**1. Introduction**

Natural products have historically been a rich source of new drugs or drug candidates. Strikingly, deep-sea-derived microorganisms survive under extreme environments, leading to special biological diversity and prolific metabolisms differing from those of terrestrial microorganisms. Recently, deep-sea-sourced microbial natural products have been reported with high hit-rates from bioactivity screening, particularly in the antitumor area [1,2].

*Epicoccum nigrum* is a chemically distinct fungal species with potential to produce structurally unique secondary metabolites including thiodiketopiperazines (TDKPs) [3,4], polyketides [5], and polysaccharides [6]. Some of these metabolites exhibited intriguing biological properties, such as antimicrobial [3], cytotoxic [4], and antioxidant activities [5,6].

The TDKP derivatives are a family of diketopiperazines which have been isolated from several fungal sources, such as *Epicoccum nigrum* [3], *Exserohilum rostratum* [7], *Penicillium brocae* [8], and *Penicillium adametzioides* [9].

In continuation of our research aimed at discovery of bioactive metabolites from marine-derived microorganisms [10–12], a fungal strain of *Epicoccum nigrum* SD-388 was isolated from a deep-sea sediment sample collected at a depth of 4500 m. Chemical investigation of the fungus resulted in the isolation of spiroepicoccin A, an unusual spiro-TDKP derivative, whose stereochemistry could not be elucidated by conventional NMR methods and was solved based on residual chemical shift anisotropies [12]. This result encouraged us to perform a further study of the fungus and has led to the isolation of four new TDKPs including 5'-hydroxy-6'-ene-epicoccin G (**1**), 7-methoxy-7'-hydroxyepicoccin G (**2**), 8'-acetoxyepicoccin D (**3**), and 7'-demethoxyrostratin C (**4**), as well as a pair of new enantiomeric diketopiperazines (DKPs), (±)-5-hydroxydiphenylalazine A ((±)-**5**), together with five known analogues including diphenylalazine A (**6**) [4], emeheterone (**7**) [13], epicoccins E (**8**) and G (**9**) [4], and rostratin C (**10**) [7] (Figure 1). Details of the isolation and purification, structural elucidation, and cytotoxic potency against Huh7.5 liver tumor cells of compounds **1**–**10** are described herein.

**Figure 1.** Structures of the isolated compounds **1**–**10.**

#### **2. Results and Discussion**

#### *2.1. Structure Elucidation of the New Compounds*

The fungal strain *E. nigrum* SD-388 was cultured on the rice solid medium, which was further exhaustively extracted with EtOAc to afford an extract. Fractionation of the extracts by a combination of column chromatography (CC) over silica gel, Lobar LiChroprepRP-18, SephadexLH-20, as well as semi-preparative HPLC, yielded compounds **1**–**10**.

Compound **1**, initially obtained as colorless gum, gave a pseudomolecular ion peak at *m*/*z* 455.1293 [M + H]<sup>+</sup> by HR-ESI-MS, consistent with a molecular formula of C20H26N2O6S2, indicating 9 degrees of unsaturation. The 1H-, 13C-NMR, and DEPT spectroscopic data (Tables 1 and 2) revealed the presence of two methyls, four sp3 hybridized methylenes, nine methines (with five oxygenated/nitrogenated and two olefinic), five nonprotonated carbons (with one ketone and two amide carbonyls), as well as three exchangeable protons. Detailed analysis of the NMR data disclosed that the structure of **1** was similar to that of epicoccin G (**9**), a well described TDKP derivative identified from a *Cordyceps*-colonizing fungus *Epicoccum nigrum* XZC04-CS-302 in 2009 [4]. However, signals for two CH2 groups at δH/δ<sup>C</sup> 2.20 and 2.59/33.8 (C-6') and at δH/δ<sup>C</sup> 1.88 and 2.12/25.8 (C-7') in compound **9**, were replaced by two olefinic CH groups at δH/δ<sup>C</sup> 5.68/133.3 (C-6') and δH/δ<sup>C</sup> 5.53/129.9 (C-7') in the NMR spectra of **1**. Furthermore, the signal for the ketone group (C-5') of **9** (δ<sup>C</sup> 207.7) was replaced by an oxygenated methine (δH/δ<sup>C</sup> 4.11/71.3) in **1**. The COSY correlations for the spin system from H-3' through H-9' via H-4'~H-8' and the HMBC correlations from H-5' to C-7' and C-9', from H-6' to C-4' and C-5', and from H-7' to C-9', confirmed the proposed structure of **1** (Figure 2).


