**2. Results**

#### *2.1. Structure Elucidation*

Asperchalasine I (**1**), the molecular formula, C33H41O7N, was determined on the basis of the HRESIMS ion at *m*/*z* 562.2798 ([M − H]− calcd. for C33H40O7N: 562.2799). As shown in Table 1, the 1H NMR data indicated characteristic signals of two double-bond protons (δH 5.93 and 5.36), nine methine protons (δH 5.52, 5.06, 4.46, 3.76, 3.21, 3.05, 2.88, 2.54 and 1.62), four methylene protons (δH 2.09, 1.90, δH 2.01, 1.58, δH 1.35 and δH 1.21) and six methyl groups (δH 2.05, 1.78, 1.26, 1.17, 0.96 and 0.94). Subsequently, the 13C NMR data showed the presence of 33 carbon signals, according to the DEPT and HSQC data, which were identified as three carbonyls (δC 211.6, 203.6 and 176.3), an aromatic ring (δC 141.3, 133.7, 132.6, 132.5, 123.7 and 111.9), two trisubstituted double bonds (δC 141.8, 137.4, 125.8 and 125.1), nine methines, four methylenes, six methyls and a quaternary carbon (δC 67.3). The 1H-1H COSY (Figure 2) correlations of H3-23/H-22/H2-10/H-3/H-4/H-5/H3-11, H-7/H-8/H-13, H2-15/H2-16/H2-17 and

H-19/H-20, together with the HMBC correlations from H-4 to C-1, C-9 and C-21, from H3-12 to C-5, C-6 and C-7, from H-13 to C-25, from H3-25 to C-15, from H2-17 and H-19 to C-18, and from H-20 to C-21, suggested the presence of an cytochalasan moiety. Meanwhile, the epicoccine moiety was inferred by HMBC correlations from H-1- to C-3-, from H-8- to C-2- and from H3-9- to C-5-, C-6- and C-7-. The key linking relation of the cytochalasan moiety and epicoccine moiety through C-19/C-8- and C-20/C-1- C-C bonds was suggested from the 1H-1H COSY cross-peak of H-19/H-8- and the HMBC correlation from H-1- to C-20 and C-21. Moreover, the structure of **1** was further confirmed by a detailed comparison of the NMR data of **1** and asperchalasine B [26], which suggested that they shared the same skeleton. The upfield shifted of H2-17 (δH 1.21) in **1** (δH 4.10 in asperchalasine B) and the disappearance of a methoxy group at C-4- (δH 3.70, δC 61.0), suggesting the replacements of methine at C-17 and a methoxy group at C-4- in asperchalasine B with a methylene and hydroxy group in **1**, respectively.


**Table 1.** 1H (500 MHz) and 13C (125 MHz) NMR data for **1** in CDCl3.

**Figure 2.** The key 2D NMR correlations of **1**–**4**.

Detailed analysis of NOESY data determined the relative configuration of chiral carbons in compound **1**. The NOESY correlations of H-4/H2-10, H-3/H3-11, H-5/H-8/H3-25, H-13/H-20 and H-19/H3-25 suggested that these protons were cofacial. Neither 1H-1H coupling nor a 1H-1H COSY correlation was observed between the protons of H-20 (δH 4.46, d, *J* = 5.8 Hz) and H-1- (δH 5.06, s), which suggested that the dihedral angle of those protons was approximately 90◦ (Figure 3) [26]. The electron circular dichroism (ECD) spectrum (Figure S8) showed two positive Cotton effects (CE) at 228 nm and 306 nm, which were also consistent with those of asperchalasine B. Therefore, the absolute configuration of **1** was suggested to be 3*S*, 4*R*, 5*S*, 8*S*, 9*S*, 19*S*, 20*S*, 1-*S*, 8-*R*.

