Bisalolane-type sesquiterpenoids are a very important family of natural products with various bioactivities, such as cytotoxicity [1], antibacterial [2], and antioxidant [3]. Several phenolic bisabolane-type sesquiterpenes have been isolated from different organisms, such as Pseudopterogorgia rigida [4], Didiscus aceratus [5], Myrmekioderma styx [6], Verticillium tenerum [7], Glonium sp. [8], Penicillium expansum [9], Penicillium aculeatum [10], and Aspergilus sydowii [3].

Fungi of genus Aspergillus are known to produce a wide array of bioactive secondary metabolites. Our previous study of searching for bioactive metabolites from Aspergillus tennesseensis, a strain isolated from the surface of an unidentified plant leaf, yielded ten prenylated indole alkaloids, including three new natural product hybrids with unprecedented chemical skeletons [11, 12]. In our continuing study on this fungus, three new polyphenols, aspertenols A–B (1–3) and six known compounds (4–9) were isolated and identified. Here, we reported the isolation, structural elucidation, and cytotoxicity evaluation of compounds 1–9.

This fungus was fermented on rice and extracted with ethyl acetate to afford the organic solvent extract. The obtained extract was subjected to chromatographic separation using silica gel, ODS, Sephadex LH-20, and preparative HPLC to yield nine compounds (Fig. 1), including three new bisabolane sesquiterpenoid esters aspertenols A–C (1–3), as well as six known compounds that identified as peniciaculin B (4) [10], hydroxysydonic acid (5) [13], sterigmatocystin (6) [14], 5-methoxysterigmatocystin (7) [15], methyl 2-hydroxy-4-(3-hydroxy-5-methylphenoxy)-6-methylbenzoate (8) [16], diorcinol D (9) [17]. The structures of known compounds (4–9) were determined by comparison of their spectroscopic data with the literature data.

Fig. 1
figure 1

Structures of compounds 1–9

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Aspertenol A (1) was isolated as colorless oil. The molecular formula was determined as C30H36O7 on the basis of HRESIMS with the [M+Na]+ peak at m/z 531.2356, indicating 13 degrees of unsaturation. Analysis of its 1H, 13C, and HSQC NMR data (Table 1) revealed the presence of five methyl, four methylene (an oxygenated), nine methine (eight olefinic), eleven quaternary carbons (nine substituted benzenoid carbons, an ester carbonyl, and an oxygenated). Its 2D NMR showed that 1 possessed a diphenyl ether substructure, similar with those of methyl 2-hydroxy-4-(3-hydroxy-5-methylphenoxy)-6-methylbenzoate (8), except for the absence of a methoxyl group, which was verified by the key HMBC correlations (Fig. 2) from H-1 (δH 6.45) to C-3 (δC 112.7) and C-5 (δC 105.1), CH3-2 (δH 2.28) to C-1 (δC 113.7), C-2 (δC 141.4) and C-3, H-3 (δH 6.48) to C-5, H-5 (δH 6.35) to C-6 (δC 156.4), H-7 (δH 6.34) to C-9 (δC 107.1) and C-11 (δC 103.4), CH3-8 (δH 2.51) to C-8 (δC 144.0) and C-9, 10-OH (δH 11.65) to C-9, C-10 (δC 165.4) and C-11 (δC 103.4), H-11 (δH 6.33) to C-9 and C-12 (δC 162.5). Further analysis of the remaining chemical shift in 1, confirmed a bisabolane sesquiterpenoid substructure, similar to that of hydroxysydonic acid (5), except for the presence of an oxygenated methylene and a methine, and the absence of an ester carbonyl and an oxygenated quaternary carbon, which was further determined by 1H–1H COSY correlations (Fig. 2) of H-3′/H-4′, H2-8′/H2-9′/H2-10′/H-11′/H3-12′, H-11′/H3-13′, and the key HMBC correlations (Fig. 2) from H-3′ (δH 7.00) to C-1′ (δC 156.8), C-5′ (δC 136.2) and C-7′ (δC 79.1), H-4′ (δH 6.86) to C-2′ (δC 129.8), C-6′ (δC 117.5) and C-15′ (δC 66.7), H2-8′ (δH 1.79, 1.89) to C-2′ and C-7′, H-6′ (δH 6.91) to C-4′ (δC 119.3) and C-15′, H3-14′ (δH 1.65) to C-2′ and C-7′. The linkage between two substructures via an ester carbonyl group (δC 171.5) was determined by HMBC correlations from CH3-8 (δH 2.51) to C-13 (δC 171.5) and H-15′ (δH 5.31) to C-13. Thus, the planar structure of 1 was confirmed. The CD spectrum (Figure S1) of 1 showed two positive Cotton effect around 225 and 282 nm, similar to that of S-(+)-sydonol [8] and S-(+)-curcutetraol [18]. Thus, the absolute configuration at C-7′ of 1 was assigned as 7′S.