**Table 1.** 1H NMR spectroscopic data for compounds **1**–**5** a.

*<sup>a</sup>* Data collected at 500 MHz in DMSO-*d*6. *<sup>b</sup>* Data not detected.

The relative configuration of **1** was deduced from analysis of the NOESY spectrum. NOE correlations from H-9 to H-3β and H-4, and from the proton of 8-OH to H-4, H-6β, and H-7β, indicated the cofacial orientation of these groups (Figure 3). Besides, NOEs from H-3α to 2-SMe placed them on another face, opposite to that of H-4, H-9, and 8-OH. Moreover, NOE cross-peaks from H-8' to H-3'α and H-4', and from H-3'α to 2'-SMe, confirmed them on the same spatial orientation, while NOE correlations from H-5' to H-9' and H-3'β placed these groups on the opposite face. On the basis of the above observation, the relative configurations for rings A/B and D/E were determined respectively. However, the relationship between these two units could not be correlated based on the NOESY experiment, because no diagnostic NOE cross-peak could be detected between rings A/B and D/E.


**Table 2.** 13C NMR spectroscopic data for compounds **1**–**5** a.

*<sup>a</sup>* Data collected at 125 MHz in DMSO-*d*6. *<sup>b</sup>* Assigned by HSQC experiment.

**Figure 2.** Key 1H-1H COSY (bold lines) and HMBC (red arrows) correlations of compounds **1**–**5**.

To fully assign the configuration of compound **1**, efforts toward a single crystal X-ray study were performed. By slow evaporation of the solvent (MeOH–H2O, 100:1) under refrigeration, quality crystals of **1** were obtained, making it feasible for an X-ray crystallographic experiment which confirmed not only the planar structure, but also the relative configuration of compound **1** (Figure 4). The defined Flack parameter 0.01(3) determined the absolute configuration of **1** as 2*R*, 4*R*, 8*S*, 9*S*, 2'*R*, 4'*S*, 5'*S*, 8'*S*, and 9'*S*, and the trivial name 5'-hydroxy-6'-ene-epicoccin G was assigned to compound **1**.

**Figure 3.** Key NOE correlations of compounds **1**–**4** (black solid lines: β-orientation; red dashed lines: α-orientation).

**Figure 4.** X-ray crystallographic structures of compounds **1***–***3**.

The elemental composition of **2** was established to be C21H28N2O8S2 by analysis of HR-ESI-MS and NMR data, indicating nine degrees of unsaturation. The 1H- and 13C-NMR data of **2** were similar to those of epicoccin G (**9**), a symmetrical TDKP derivative characterized from *E. nigrum* XZC04-CS-302 [4], except that the signals of two methylene groups at δH/δ<sup>C</sup> 1.88 and 2.12/25.8 (CH2-7 and CH2-7') in **9** were replaced by two oxygenated methine groups at δH/δ<sup>C</sup> 3.82/75.8 (CH-7) and δH/δ<sup>C</sup> 4.12/65.7 (CH-7') in **2**, respectively. Moreover, signals for a methoxy group at δH/δ<sup>C</sup> 3.25/55.8 (7-OMe) were also observed (Tables 1 and 2). The methoxy group was assigned at C-7 based on the observed HMBC correlation from 7-OMe to C-7. Supported by key COSY correlations from H-6 to H-7, and from H-6' to H-7', as well as by HMBC correlations from H-6 and H-9 to C-7 and from H-6' and H-9' to C-7' (Figure 2), the planar structure of compound **2** was determined.

