*2.2. Structural Elucidation*

Compound **1** was isolated as a yellowish amorphous powder. Its molecular formula, C31H26O12, was determined on the basis of the positive HR-ESI-MS ion at *m*/*z* 613.13200 [M + Na] <sup>+</sup> (calcd. 613.13219) and supported by the 1H- and 13C- NMR data. The IR spectrum showed the presence of hydroxyl (3428 cm−1), ester carbonyl (1744 cm−1), and conjugated carbonyl (1655 cm−1) groups. The 1H- and 13C-NMR data of **1** showed the presence of two hydroxy groups, two methyl groups, three methoxy groups, two pairs of meta-coupling aromatic protons, two methylene protons, and three methine protons. The signals at δ 12.11 and 12.30 exhibited two chelated hydroxyl groups with the carbonyl group. Comparison of the 1H and 13C NMR data of **1** with those of epiremisporine B [16] suggested that their structures were closely related, except that the 2 -methoxyl group of **1** replaced the 2 -hydroxy group of epiremisporine B [16]. This was supported by both

HMBC correlations between OMe-2 (δ<sup>H</sup> 3.50) and C-2 (δ<sup>C</sup> 111.1) and ROESY correlations between OMe-2 (δ<sup>H</sup> 3.50) and H-3 (δ<sup>H</sup> 2.86). The relative configuration of **1** was elucidated on the basis of ROESY experiments. The ROESY cross-peaks between H-3/H-4, H-3/H-3 , H-3/Hα-4 , OMe-2 /H-3 , and H-3/H-16 suggested that H-3, H-4, H-3 , OMe-2 , and COOMe-2 are α-oriented, and COOMe-2 is β-oriented. To further confirm the relative configuration of **1**, a computer-assisted 3D structure was obtained by using the molecularmodeling program CS CHEM 3D Ultra 16.0, with MM2 force-field calculations for energy minimization. The calculated distances between H-3/H-4 (2.210 Å), H-3/H-3 (2.491 Å), OMe-2 /H-3 (2.305 Å), and H-3/H-16 (2.371 Å) were all less than 4 Å (Figure 2). This was consistent with the well-defined ROESY observed for each of these H-atom pairs. The absolute configuration of **1** was evidenced by the CD Cotton effects at 332.5 (Δ*ε* +8.81), 293.0 (Δ*ε* −1.23), 258.5 (Δ*ε* +15.60), 239.5 (Δ*ε* −4.12), and 206.5 (Δ*ε* +4.09) nm, in analogy with those of epiremisporine B [16]. The 1H- and 13C-NMR resonances were fully assigned by the 1H–1H COSY, HSQC, ROESY, and HMBC experiments (Figure 3). On the basis of the above data, the structure of **1** was elucidated, as shown in Figure 1, and named epiremisporine C.

**Figure 2.** Selected ROESY correlations and relative configuration of **1**.

**Figure 3.** Key ROESY (**a**) and HMBC (**b**) correlations of **1**.

Compound **2** was obtained as an amorphous powder. The ESI–MS demonstrated the quasi-molecular ion [M + Na]<sup>+</sup> at *m*/*z* 627, implying a molecular formula of C32H28O12, which was confirmed by the HR-ESI-MS (*m*/*z* 627.12902 [M + Na]+, calcd. 627.14784) and by the 1H- and 13C-NMR data. The IR spectrum showed the presence of hydroxyl (3480 cm<sup>−</sup>1), ester carbonyl (1763 cm−1), and conjugated carbonyl (1657 cm−1) groups. The signal at δ 12.36 exhibited a chelated hydroxyl group with the carbonyl group. Comparison of the 1H and 13C NMR data of **2** with those of epiremisporine C (**1**), suggested that their structures were closely related, except that the 11-methoxyl group of **2** replaced the 11 hydroxy group of **1**. This was supported by both HMBC correlations between OMe-11 (δ<sup>H</sup> 3.91) and C-11 (δ<sup>C</sup> 160.0) and ROESY correlations between OMe-11 (δ<sup>H</sup> 3.91) and H-10 (δ<sup>H</sup> 6.58). The relative configuration of **2** was elucidated on the basis of ROESY experiments. The ROESY cross-peaks between H-3/H-4, H-3/H-3 , H-3/Hα-4 , OMe-2 /H-3 , and H-3/H-16 suggested that H-3, H-4, H-3 , OMe-2 , and COOMe-2 were α-oriented, and COOMe-2 was β-oriented. To further confirm the relative configuration of **2**, a computer-assisted 3D structure was obtained by using the molecular-modeling program CS CHEM 3D Ultra 16.0, with MM2 force-field calculations for energy minimization. The calculated distances between H-3/H-4 (2.200 Å), H-3/H-3 (2.484 Å), OMe-2 /H-3 (2.306 Å), and H-3/H-16 (2.329 Å) were all less than 4 Å (Figure 4). This was consistent with the well-defined ROESY observed for each of these H-atom pairs. Compound **2** showed similar CD Cotton effects [330.5 (Δ*ε* +5.39), 290.5 (Δ*ε* –6.24), 262.5 (Δ*ε* +19.72), 238.5 (Δ*ε* –2.06), and 207.0 (Δ*ε* +13.72) nm] compared with **1** and epiremisporine B [16]. Thus, **2** possessed a 2*S*,3*R*,2 *R*,3 *S*-configuration. On the basis of the above data, the structure of **2** was elucidated, as shown in Figure 1, and named epiremisporine D, which was further confirmed by the 1H-1H COSY, ROESY (Figure 5a), DEPT, HSQC, and HMBC (Figure 5b) experiments.

