**2. Results and Discussion**

The EtOAc extract of a PDA (potato dextrose agar) culture of *Teratosphaeria* sp. AK1128 exhibiting cytotoxic activity, on bioassy-guided fractionation involving solvent-solvent partitioning, Sephadex LH-20 size-exclusion and silica gel chromatography followed by HPLC purification, afforded metabolites **1**–**10** (Figure 1). Of these, **4**–**10** were previously known and were identified as naphtho-γ-pyrones, aurasperone B (**4**) [9], aurasperone C (**5**) [10], aurasperone F (**6**) [11], nigerasperone A (**7**) [12], and fonsecin B (**8**) [9], and diketopiperazines, asperazine (**9**) [13] and isorugulosuvine (**10**) [14], by comparison of their spectroscopic (1H NMR, 13C NMR, and LR-MS) data with those reported.

**Figure 1.** Structures of metabolites isolated from *Teratosphaeria* sp. AK1128.

Spectroscopic (1H and 13C NMR, HRESIMS, and UV) data of teratopyrones A–C (**1**–**3**), together with their common molecular formula, C31H26O11, suggested that they are dimeric naphtho-γ-pyrones [11]. In their 1H and 13C NMR spectra, two sets of signals were observed in different intensity ratios and this was suspected to be due to the atropisomerism around the C7–C10 axis [10] and/or the presence of C-2 and C-2 hemi-ketal stereoisomeric mixtures as a result of non-enzymatic formation of this moiety during the biosynthesis of naphtho-γ-pyrones [15]. Although several recent publications on dimeric naphtho-γ-pyrones report only one set of the NMR data, careful examination of the spectra of these dimeric naphtho-γ-pyrones provided in the Supporting Information of these papers indicated the presence of two sets of signals, corresponding to two possible tautomers [16–18]. The atropisomerization around the C7–C10 axis is known to be restricted under mild conditions [10], and the atropisomer ratio for a given dimeric naphtho-γ-pyrone may depend on its producer fungus [19]. Although it is not possible to obtain the atropisomer ratio directly, the use circular dichroism (CD) data for the assignment of stereochemistry of the binaphthyl moiety of the major atropisomer of dimeric naphtho-γ-pyrones by the exciton chirality method has been reported [20,21].

In its 1H NMR spectrum, teratopyrone A (**1**) exhibited signals due to two methyls (δ<sup>H</sup> 1.43 (s) and 2.36 (s)), three methoxyls (δ<sup>H</sup> 3.39 (s), 3.60 (s), and 3.90 (s)), five aromatic protons (δ<sup>H</sup> 5.99 (s), 6.12 (br. s), 6.26 (br. s), 7.06 (s), and 7.07 (s)), and two hydrogen-bonded phenolic hydroxy groups (δ<sup>H</sup> 14.48 (s) and 14.97 (s)). The 13C NMR spectrum of **1** (Table 1) contained 31 carbon signals due to its major tautomer and these were assigned with the help of HSQC and HMBC spectra. The two carbonyl signals at δ<sup>C</sup> 197.9 and 184.6 revealed that **1** was made up of two naphtho-γ-pyrone monomers, of which one was hydrated at C2-–C3-. The HMBC data for **1** (Figure 2) revealed that the linkage of two monomers of **1** was the same as that of nigerasperone C (**11**; Figure 1)) in which the hydrated monomer consisted of the upper unit [12]. The 13C NMR data of **1** closely resembled those of **11** [12], except for the signals in the vicinity of C-5–C-8. However, some differences were observed for C-5 (δ<sup>C</sup> 162.5 for **1**; 166.1 for **11**), C-6 (δ<sup>C</sup> 107.1 for **1**; 111.8 for **11**), and C-8 (δ<sup>C</sup> 157.3 for **1**; 163.1 for **11**), suggesting that **1** differed from **11** in the attachment of hydroxy/methoxy groups at C-6/C-8. The attachment of the methoxy group to C-8 in **1** was confirmed by the HMBC correlations of 8-OCH3 (δ<sup>H</sup> 3.38) and H-9 (δ<sup>H</sup> 7.06) to C-8 (δ<sup>C</sup> 157.3). The ECD spectrum of **<sup>1</sup>** (Figure 3) showed Davydov-split Cotton effects as negative ([θ] <sup>−</sup>1.46 <sup>×</sup> <sup>10</sup>5, 289.5 nm) first and positive ([θ] +1.78 <sup>×</sup> 105, 272 nm) second, suggesting negative chirality [21] and M-configuration of the 7- –10 bond [20]. Thus, the structure of teratopyrone A was determined as (10- *S*)-2- ,5,5--,6-tetrahydroxy-6- ,8,8- -trimethoxy-2,2- -dimethyl-2- ,3- -dihydro-4*H*,4- *H*-[7,10- -bibenzo[g] chromene]-4,4- -dione (**1**) (Figure 1).