The relative configuration of **2** was assigned by analysis of *J*-coupling constants and NOESY data. A coupling constant of 8.5 Hz between H-4 and H-9 as well as between H-4' and H-9' suggested their *cis* relationships, as reported in the previous literature [7]. NOE correlations from the proton of 7-OMe to H-4 and H-9 indicated the cofacial orientation of these groups. However, the relative configurations of **2** could not be fully assigned due to the lack of some key NOE correlations.

A single crystal of **2** was cultivated, after attempts by dissolving the samples in MeOH–H2O (100:1) followed by slow evaporation under refrigeration for two weeks. Once the Cu/Kα X-ray crystallographic experiment was conducted (Figure 4), the structure and absolute configuration of

**2** were assigned as 2*R*, 4*R*, 7*S*, 8*R*, 9*S*, 2'*R*, 4'*R*, 7'*S*, 8'*R*, and 9'*S*, with a Flack parameter of 0.02(4). Compound **2** was named 7-methoxy-7'-hydroxyepicoccin G.

The accurate mass data measured by HR-ESI-MS of compound **3** assigned its molecular formula, C20H20N2O7S2 (12 degrees of unsaturation), and was supported by the NMR data. The 1H- and 13C-NMR data of **3** (Tables 1 and 2) showed close similarity to those of epicoccin D, a TDKP derivative isolated from the fungal strain *E. nigrum* (2203) in 2007 [3]. However, resonances for an ester carbonyl carbon (δ<sup>C</sup> 168.8, C-1") and a methyl group (δH/δ<sup>C</sup> 2.04/20.6, CH3-2") were observed in the NMR spectra of **3**. Deshielded shift at δ<sup>H</sup> 5.04 for H-8' in **3** was detected, compared to that of δ<sup>H</sup> 4.00 in epicoccin D. The above observation suggested that compound **3** was a C-8' acetylated derivative of epicoccin D. The relative configuration of **3** was assigned on the basis of the NOESY experiment and *J*-coupling constants. For ring A of **3**, NOE correlations from the proton of OH-8 to H-7 and H-9 revealed the same orientation of these groups (Figure 3). In addition, the *cis* relationship between H-4 and H-9 was established by the coupling constant (*J* = 8.3 Hz) which is in agreement with that of rostratin B (*J* = 7.2 Hz) [7]. However, the relative configurations of ring E could not be solved as it lacked some key NOE correlations. To unequivocally determine the relative and absolute configurations, single crystals for **3** were cultivated upon slow evaporation of the solvent (MeOH) and a Cu/Kα X-ray diffraction analysis was conducted (Figure 4). The final refinement of the X-ray data resulted in a 0.02(3) Flack parameter, allowing for the assignment of the absolute configuration as 2*R*, 4*R*, 7*R*, 8*R*, 9*S*, 2'*R*, 4'*R*, 7'*R*, 8'*R*, and 9'*S*.

Compound **4** was initially isolated as a colorless powder. Its molecular formula was postulated as C19H22N2O7S2 through HR-ESI-MS analysis, indicating 10 degrees of unsaturation. The 1D NMR data of **4** were similar to those of rostratin C (**10**), a DKP derivative isolated from the marine-derived fungal strain *Exserohilum rostratum* CNK-630 [7], with the exception of the disappeared signals for the oxygenated methine (C-7') and the methoxy group attached to C-7'. In contrast, signals for a methylene group at δ<sup>H</sup> 1.62/1.89 and δ<sup>C</sup> 25.4 (CH2-7') were observed in the NMR spectra of **4** (Tables 1 and 2), indicating that **4** is a 7'-demethoxy derivative of **10**. The 2D NMR correlations supported this inference by the COSY correlations from H-7*'* to H-6*'* and H-8*'*, and HMBC correlations from H-7*'* to C-5*'* and from H-9*'* to C-7*'* (Figure 2).