**Figure 4.** Selected ROESY correlations and relative configuration of **2**.

**Figure 5.** Key ROESY (**a**) and HMBC (**b**) correlations of **2**.

Compound **3** was isolated as an amorphous powder. The ESI–MS demonstrated the quasi-molecular ion [M + Na]<sup>+</sup> at *m*/*z* 627, implying a molecular formula of C32H28O12, which was confirmed by the HR–ESI–MS (*m*/*z* 627.12919 [M + Na]+, calcd. 627.14784) and by the 1H- and 13C-NMR data. The IR spectrum showed the presence of hydroxyl

(3466 cm<sup>−</sup>1), ester carbonyl (1761 and 1740 cm−1), and the conjugated carbonyl (1657 cm−1) groups. The signal at δ 12.50 exhibited a chelated hydroxyl group with the carbonyl group. Comparison of the 1H and 13C NMR data of **3** with those of epiremisporine D (**2**) suggested that their structures were closely related, except that the 2 β-methoxyl group of **3** replaced the 2 α-methoxyl group of **2**. This was supported by both HMBC correlations between OMe-2 (δ<sup>H</sup> 3.11) and C-2 (δ<sup>C</sup> 107.6), and the ROESY correlations between OMe-2 (δ<sup>H</sup> 3.11) and Hβ-4 (δ<sup>H</sup> 2.85). The relative configuration of **3** was elucidated on the basis of ROESY experiments. The ROESY cross-peaks between H-3/H-4, H-3/H-3 , H-3/Hα-4 , OMe-2 /H-4 , and H-3/H-16 suggested that H-3, H-4, H-3 , COOMe-2, and COOMe-2 were α-oriented, and OMe-2 was β-oriented. To further confirm the relative configuration of **3**, a computer-assisted 3D structure was obtained by using the molecular-modeling program CS CHEM 3D Ultra 16.0, with MM2 force-field calculations for energy minimization. The calculated distances between H-3/H-4 (2.169 Å), H-3/H-16 (2.285Å), H-3/H-3 (2.445 Å), and OMe-2 /Hβ-4 (3.682Å) were all less than 4 Å (Figure 6). This was consistent with the well-defined ROESY observed for each of these H-atom pairs. Compound **3** showed similar CD Cotton effects [331.0 (Δ*ε* +4.01), 286.5 (Δ*ε* −7.51), 261.5 (Δ*ε* +19.77), 230.5 (Δ*ε* −4.98), and 207.5 (Δ*ε* +12.68) nm], compared to the literature data [16]. Thus, **3** possessed a 2*S*,3*R*,2 *S*,3 *S*-configuration. The 1H- and 13C-NMR resonances were fully assigned by 1H–1H COSY, ROESY (Figure 7a), HSQC, and HMBC (Figure 7b) experiments. On the basis of the above data, the structure of **3** was elucidated, as shown in Figure 1, and named epiremisporine E.

**Figure 6.** Selected ROESY correlations and the relative configuration of **3**.

**Figure 7.** Key ROESY (**a**) and HMBC (**b**) correlations of **3**.

The correlations between the dihedral angles (H3 -C3 -C4 -H4 α and H3 -C3 -C4 - H4 β) and the vicinal coupling constants (*J*3 , 4 <sup>α</sup> and *J*3 , 4 <sup>β</sup>) of compounds **1**–**3** and related analogues [16] are summarized in Table 1. The dihedral angles were calculated by using the molecular-modeling program CS CHEM 3D Ultra 16.0, with the MM2 force-field calculations for energy minimization. The correlations between dihedral angles (H3 -C3 - C4 -H4 α and H3 -C3 -C4 -H4 β) and vicinal coupling constants (*J*3 , 4 <sup>α</sup> and *J*3 , 4 <sup>β</sup>) of compounds **1**–**3** were consistent with the Karplus relationship. The 2 *S,*3 *S*-configuration slightly decreased the *J*3 , 4 <sup>β</sup> value from 11.3~12.8 to 8.3~10.3 compared to the 2 *R,*3 *S*configuration. These data could also support the structural confirmation of the new compounds **1**–**3**.


**Table 1.** The correlations between dihedral angles and vicinal coupling constants of compounds **1**–**3** and related analogues [16].

## *2.3. Structure Identification of the Known Isolated Compounds*

The known isolated compounds were readily identified by a comparison of physical and spectroscopic data (UV, IR, 1H-NMR, [α]D, and MS) with corresponding authentic samples or literature values. They included epiremisporine B (**4**) [16] (Figures S31–S34; Tables S1 and S2), penicitrinone A (**5**) [15,28] (Figures S35 and S36), 8-hydroxy-1-methoxycarbonyl-6-methylxanthone (**6**) [10] (Figures S37 and S38), and isoconiochaetone C (**7**) [16] (Figures S39 and S40).
