**2. Results**

#### *2.1. Structure Elucidation*

Compound **1** was isolated as a light-yellow amorphous powder. The molecular formula of **1** was deduced to be C24H24N6O6S4 by the high-resolution electrospray ionization mass spectroscopy (HRESIMS) measurement (*m*/*z* [M+H]+ 621.0712, calcd for C24H25N6O6S4, 621.0713), accounting for sixteen degrees of unsaturation (Figure S1). The 1H NMR spectrum (Table 1, Figure S2) showed the presence of three exchangeable singlets (*δ*H 10.27, s, 7-*NH*; 10.21, s, 7--*NH*; 10.17, s, 7"-*NH*), three olefinic protons (*δ*H 6.58, s, H-3; 6.36, s, H-3-; 6.54, s, H-3"), three *N*-Me groups (*δ*H 3.19, s, Me-10; 3.47, s, Me-10-; 3.40, s, Me-10"), as well as three methyl singlets for acetyl groups (*δ*H 2.07, s, Me-9 and Me-9"; 2.06, s, Me-9-). The 13C NMR spectrum (Figure S3), in association with the heteronuclear single quantum correlation (HSQC) spectrum (Figure S4), indicated 24 carbon signals (Table 1), including six methyls (*δ*C 29.9, C-10; *δ*C 29.2, C-10-/C-10"; *δ*C 22.9, C-9; *δ*C 22.8, C-9-/C-9"), three sp<sup>2</sup> methines (*δ*C 111.9, C-3; *δ*C 112.5, C-3-; *δ*C 112.8, C-3"), six amide carbonyls (*δ*C 164.0/163.7/163.8, C-5/C-5-/C-5"; *δ*C 168.4/168.3/167.9, C-8/C-8-/C-8"), and nine sp<sup>2</sup> quaternary carbons (*δ*C 137.1/131.9/133.8, 3a/3a-/3a"; *δ*C 130.7/129.8/132.7, 6/6-/6"; *δ*C 124.6/124.6/126.4, 6a/6a-/6a"). The amide carbonyls and olefinic carbons accounted for twelve degrees of unsaturation, which indicated compound **1** was a tetracyclic molecule. Comparison of the NMR data with those of the known compound thiolutin (**3,** Table 1) [25] revealed that **1** was an analogue of thiolutin with a pseudo trimer structure. Furthermore, the heteronuclear multiple bond correlation (HMBC) correlations (Figures 2, S5 and S6) from H3-9 and H-7-*NH* to C-8, H3-9- and H-7--*NH* to C-8-, and H3-9" and H-7"-*NH* to C-8" revealed the presence of three acetamides. The HMBC correlations from H3-10 to C-3a and C-5, H3-10- to C-3a- and C-5-, H3-10" to C-3a" and C-5" confirmed the presence of three *N*-Me amides. The moieties of 1,5-dihydro-2*<sup>H</sup>*-pyrrol-2-one were determined by HMBC correlations from H-3 to C-3a and C-6a, H-7-*NH* to C-6 and C-6a, H-3- to C-3a- and C-6a-, H-7--*NH* to C-6- and C-6a-, H-3" to C-3a" and C-6a", H-7"-*NH* to C-6" and C-6a". The monosulfur bonds between C-6a and C-3-, C-6a- to C-3" were revealed by the HMBC

correlations from H-3- to C-6a, and H-3" to C-6a-. The absence of HMBC correlation from H-3 to C-6a", together with molecular formula of **1**, indicated that the C-6a and C-3 were linked through a disulfur bond. Thus, the structure of **1** was established (Figure 1) and named thiolopyrrolone A.


**Table 1.** 1H (500 MHz) and 13C NMR (125 MHz) data of **1**–**3** (DMSO-*d*6).

**Figure 2.** Key HMBC (arrows) correlations in **1** and **2**.

Compound **2** was isolated as a yellow amorphous powder. The molecular formula of **2** was deduced to be C8H8N2O4S2 by the HRESIMS measurement (*m*/*z* [M + H]+ 260.9996, calcd for C8H9N2O4S2, 260.9998), accounting for six degrees of unsaturation (Figure S7). The 1H 13C and HSQC spectra (Figures S8–S10) displayed similar signals to those of **3**, including one sp<sup>2</sup> methine (*δ*H 7.56, s, H-3; *δ*C 109.6, C-3), one *N*-Me group (*δ*H 3.10, s, H3-10; *δ*C 27.9, C-10), and one methyl singlet for acetyl group (*δ*H 2.10, s, H3-9; *δ*C 22.6, C-9), two amide carbonyls (*δ*C 164.3, C-5; *δ*C 170.5, C-8), and three sp<sup>2</sup> quaternary carbons (*δ*C 145.5, C-3; *δ*C 114.1, C-6, *δ*C 123.1, C-6a). The HMBC spectrum (Figure S11) showed correlations from H3-9 to C-8, H3-10 to C-3a and C-5, H-3 to C-3a and C-6a (Figure 2). Together with the molecular formula calculated by HRESIMS, there are two more oxygen atoms in compound **2**. So there are four possible structures for **2** as shown in Figure 3. The two oxygen atoms for sulfoxides formed *cis* and *trans* conformations, but the optical rotation did not reveal any solid data because of the decomposition of **2**. So, both of the conformations were subjected to quantum chemical calculation. By comparing the experimental and calculated ultraviolet spectra of **2a**–**2d** (Figure 4), the structures of **2b2** and **2d** are consistent with those of experimental data. In order to confirm the structure of **2**, the 13C NMR data of the four possible structures were also calculated by density functional theory (DFT). The

data were evaluated based on the statistical parameters including correlation coefficient (*R*2) between experimental and calculated 13C NMR spectroscopic data with a linear regression, the maximum error (MaxErr), and mean absolute error (MAE). Comparison of all these parameters for calculated 13C chemical shifts of the four possible isomers with experimental data revealed the best fit was **2d** (Table 2). Thus, the structure of **2** was determined and named 2,2-dioxidothiolutin.

**Figure 3.** Four possible structures of **2** for calculating 13C NMR data in DMSO-*d*6.

**Figure 4.** Calculated UV spectra for **2a**–**2d** and UV spectrum for compound **2**.


**Table 2.** Comparison of calculated (TMS as a reference standard) and experimental 13C data for **2**.

Compound **3** was isolated as a yellow amorphous powder. The molecular formula of **3** was deduced to be C8H8N2O2S2 by the HRESIMS measurement (*m*/*z* [M + H]+ 229.0100, calcd for C8H9N2O2S2, 229.0100), accounting for six degrees of unsaturation (Figure S12). The 1H 13C and HSQC spectra (Figures S13–S15) displayed almost the same signals as those of reported thiolutin **3 [21]**. In the HMBC spectrum (Figure S16), the correlations from H3-10 (*δ*H 3.25) to C-3a (*δ*C 136.0) and C-5 (*δ*C 166.1), H-3 to C-3a and C-6a (*δ*C 132.4), H-7-*NH* to C-6 (*δ*C 114.8) and C-6a determined the chemical shifts of C-3, C-6, and C-6a. Thus, the chemical shifts for C-3a and C-6a should be swapped [21].