The relative configuration for rings A and E of **4** were determined by analysis of NOESY data. NOE correlations, with respect to ring A from H-8 to H-3α and 7-OMe, placed them on the same face. Meanwhile, NOEs from the proton of 8-OH to H-4, and from H-3β to H-9, disclosed the cofacial orientation of these groups. As for ring E, the coupling constant (*J* = 8.0 Hz) observed between H-4' and H-9' revealed their *cis* relationship [7]. In addition, NOEs from the proton of 8'-OH to H-4', and from H-3'β to H-9', revealed them on the cofacial orientation. Whereas NOE from H-3'α to H-8' revealed that these groups were on the other face (Figure 3).

The assignment of the absolute configurations at C-2/C-2' were established by analysis of ECD cotton effects (CEs) following the rules reported by the previous reference [14]. The ECD spectrum of **4** showed a positive CE near 265 nm, which was characteristic for the 2*R*/2'*R* configurations in TDKPs. The whole absolute configuration of **4** was further studied using the time-dependent density functional (TDDFT)-ECD calculation in Gaussian 09. The ECD spectra of four possible stereoisomers of **4**, including (2*R*, 4*R*, 7*R*, 8*R*, 9*S*, 2'*R*, 4'*R*, 8'*S*, 9'*S*)-**4**, (2*R*, 4*R*, 7*R*, 8*R*, 9*S*, 2'*R*, 4'*S*, 8'*R*, 9'*R*)-**4**, (2*R*, 4*S*, 7*S*, 8*S*, 9*R*, 2'*R*, 4'*R*, 8'*S*, 9'*S*)-**4**, and (2*R*, 4*S*, 7*S*, 8*S*, 9*R*, 2'*R*, 4'*S*, 8'*R*, 9'*R*)-**4**, were calculated. The experimental ECD spectrum for **4** showed agreement with that calculated for (2*R*, 4*R*, 7*R*, 8*R*, 9*S*, 2'*R*, 4'*R*, 8'*S*, 9'*S*)-**4** (Figure 5a), allowing the elucidation of whole chiral centers as 2*R*, 4*R*, 7*R*, 8*R*, 9*S*, 2'*R*, 4'*R*, 8'*S*, and 9'*S*.

Compound **5**, obtained as a yellow oil, was assigned the molecular formula C19H18N2O3 by HR-ESI-MS, and required 12 degrees of unsaturation. Analysis of the 1H- and 13C-NMR data (Tables 1 and 2) revealed that compound **5** had same basic structure as that of the previously reported diphenylalazine A (**6**), which was identified from the fungus *E. nigrum* XZC04-CS-302 [4]. However, the aromatic methine at C-5 in **6** (δH/δ<sup>C</sup> 7.18/129.82) was replaced by a nonprotonated and hydroxylated

carbon (δ<sup>C</sup> 156.1) in **5**. HMBC correlations from H-3, H-7, and H-9 to C-5, supported this deduction. The planar structure of **5** was thus established as 5-hydroxydiphenylalazine A. However, the specific optical rotation value of [α] D <sup>25</sup> = 0 (c 0.10, MeOH) revealed the racemic nature of compound **5**, which was also confirmed by the fact that no cotton effects were observed in the ECD spectrum (Figure S36). Separation of **5** by HPLC using the Daicel Chiral-pak IC column yielded (+)-**5** and (–)-**5** (Figure S37), which were individually determined absolute configurations by experimental and calculated ECD spectra (Figure 5b), and assigned (+)-**5** as 2*R* and (–)-**5** as 2*S*.

**Figure 5.** Experimental and calculated ECD spectra of compounds **4** (**a**) and **5** (**b**).

In addition to compounds **1**–**5**, five known analogues (**6**–**10**) were also isolated. By detailed spectroscopic analysis as well as comparison with reported data, the structures of compounds **6**–**10** were identified as diphenylalazines A (**6**) [4], emeheterone (**7**) [13], epicoccins E (**8**) and G (**9**) [4], and rostratin C (**10**) [7].