**Figure 3.** Molecular model of **1** (1β, 8- β-oxygen bridge).

Dibefurin B (**2**), a colorless monocrystal, was assigned a molecular formula of C18H20O6 by the HRESIMS ion at *m*/*z* 331.1187 ([M − H]− calcd. for C18H19O6: 331.1187). For the monomer, the 1H, 13C NMR (Table 2) and HSQC data of **2** revealed diagnostic signals for nine carbons, including two carbonyls (δC 200.6 and 192.6), two disubstituted olefin carbons (δC 157.6 and 131.4), an oxygen-bearing carbon [δH 6.67 (OH-6), δC 91.7 (C-6)], three methyls (δC 19.1, 12.2 and 12.1) and a quaternary carbon (δC 60.4). The HMBC correlations from H3-7 to C-1, C-2, C-3 and C-6-, from H3-8 to C-2, C-3 and C-4, from H3-9 to C-3, C-4 and C-5, revealed that Me-7, Me-8 and Me-9 were connected at C-2, C-3 and C-4, respectively. Similarly, the hydroxyl was attached to C-6, as evidenced by the HMBC correlations from OH-6 to C-1, C-2-, C-5 and C-6. In view of the HRESIMS data and X-ray (Figure 4), it could confirm

that compound **2** was a symmetric dimer and the absolute configuration of **2** was assigned as 2*S*, 6*R*, 2-*R*, 6-*S*.


**Table 2.** 1H (400 MHz) and 13C (100 MHz) NMR data for **2** in DMSO-*d*6.

**Figure 4.** Single-crystal X-ray structures of **2**–**4**.

Compound **3** was purified as a colorless crystal whose molecular formula was deduced as C10H12O3 based on HRESIMS data (179.0716 [M − H]<sup>−</sup>, calcd. 179.0714). Analysis of the 1H NMR spectrum of **3** (Table 3) displayed three aromatic proton resonances (δH 7.13, 6.82 and 6.69), an oxygenated methylene (δHa 5.12, δHb 5.02), two oxygenated methines (δH 5.06 and 3.97), and a methyl (δH 1.20). The 13C spectrum revealed 10 signals, indicating an aromatic ring, a methylene, two methines and a methyl. In the 1H-1H COSY spectrum, the ortho-trisubstitution on the aromatic ring was confirmed by the cross-peaks of H-4/H-5/H-6. Moreover, the 1H-1H COSY spectrum showed correlations from H-10 to H-9 and H-11, and the chemical shift of C-10 (δC 70.6) showed the hydroxyl was attached to C-10. Subsequently, the HMBC correlations between H-6 and C-1/C-2 determined the linkage of 1-OH to C-1, and the correlations between H-7 and C-1/C-2/C-3, H-9 and C-2/C-3/C-10 established the presence of a phthalan ring. The same relative configuration of C-9 and C-10 was clearly deduced under the guidance of single-crystal X-ray (Figure 4). Hence, the absolute configuration of **3** was determined as 9*R*, 10*R*.

Compound **4** was deduced to have a molecular formula of C10H10O6 from its HRESIMS spectrum with a deprotonated molecular ion at *m*/*z* 225.0407. The 1H NMR (Table 3) in MeOH-*d*4 showed three singlets at δH 9.71, 3.90 and 2.08, according to the 13C NMR and HSQC data, which were attributed to an aldehyde group (δC 194.7), a methoxy group (δC 52.9) and a methyl (δC 12.4), respectively. In addition, resonances of a carbonyl and an aromatic ring were observed in the 13C NMR data. In the HMBC spectrum, the correlations of H-9 to C-2, C-3 and C-4 supported the connection of Me-9 to C-3, the correlations of H-7 to C-1 and C-6 indicated the linkage of aldehyde group and C-1. Meanwhile, the carbonyl (δC 169.9, C-8) had the HMBC correlation from H-10 (δH 3.90), further indicated the presence of methyl ester. With the assistance of single-crystal X-ray (Figure 4), the structure of compound **4** was clearly confirmed.


**Table 3.** 1H and 13C NMR data for **3** and **4** in MeOH-*d*4.

a 1H and 13C NMR recorded at 500 MHz and 125 MHz; b 1H and 13C NMR recorded at 400 MHz and 100 MHz.
