**1. Introduction**

Marine fungi, and particularly the genus *Aspergillus*, have proven to be a prolific source of structurally novel and biologically active secondary metabolites that play an eminent role in drug discovery progress [1,2]. A wide array of bioactive compounds, including xanthones, alkaloids, cyclic peptides, and terpenes, have been isolated from marine-derived fungi of *Aspergillus* species [3,4]. Among them, thiodiketopiperazines alkaloids (TDKPs) are an important class of secondary metabolites divided into nearly twenty distinct families, and characterized by the presence of a diketopiperazine core featuring thiomethyl groups and/or transannular sulfide bridges [5]. These compounds have been reported to exhibit a broad range of biological properties, including immunosuppressive [6], cytotoxic [7], antibacterial [8], antiviral [9], and anti-angiogenic activities [10]. Specifically, *Aspergillus terreus* has been reported to produce diverse secondary metabolites that display multiple bioactivities, such as antibiotic, hypoglycemic, and lipid-lowering activities [3,4,11].

During our ongoing research for novel bioactive secondary metabolites from marine-derived *Aspergillus* species, we found a series of bioactive natural products with antifungal, antibacterial, antiviral, antifouling, and cytotoxic activities [12]. In the present study, the chemical investigation of the ethyl acetate (EtOAc) extract of *Aspergillus terreus* RA2905, isolated from the fresh inner tissue of the sea hare *Aplysia pulmonica*, resulted in the identification of two new thiodiketopiperazines, emestrins L (**1**) and M (**2**), as well as five known analogues (**3**–**7**), and five known dihydroisocoumarins (**8**–**12**) (Figure 1). Herein, we report the isolation, structural determination, and bioactivity evaluation of these compounds.

**Figure 1.** The structures of the compounds.

#### **2. Results**

The marine-derived fungus *Aspergillus terreus* RA2905 demonstrated a rapid growth rate on the potato dextrose agar (PDA) plate and produced mature colonies in 3 days. The colonies were characterized by a brown velvety surface (Figure S1). They were cultivated in starch liquid medium at 180 rpm and 28 ◦C for 7 days. The EtOAc extract (12.5 g) was subjected to column chromatography and semi-preparative high-performance liquid chromatography (HPLC) to yield compounds **1**–**12**, which consisted of two new thiodiketopiperazines, emestrins L (**1**) andM (**2**), five known thiodiketopiperazines, emethacin C (**3**) [13], emethacin B (**4**) [14], bisdethiobis(methylsulfanyl)acetylapoaranotin (**5**) [15], bisdethiobis(methylsulfanyl)acetylaranotin (**6**) [16], and alternarosin A (**7**) [17], and five known dihydroisocoumarins, (3R)-8-methoxy-6-hydroxymellein (**8**) [18], (3R)-6,7,8-trihydroxymellein (**9**) [19], cis-4,6-dihydroxymellein (**10**) [20], (3R)-6,7-dimethoxymellein (**11**) [21], and (3R)-6-hydroxymellein (**12**) [22].

## *2.1. Structure Elucidation*

Emestrin L (**1**) was obtained as a white powder with the molecular formula C22H24N2O6S2 established by the HRESIMS spectrum, indicating 12 degrees of unsaturation. The stretch signals at 3600, 3395, 2998, 2913, 1646, 1436, and 1314 cm-1 in the infrared (IR) spectrum suggested the presence of aromatic and carbonyl groups in **1**. The 1H NMR spectroscopic data revealed the signal characteristics of the *ortho*-substituted phenyl group (H-6 to H-9- ) (Table 1), which were supported by the corresponding 1H-1H correlation spectroscopy (COSY) and heteronuclear multiple bond correlation (HMBC) correlations, as shown in Figure 2. A total of 22 carbon atoms, including two thiomethyl carbons (δ<sup>H</sup> 2.13, δ<sup>C</sup> 14.4; δ<sup>H</sup> 2.25, δ<sup>C</sup> 13.8), one acetyl methyl (δ<sup>H</sup> 1.99, δ<sup>C</sup> 21.3), two heteroatom-substituted methane carbons, two methylene carbons, two saturated quaternary carbons (heteroatom-substituted), ten olefinic carbons (seven protonated), and three carbonyl carbons were observed in the 13C NMR spectrum (Table 1). The HMBC correlations from H-10 to C-2, from H-10 to C-2- , from H-3 to C-1, and from H-3 to C-1 indicated the presence of a disulfide diketopiperazine skeleton in **1** (Figure 2). The *ortho*-substituted aromatic ring was connected with the diketopiperazine moiety via a methylene bridge of C-3 based on the HMBC correlations from H-3 to C-4- /C-5- /C-9- . The 1H−1H COSY data showed the presence of an isolated spin system corresponding to the C-6−C-7−C-8−C-9 fragment. A 4,5-dihydrooxepine existed in **1** based on the key HMBC correlations from H-5 to C-3/C-6/C-9 and from H-6 to C-5/C-7/C-8, along with the chemical shifts of C-5 (δ<sup>C</sup> 137.6), C-6 (δ<sup>C</sup> 140.3), and C-9 (δ<sup>C</sup> 61.5). The HMBC correlations from H-3 to C-1/C-5/C-9 indicated that the 4,5-dihydrooxepine moiety was located at C-2 of the diketopiperazine core (Figure 2). As the presence of cyclic dipeptide, aromatic ring, 4,5-dihydrooxepine, and the other carbonyl in the molecule occupied 11 degrees of unsaturation, C-9 should be attached to the 2-N atom to form a pyrrolidine ring on the basis of the chemical shifts of C-9 (δ<sup>H</sup> 4.79, δ<sup>C</sup> 61.5). The carbethoxy was anchored at C-8 on the basis of the HMBC correlation from H-8 to C-11. Collectively, these data permitted the assignment of the planar structure of **1**.