Table 1 1H and 13C NMR data for compounds 1–3a
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Fig. 2
figure 2

a Key 1H-1H COSY and HMBC correlations of 1–3. b Key NOE correlations of 2 and 3 (Color figure online)

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Aspertenol B (2) was obtained as yellow powder with a molecular formula of C34H36O12, as established by HRESIMS data at m/z [M+H]+ 637.2284. The 1H and 13C NMR signals in bisabolane sesquiterpenoid moiety of 2 were similar to those of compound 5. Further detailed analysis of 1D NMR (Table 1) revealed that the remaining chemical shift in 2 showed similarity with xanthones 5-methoxysterigmatocystin (7), except for the presence of a methylene (δH 2.64, 2.82, δC 36.9) and an oxygenated methine (δH 6.73, δC 99.4), and the lack of two olefinic methines. Thus, we inferred that this unit of 2 was from a 3′,4′-dihydro-5-methoxysterigmatocystin residue, which was confirmed by the 1H–1H COSY correlations (Fig. 2) between H-6/H-7, H-1′/H-2′/H2-3′/H-4′, along with the key HMBC correlations (Fig. 2) from H-2 (δH 6.47) to C-3 (δC 165.2), C-4 (δC 107.2), C-9 (δC181.5), C-12 (δC 106.1), and C-13 (δC 154.1), H-6 (δH 7.17) to C-8 (δC 155.1) and C-10 (δC 144.7), H-7 (δH 6.67) to C-5 (δC 139.6), C-9 and C-11 (δC 109.7), H3-14 (δH 3.91) to C-5, H3-15 (δH 4.01) to C-1 (δC 163.9) and C-2 (δC 91.0), H-1′ (δH 6.62) to C-3, C-4 and C-4′ (δC 99.4), H-2′ (δH 4.39) to C-3 and C-13, H-3′ (δH 2.64, 2.82) to C-4 and C-1′ (δC 113.9), H-4′ (δH 6.73) to C-2′ (δC 42.7). The connection between the two substructures of 2 was verified by HMBC correlations from H-4′ (δH 6.73) to C-15′′ (δC 164.8). The relative configurations of 2 was established by the NOE correlations (Fig. 2) of H-1′ (δH 6.62) with H-2′ (δH 4.39), H-2′ with H-3′α (δH 2.64), H-3′α with H-4′ (δH 6.73), which indicated that H-1′, H-2′, H-3′α, and H-4′ were on the same side and H-3′β was on the opposite side. From a biosynthetic perspective, it seemed reasonable to assume that compound 2 possessed the same R absolute configuration at C-1′ and C-2′ as that of 6 and 7, the S absolute configuration at C-7′′ that is identical with that in 1 and 5. Thus, the absolute configuration of 2 was proposed to be 1′R, 2′R, 4′R, 7′′S.

Aspertenol C (3) was obtained as yellow powder with the molecular formula C34H36O12. Careful analysis of 1H and 13C NMR spectra (Table 1), particularly 1H-1H COSY, HSQC, and HMBC (Fig. 2) revealed a same planar structure with 2. The relative configuration of 3 was elucidated on the basis of the NOE correlations (Fig. 2). The NOE correlations of H-1′ (δH 6.60) with H-2′ (δH 4.45), and H-2′ with H-3′α (δH 2.80) placed them on the same side. The NOE correlations of H-3′β (δH 2.67) with H-4′ (δH 6.63) indicated that H-3′β and H-4′ were on the opposite side. Compound 3 was determined to be a C-4′ isomer of 2.

All isolated compounds were evaluated for cytotoxicities against A549, K562, and ASPC cell lines using CCK8 method. The results were summarized in Table 2. Compounds 1, 3, 4, 6, 7, and 9 showed inhibition on K562 cell line with IC50 value in the range from 16.6 to 72.7 μM. In A549 inhibition assay, 1, 4, and 9 showed inhibitory activity with IC50 of 43.5, 70.2, and 61.1 μM, respectively. The cytotoxicity of compound 3 (IC50 = 48.53 μM) against K562 was much stronger than that of 2 (IC50 > 100 μM). Structures of 3 and 2 differ in the absolute configuration at C-4′. Thus, we infer that S absolute configuration at C-4′ in 4 might make great contribution to its inhibitory activity against K562 cell line.

Table 2 Cytotoxicities of compounds 1–9
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In conclusion, three new phenolic bisabolane sesquiterpenoid esters, aspertenols A–C (1–3), together with six known compounds (4–9) were isolated and identified from the solid culture of A. tennesseensis. The cytotoxicities of 1–14 against A549, K562, and ASPC cell lines were evaluated in vitro. As a result, compounds 1, 3, 4, 6, 7, and 9 were found to have inhibitory activity against K562 or A549 cell lines. Compounds 1–3 represent the new type of phenolic bisabolane sesquiterpenoid esters.