**Table 1.** 13C NMR data (100 MHz, δ) for teratopyrones A–C (**1**–**3**).

\* The position number in parentheses is for teratopyrone C (**3**).

**Figure 2.** Selected HMBC correlations of teratopyrones A–C (**1**–**3**).

**Figure 3.** ECD spectra (**A**) and negative exciton chirality (**B**) of teratopyrones A–C (**1**–**3**).

Teratopyrone B (**2**) also exhibited UV adsorption bands at 202, 238, 281.5, and 386 nm typical for naphtho-γ-pyrones [11]. Similar to **1**, the 1H NMR spectrum of **2** in DMSO-d6 solution showed two sets of signals due to the presence of two tautomeric forms in almost equal amounts (10:8 ratio) and these consisted of two methyl singlets (δ<sup>H</sup> 1.65 (1.64 for the other conformer) and 2.55), three methoxy singlets (δ<sup>H</sup> 3.41 (3.40), 3.60 (3.59), and 4.00), five aromatic signals (δ<sup>H</sup> 6.19 (6.17), 6.55, 6.61 (2H), and 6.90), and two singlets due to hydrogen-bonded phenolic hydroxy groups (δ<sup>H</sup> 14.27 (14.25), and 13.19 (13.18)). The 13C NMR spectrum of **2** (Table 1) resembled closely that of aurasperone F (**6**). The 13C NMR signals belonging to each of the conformers of **2** were recognized and assigned with the help of HSQC and HMBC data. The presence of two carbonyl signals at δ<sup>C</sup> 198.4 and 182.3 indicated that **2** is a dimer consisting of a naphtho-γ-pyrone monomer and its hydrated form. The positions of attachment of OCH3 groups in **2** were determined by the analysis of its HMBC spectrum (Figure 2). The presence of 8-OCH3 in **2** was confirmed by the HMBC correlations of H-9 (δ<sup>H</sup> 6.90) to C-8 (δ<sup>C</sup> 159.3) and 8-OCH3 (δ<sup>H</sup> 4.00) to C-8 (δ<sup>C</sup> 159.3). The ECD spectrum of **<sup>2</sup>** (Figure 3) showed Davydov-split Cotton effects as negative ([θ] <sup>−</sup>5.70 <sup>×</sup> <sup>10</sup>4, 297 nm) first and positive ([θ] +1.63 <sup>×</sup> 105, 278 nm) second, suggesting the negative chirality [21] and M-configuration of the 7–10 bond [20]. Therefore, the structure teratopyrone B was determined as (10- *S*)-2,5,5- ,6-tetrahydroxy-6- ,8,8- -trimethoxy-2,2- -dimethyl-2,3-dihydro-4*H*,4- *H*-[7,10- -bibenzo[g]

chromene]-4,4- -dione (**2**) (Figure 1), suggesting that it is the isomer of aurasperone F (**6**) with different methyl ether positions at C-6 and C-8.

