*3.3. Extraction and Isolation*

Sixty 500-mL Erlenmeyer flasks of the fungal strain were cultivated in a starch liquid medium (soluble starch 10 g/L, peptone 1 g/L, artificial sea salt 30 g/L, 200 mL each flask) at 150 rpm and 28 ◦C for 7 days. The fermentation broth was filtered through a cheesecloth and extracted repeatedly with an equal amount of EtOAc three times, and then it was evaporated in vacuo to obtain an EtOAc extract (12.5 g). The crude extract was isolated on silica gel CC using a step gradient elution with petroleum ether/EtOAc (10:1 to 1:4, v/v) to provide five fractions (Fr.1−Fr.5). Fr.3 was subjected to a silica gel CC eluted with Hexane/CH2Cl2/MeOH (10:1:0 to 0:1:1) to obtain four subfractions (Fr.3.1−Fr.3.4). Fr.3.2 was further separated by the semi-preparative HPLC with MeOH/H2O (60:40) to give compounds **8** (5.5 mg) and **12** (7.1 mg). Fr.3.3 was separated by Sephadex LH-20 CC eluted with CH2Cl2/MeOH (1:1) and the semi-preparative HPLC with MeOH/H2O (45:55) further, to obtain compounds **9**−**11** (3.2 mg, 3.7 mg, and 2.1 mg, respectively). Fr.4 was separated on a silica gel CC eluted with CH2Cl2/MeOH (100:1 to 1:1) to provide four subfractions (Fr.4.1−Fr.4.4). Fr.4.1 was further subjected to Sephadex LH-20 CC eluted with CH2Cl2/MeOH (1:1) to obtain compounds **6** and **7**. Fr.4.3 was separated by the semi-preparative HPLC with MeOH/H2O (45:55) to give compounds **1**−**5** (3.3 mg, 3.1 mg, 4.2 mg, 4.5 mg, and 4.3 mg, respectively).

*Emestrin L* (**1**). White powder; [α] 20 <sup>D</sup> -70.4 (*c* 0.42, MeOH); UV (MeOH) λmax (log ε) 229 (3.83), 275 (1.61) nm; IR (KBr) *v*max 3600, 3395, 2998, 2913, 1646, 1436, 1314 cm<sup>−</sup>1; 1H and 13C NMR (see Table 1); (–)-HRESIMS *m*/*z* 475.1007 [M − H]<sup>−</sup> (calcd for C22H23N2O6S2, 475.1003).

*Emestrin M* (**2**). White powder; [α] 20 <sup>D</sup> -85.1 (*c* 0.43, MeOH); UV (MeOH) λmax (log ε) 229 (3.75), 275 (1.75) nm; IR (KBr) *v*max 3600, 3000, 2912, 2138, 1663, 1438, 1315, 1043 cm<sup>−</sup>1; 1H NMR (500 MHz, DMSO-*d*6) and 13C NMR (125 MHz, DMSO-*d*6) (see Table 1); (–)-HRESIMS *m*/*z* 433.0896 [M <sup>−</sup> H]<sup>−</sup> (calcd for C20H21N2O5S2, 433.0897).

*Emethacin C* (**3**). White powder; [α] 20 <sup>D</sup> -64.4 (*c* 0.52, MeOH); UV (MeOH) λmax (log ε) 229 (3.89), 275 (2.54) nm; IR (KBr) *v*max 2952, 2868, 1802, 1715, 1459, 1200, 1139 cm<sup>−</sup>1; 1H NMR (500 MHz, DMSO-*d*6): 8.97 (1H, s, 2-NH), 8.36 (1H, br s, 2- -NH), 7.18 (1H, m, H-7), 7.10 (4H, m, H-5, H-6, H-8, H-9), 6.89 (1H, td, *J* = 7.8, 1.7 Hz, H-7- ), 6.67 (1H, td, *J* = 7.8, 1.2 Hz, H-8- ), 6.26 (1H, dd, *J* = 7.8, 1.7 Hz, H-9- ), 6.22 (1H, dd, *J* =7.8, 1.2 Hz, H-6- ), 3.47 (1H, d, *J* = 13.6 Hz, H-3a), 3.05 (1H, d, *J* = 15.4 Hz, H-3- a), 3.01 (1H, d, *J* = 15.4 Hz, H-3- b), 2.96 (1H, d, *J* = 13.6 Hz, H-3b), 2.25 (3H, s, H-10), 2.17 (3H, s, H-10- ); 13C NMR (125 MHz, DMSO-*d*6): 166.3 (C, C-1- ), 165.6 (C, C-1), 155.6 (C, C-5- ), 135.4 (C, C-4), 130.8 (CH × 2, C-5, C-9), 129.9 (CH, C-9- ), 128.3(CH×2, C-6, C-8), 127.7 (CH, C-7- ), 127.2 (CH, C-7), 122.3 (C, C-4- ), 119.4 (CH, C-6- ), 115.2 (CH, C-8- ), 65.5 (C, C-2), 65.2 (C, C-2- ), 43.5 (CH2, C-3), 37.0 (CH2, C-3- ), 14.0 (CH3, C-10), 13.9 (CH3, C-10- ); (–)-HRESIMS *m*/*z* 401.1003 [M − H]<sup>−</sup> (calcd for C20H21N2O3S2, 401.0999).

