq. MeOH as eluant) to afford **1** (7.4 mg, tR 16.5 min) and **8** (2.7 mg, tR 10.5 min). Reversed-phase HPLC separation of a portion (55.6 mg) of fraction 2 (135.8 mg) using 65% aq. MeOH gave additional amounts of **8** (3.7 mg, tR 14.4 min) and **4** (29.6 mg, tR 25.6 min). Compounds **3** (16.9 mg, tR 26.2 min), **4** (10.8 mg, tR 18.1 min), and **7** (4.2 mg, tR 15.5 min) were obtained from fraction 3 (70.8 mg) by reversed-phase HPLC using 65% aq. MeOH as the eluent. Fraction 4 (29.1 mg) was further purified by reversed-phase HPLC (65% aq. MeOH) to afford **2** (8.3 mg, tR 27.7 min) and **6** (9.8 mg, tR 21.6 min). A portion (78.5 mg) of fraction 5 (299.7 mg) was subjected to reversed-phase HPLC. Elution with 65% aq. MeOH yielded metabolites **5** (54.5 mg, tR 15.3 min) and **6** (10.3 mg). Reversed-phase HPLC separation of fraction 6 (106.0 mg) using 60% aq. MeOH as eluent gave two crude fractions, A (tR 10.8 min) and B (tR 27.7 min). Further purification of fractions A and B by normal-phase SiO2 HPLC (eluent: 97.5:2.5 CHCl3-MeOH) afforded **10** (2.3 mg, tR 15.0 min) and **9** (26.0 mg, tR 17.5 min), respectively.

Teratopyrone A (**1**). Yellow amorphous solid; [α] 25 <sup>D</sup> −54.4 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 203 (4.45), 229 (4.63), 281.5 (4.84), 403 (4.09); ECD (MeOH) [θ] +1.78 <sup>×</sup> 105 (272 nm), <sup>−</sup>1.46 <sup>×</sup> 105 (289.5 nm); 1H NMR (400 MHz, CDCl3) δ 14.97 (s, 1H, 5-OH), 14.48 (s, 1H, 5- -OH), 7.08/7.03 (br s, 1H, H-10), 7.07/7.04 (br s, 1H, H-9), 6.26/6.28 (br s, 1H, H-7- ), 6.12 (br s, 1H, H-9- ), 5.98 (s, 1H, H-3- ), 3.90/3.94 (s, 3H, 6- -OMe), 3.60/3.59 (s, 3H, 8- -OMe), 3.39/3.55 (s, 3H, 8-OMe), 3.35 (m, 1H, H-3- ), 2.80/2.81 (m, 1H, H-3- ), 2.35 (s, 3H, 2-Me), 1.42/1.47 (s, 3H, 2- -Me); 13C NMR data, see Table 1; HRESIMS *m*/*z* 575.1546 [M + H]<sup>+</sup> (calcd. for C31H27O11, 575.1548).

Teratopyrone B (**2**). Yellow amorphous solid; [α] 25 <sup>D</sup> +39.7 (*c* 0.105, CHCl3); UV (MeOH) λmax (log <sup>ε</sup>) 202 (4.36), 238 (4.66), 281.5 (4.73), 386 (3.95); ECD (MeOH) [θ] <sup>+</sup> 1.63×105 (278 nm), <sup>−</sup>5.70 <sup>×</sup> 104 (297 nm); 1H NMR (400 MHz, DMSO-d6) δ 14.27/14.25 (s, 1H, 5-OH), 13.19/13.18 (s, 1H, 5- -OH), 6.90/6.85 (br s, 1H, H-9), 6.61 (br s, 2H, H-10, H-7- ), 6.54 (s, 1H, H-3- ), 6.19/6.17 (d, 1H, *J* = 2.0, H-9- ), 4.00 (s, 3H, 8-OMe), 3.60/3.59 (s, 3H, 8- -OMe), 3.41/3.40 (s, 3H, 6- -OMe), 3.25/3.23 (d, 1H, *J* = 12.6, H-3- ), 2.78/2.79 (d, 1H, *J* = 12.6, H-3- ), 2.55 (s, 3H, 2- -Me), 1.65/1.64/1.69 (s, 3H, 2-Me); 13C NMR data, see Table 1; HRESIMS *m*/*z* 575.1543 [M + H]<sup>+</sup> (calcd. for C31H27O11, 575.1548).

Teratopyrone C (**3**). Yellow amorphous powder; [α] 25 <sup>D</sup> +19.3 (*c* 0.07, CHCl3); UV (MeOH) λmax (log <sup>ε</sup>) 204 (4.52), 237.5 (4.80), 281 (4.80), 395 (4.03); ECD (MeOH) [θ] <sup>+</sup>8.82 <sup>×</sup> 104 (278 nm), <sup>−</sup>5.14 <sup>×</sup> <sup>10</sup><sup>4</sup> (292 nm); 1H NMR (400 MHz, DMSO-d6) δ 14.42 (s, 1H, 5- -OH), 12.89/12.88 (s, 1H, 5-OH), 7.00/7.02 (br s, 1H, H-7), 6.94 (br s, 1H, H-6), 6.50 (s, 1H, H-3), 6.44/6.39 (br s, 1H, H-7- ), 5.99/5.95 (br s, 1H, H-9- ) 3.91/3.89 (s, 3H, 6- -OMe), 3.55 (s, 3H, 8- -OMe), 3.46/3.53 (s, 3H, 10-OMe), 3.09 (m, 1H, H-3- ), 2.86 (m, 1H, H-3- ), 2.47 (s, 3H, 2-Me), 1.40/1.78 (s, 3H, 2- -Me); 13C NMR data, see Table 1; HRESIMS *m*/*z* 575.1551 [M + H]<sup>+</sup> (calcd for C31H27O11, 575.1548).