**Table 1.** 1H Nuclear Magnetic Resonance (NMR) and 13C NMR Data for **1** and **2** a.

<sup>a</sup> 500 MHz for 1H NMR and 125 MHz for 13C NMR in Dimethyl Sulfoxide (DMSO)-*d*6.

**Figure 2.** The 1H-1H Correlation Spectroscopy (COSY) and Key Heteronuclear Multiple Bond Correlation (HMBC) Correlations for **1** and **2**.

The coupling constants of <sup>3</sup>*J*H-6–H-7 = 8.3 Hz indicated that the double bond was a *Z*-configuration. The 7.9 Hz coupling constants between H-8 and H-9 suggested their *anti*-relationship. The nuclear Overhauser effects (NOE) were observed for H-8 and H-10 when on irradiation of H-10, which demonstrated that they should be on the same face (Figure 3). The absolute configurations of C-2 and C-2 were assigned as *R* and *R*, respectively, determined by the similar electronic circular dichroism (ECD) data and the same biogenetic pathway as the co-isolated known compound **6** (Figures 4 and 5), for which the absolute configuration was confirmed by X-ray data (Figure S41). Thus, the absolute configurations of **1** were established as 2*R*,2- *R*,8*S*,9*S*.

**Figure 3.** The Nuclear Overhauser Effect (NOE) Correlations for **1** and **2**.

**Figure 4.** The Electronic Circular Dichroism (ECD) Spectra of **1**–**3** and **6**.

Emestrin M (**2**) was assigned the molecular formula C20H22N2O5S2, with 11 degrees of unsaturation, on the basis of its HRESIMS data, less 42 Da compared with **1**. The 1H and 13C NMR data were very similar to those of **1** (Table 1), which revealed the presence of a thiodiketopiperazine, an *ortho*-substituted phenyl, and a 4,5-dihydrooxepine structure. The main differences were the disappearance of the carbethoxy (δ<sup>H</sup> 1.99, δ<sup>C</sup> 21.3; δ<sup>C</sup> 169.9) in **1**, and the appearance of one hydroxyl (δ<sup>H</sup> 5.44). The hydroxyl was anchored at C-8 based on the HMBC correlations from 8-OH to C-7/C-8/C-9. Thus, the planar structure of **2** was established. The similar coupling constants of **2** and **1** between H-6 and H-7, and between H-8 and H-9, respectively, suggested that the relative configurations of the 4,5-dihydrooxepine of **2** were consistent with those of **1** (Figure 3). The absolute configurations of **2** were identical to those of **1** on the basis of the similar ECD data and of the same biogenetic pathway (Figures 4 and 5).

Emethacin C (**3**) was isolated as a white powder. Its molecular formula was defined as C20H22N2O3S2 by HRESIMS, with more than 16 Da compared with the known compound emethacin B (**4**). The 1H NMR and 13C NMR spectroscopic data of **3** were closely related to those of **4**, which revealed the presence of a thiodiketopiperazine structure. The distinction was that one aromatic hydrogen in **4** was replaced by one hydroxyl in **3**. The hydroxyl was anchored at C-5 based on the 1H−1H COSY correlations of H-6- /H-7- /H-8- /H-9 and the HMBC correlations from H-3 to C-5- /C-9- (Figure S40). The absolute configurations of **3** were identical to those of **1** and **2** on the basis of the similar ECD data and of the same biogenetic pathway (Figures 4 and 5). It should be noted that the known compound **3** is listed in SciFinder Scholar with the CAS Registry Number 2166398-50-9, but this is the first time that its spectroscopic data have been reported.

**Figure 5.** The Possible Biosynthesis Pathway of Thiodiketopiperazines.

(3*R*)-8-methoxy-6-hydroxymellein (**8**) was obtained as a yellow solid with the molecular formula C11H12O4 determined by the HRESIMS data, with more than 14 Da compared with the co-isolated known compound (3*R*)-6-hydroxymellein (**12**). The 1H NMR data of **8** displayed the presence of the *meta*-coupled aromatic protons at δ<sup>H</sup> 5.80 (1H, d, *J* = 1.9 Hz) and at δ<sup>H</sup> 5.71 (1H, d, *J* = 1.9 Hz). The 13C NMR spectrum revealed 17 carbons, including one carbonyl, six olefinic carbons (two oxygenated), one oxygenated methine carbon, one methylene, and two methyl carbons (one oxygenated). These spectroscopic features were similar to those of (3*R*)-6-hydroxymellein (**12**) except for an additional oxygenated methyl group. The HMBC correlation from H-9 to C-8 was observed for **8** (Figure S42), suggesting that the methoxy group was attached to C-8. The negative Cotton effect at 270 nm in the ECD spectrum of **8** suggested that the absolute configuration of C-3 was *R* (Figure S43) [23]. Compound

**8** is listed in SciFinder Scholar with the CAS Registry Number 2247026-31-7, but this is the first time that its spectroscopic data have been reported.

(3*R*)-6,7,8-trihydroxymellein (**9**) was established as C10H10O5 on the basis of the HRESIMS data, with more than 16 Da compared with compound **12**. The 1H NMR and 13C NMR spectroscopic data of **9** were nearly identical to those of **12,** except that one proton at the aromatic ring in **12** was replaced by one hydroxyl in **9**. The HMBC correlations from H-5 to C-4/C-7/C-8a indicated that the hydroxyl group was located at C-7 (Figure S42). Similar to **8**, the absolute configuration of C-3 in **9** was also determined as *R* by the negative Cotton effect at 270 nm (Figure S44). Compound **9** is also listed in SciFinder Scholar with the CAS Registry Number 2407423-58-7, but this is the first time that its spectroscopic data have been reported.