The 1H NMR spectrum of teratopyrone C (**3**) also exhibited two sets of signals belonging to two tautomers in the ratio 3:2 as a result of the hemi-ketal tautomerism and consisted of two methyl singlets (δ<sup>H</sup> 1.40 (1.78 for another tautomer) and 2.47), three methoxy singlets (δ<sup>H</sup> 3.46 (3.53), 3.55, and 3.91 (3.89)), five aromatic proton singlets (δ<sup>H</sup> 5.99 (5.95), 6.44 (6.39), 6.50, 6.93, and 7.00 (7.02)), and two hydrogen-bonded phenolic hydroxy singlets (δ<sup>H</sup> 14.23 and 12.89 (12.88)). The hydrogen-bonded phenolic hydroxy singlet at δ<sup>H</sup> 12.89 (12.88) suggested the existence of an angular naphtho-γ-pyrone moiety [9]. The 13C NMR spectrum of **3** (Table 1) contained signals due to each tautomer that were assigned separately with the help of HSQC and HMBC data. The two carbonyl signals at δ<sup>C</sup> 198.4 and 182.4 (182.3) indicated that **3** consisted of one each of naphtho-γ-pyrone and hydrated naphtho-γ-pyrone monomers. The positions of attachment of OCH3 groups in **3** were also determined by the analysis of its HMBC spectrum (Figure 2). The HBMC correlation of 5-OH (δ<sup>H</sup> 12.89) to C-6 (δ<sup>C</sup> 104.3), further supporting the existence of angular naphtho-γ-pyrone moiety, and the correlations of H-7 (δ<sup>H</sup> 7.01) to C-8 (δ<sup>C</sup> 158.9), H-7 (δ<sup>H</sup> 7.01) to C-9 (δ<sup>C</sup> 117.9), and 10-OCH3 (δ<sup>H</sup> 3.53 (3.46)) to C-8 (δ<sup>C</sup> 156.8) established the C9–C10 linkage and 8-methoxyl group. The ECD spectrum of **<sup>3</sup>** (Figure 3) exhibited Davydov-split Cotton effects as negative ([θ] <sup>−</sup>5.14 <sup>×</sup> <sup>10</sup>4, 292 nm) first and positive ([θ] <sup>+</sup>8.82 <sup>×</sup> <sup>10</sup>4, 278 nm) second, suggesting the negative chirality [21] and M-configuration of the 9–10 bond [20]. Therefore, the structure of teratopyrone C was determined as 9-((10*R*)-2,5-dihydroxy-6,8-dimethoxy-2-methyl-4-oxo-3,4-dihydro-2*H*-benzo[g]chromen-10-yl)-5,8 dihydroxy-10-methoxy-2-methyl-4*H*-benzo[h]chromen-4-one (**3**) (Figure 1).

Previous studies have shown that some naphtha-γ-pyrone dimers such as chaetochromins exhibit cytotoxic activity [22]. Thus, teratopyrones A–C and other metabolites encountered were evaluated for their cytotoxic activity, employing a panel of five cancer cell lines and normal cells. Among these, only nigerasperone A (**7**) showed cytotoxic activity at a concentration <5.0 μM. The IC50s of **7** for these cancer cell lines were determined as 4.12 ± 0.32 μM (PC-3M (human metastatic prostate cancer)), 3.01 ± 0.11 μM (NCI-H460 (human non-small lung cancer)), 2.37 ± 0.15 μM (SF-268 (human central nervous system glioma)), 3.90 ± 0.33 μM (MCF-7 (human breast cancer)), and >5.0 μM for MDA-MB-231 (human metastatic breast cancer) and WI-38 (normal human lung fibroblast cells). Interestingly, nigerasperone A (**7**) has been reported previously to be inactive against A549 (human alveolar adenocarcinoma) and SMMC-7721 (human hepatocellular carcinoma) cancer cell lines [12]. These and our data suggest that nigerasperone A (**7**) may be selectively cytotoxic against some cancer cell lines.
