*3.3. Identification and Activity Evaluation of the Antifungal Compounds*

An antifungal activity-guided separation of the components of four active strains against *S. sclerotiorum*, using the in vitro antifungal assay, led to the isolation of nine compounds as their active principles (Figure 3). Out of the nine compounds, compounds **1** and **2** were from strain DAAG3-11, compound **3** was from strain DGS1-1, compounds **4**–**7** were from strain DGS3-15, and compounds **8** and **9** were from strain DDPA2-14. Compounds **1**–**8** are known compounds, which structures were elucidated as azalomycins F4a (**1**) [52], azalomycins F5a (**2**) [52], bafilomycin B1 (**3**) [53], actinolactomycin (**4**) [54], dimeric dinactin (**5**) [55], tetranactin (**6**) [56], dinactin (**7**) [56] and maremycin G (**8**) [57] by analysis of their spectroscopic data and comparison with literature values (Figure S1). Compound **9** is a new maremycin analogue.

Compound **9** was obtained as a yellow, amorphous powder. Its molecular formula C22H27N3O4S was determined by high resolution electrospray ionization mass spectrometry (HRESIMS) data (*m*/*z* 468.1555, [M + Na]+, calculated for 468.1564), corresponding to 11 degrees of unsaturation. The IR spectrum indicated the presence of hydroxy (3424 cm<sup>−</sup>1) and carbonyl (1720, 1682 cm−1) groups. The 1H NMR data (Table 2) of **9** suggested the presence of one 1, 2-disubstituted benzene system at δ<sup>H</sup> 7.43 (1H, d, *J* = 7.8 Hz, H-4), 7.12 (1H, td, *J* = 7.6, 1.0 Hz, H-5), 7.37 (1H, td, *J* = 7.7, 1.2 Hz, H-6) and 6.89 (1H, d, *J* = 7.9 Hz, H-7). The 1H NMR data of **9** also revealed the presence of two methyl signals at δ<sup>H</sup> 2.17, (3H, s, H-23) and δ<sup>H</sup> 3.23, (3H, s, H-25). 13C NMR spectrum of **9** showed 11 sp2-carbons including three carbonyls at δ<sup>C</sup> 204.84 (C-21), 178.33 (C-2), 168.5 (C-13) and eight aromatic or olefinic carbons at δ<sup>C</sup> 152.27 (C-16), 142.83 (C-9), 130.36 (C-7), 130.01 (C-4), 125.53 (C-5), 123.24 (C-6), 109.08 (C-8), 100.43 (C-17). In the sp3-carbon region, the spectrum showed three methine at δ<sup>C</sup> 42.84 (C-10), 52.94 (C-11), 52.8 (C-14), four methylene at δ<sup>C</sup> 27.04 (C-18), 21.04 (C-19), 38.79(C-20), 38.75(C-22), and three methyl carbons at δ<sup>C</sup> 16.36 (C-23), 8.92 (C-24), 26.65 (C-25).

**Figure 3.** The structures of compounds **1**–**9**.

Comparison the NMR data of **9** with FR900452 [58], an indole diketopiperazine motif linked with a cyclopentenone moiety, which was isolated from the fermentation broth of *Streptomyces* sp. B9173, implied that **9** was identified as a reduced form of FR900452 in which the cyclopentenone moiety is hydrogenated to cyclopentanone. As shown in Figure 4, the accurate assignments of all protons and carbons for compound **9** were preformed through the correlations in 2D-NMR spectra (1H–1H COSY, HSQC and HMBC, Figure S2). The HMBC couplings Me-25/C-9/C-2, H-5/C-3, and H-10/C-2/C-3/C-4, along with 1H–1H COSY correlations of H-5/H-6/H7/H-8, revealed N-methyl-2-oxindole unit. In addition, 1H–1H COSY correlations of H-18/H-19/H-20, as well as the HMBC cross peaks H-18/C-17/C-16, H-20/C-21, demonstrated that oxopiperazinyl moiety was linked to C-16/C-17 on the cyclopentenone moiety. 1H–1H COSY correlations of Me-24/H-10/H-11, together with the HMBC correlations from Me-24/C-3/C-10/C-11, indicated that N-methyl-2-oxindole unit was linked to C-10/C-5 on the oxopiperazinyl moiety. The 1H and 13C NMR spectroscopic data of **9** were also indicative of methyl mercaptomethylene moieties [δ<sup>C</sup> 38.75, δH-CH2 3.17 (1H, m), 2.83 (1H, dd, 14.0, 8.2), δC-CH3 16.36, δH-S-CH3 2.15 (3H, s)]. The HMBC cross peaks Me-23/C22, H-22/C-14/C-13, along with 1H–1H COSY correlations of H-22/H-14, evidenced that methyl mercaptomethylene moieties were linked to C-14 on the oxopiperazinyl moieties, respectively. Therefore, the planar structure of **9** was elucidated as a reduced form of FR900452, depicted in Figure 3.


**Table 2.** 1H NMR and 13C NMR data of compound **9** in CDCl3.

**Figure 4.** Key 1H–1H COSY, HMBC and ROESY correlations of compound **9**.

The relative configuration of compound **9** was determined by interpretation of its ROESY NMR spectrum. The correlations of H-14/Me-24, and H-10/H-11, indicated that H-14 and Me-24 were α-oriented, whereas H-10 and H-11 were β-oriented (Figure 4). Based on the close skeleton, the comparison of ECD spectra between **9** and N-demethylmaremycin B [57], and the largely consistent data supported that the configurations of 3-OH was α-oriented. Ultimately, the absolute configuration of was identified as 3S, 10R, 11R, 14R, resulting from the same trends of cotton effects (CEs) in the experimental ECD spectra of **9** and N-demethylmaremycin B.

The in vitro antifungal activity of compounds **1**–**9** against *S. sclerotiorum* was determined at various concentrations. All compounds showed significantly antifungal activity against *S. sclerotiorum* with the EC50 values ranging from 49.14 mg/L to 0.21 mg/L (Table 3). Thus, it further confirmed that these compounds were the main antifungal constituents produced by the four active strains.


**Table 3.** EC50 values of active compounds against *S. sclerotiorum*.

Values are the means ± SE (*n* = 9). Data within the same column followed by different letters are significantly different.