*(3R)-8-methoxy-6-hydroxymellein* (**8**). Yellow solid; [α] 20 <sup>D</sup> -47.8 (*c* 1.00, MeOH); UV (MeOH) λmax (log ε) 238 (2.11), 251 (0.95), 319 (0.41) nm; IR (KBr) νmax 3749, 3674, 2360, 1736, 1581, 1418 cm−1; 1H NMR (500 MHz, DMSO-*d*6): 5.80 (1H, d, *J* = 1.9 Hz, H-5), 5.71 (1H, d, *J* = 1.9 Hz, H-7), 4.25 (1H, m, H-3), 3.59 (3H, s, H-9), 2.57 (1H, dd, *J* = 15.8, 3.3 Hz, H-4a), 2.55 (1H, m, overlapped, H-4b), 1.26 (3H, d, *J* = 6.2 Hz, H-10); 13C NMR (125 MHz, DMSO-*d*6): 172.3 (C, C-1), 163.4 (C-6), 162.1 (C, C-8), 142.5 (C, C-4a), 109.4 (CH, C-5), 100.1 (C, C-8a), 97.1 (CH, C-7), 72.0 (CH, C-3), 54.7 (CH3, C-9), 36.2 (CH2, C-4), 20.6 (CH3, C-10); (+)-HRESIMS *m*/*z* 209.0809 [M + H]<sup>+</sup> (calcd for C11H13O4, 209.0808).

*(3R)-6,7,8-trihydroxymellein* (**9**). Yellow solid; [α] 20 <sup>D</sup> -50.1 (*c* 0.37, MeOH); UV (MeOH) λmax (log ε) 238 (2.03), 251 (1.05), 317 (0.23) nm; IR (KBr) νmax 3749, 3674, 2360, 1736, 1651, 1384 cm−1; 1H NMR (500 MHz, DMSO-*d*6): 6.26 (1H, s, H-5), 4.64 (1H, m, H-3), 2.82 (1H, dd, *J* = 16.2, 3.3 Hz, H-4a), 2.71 (1H, dd, *J* = 16.2, 1.3 Hz, H-4b), 1.37 (3H, d, *J* = 6.2 Hz, H-9); 13C NMR (125 MHz, DMSO-*d*6): 170.0 (C, C-1), 153.2 (C-6), 151.1 (C, C-8), 131.3 (C, C-7), 131.0 (C, C-4a), 106.5 (CH, C-5), 100.0 (C, C-8a), 75.9 (CH, C-3), 33.2 (CH2, C-4), 20.4 (CH3, C-9); (–)-HRESIMS *m*/*z* 209.0458 [M − H]<sup>−</sup> (calcd for C10H9O5, 209.0455).

*Crystal data for* **6**: C18H18N2O7S2, *M*r = 534.11, monoclinic, *a* = 6.8830(3) Å, *b* = 12.4383(5) Å, *c* = 14.6020(6) Å, α = 90.00◦, β = 94.415(4)◦, γ = 90.00◦, *V* = 1246.41(9) Å3, space group *P*21, *Z* = 2, *D*x = 1.135 mg/m3, <sup>μ</sup> (Cu Kα) = 0.670 mm–1, and *F* (000) = 425. Crystal dimensions: 0.12 <sup>×</sup> 0.11 <sup>×</sup> 0.11 mm3. Independent reflections: 7369/4117 (*R*int = 0.0422). The final *R*1 value was 0.0481, *wR*2 = 0.1180 (*I* > 2σ(*I*)). Flack parameter = –0.012(13). Crystallographic data for **6** are deposited at the Cambridge Crystallographic Data Centre as supplementary publication number CCDC 1911625.
