Marine-derived Aspergillus genus, belonging to ascomycetes, has been regarded as a well-known producer of new bioactive natural products encompassing structurally diverse scaffolds with fascinating biological functions [1]. In recent years, species of the genus Aspergillus have been reported to produce a wide variety of bioactive metabolites such as polyketides [2], diketopiperazines [3], ergosteroids [4], xanthonoids [5], alkaloids [6], and lipopeptides [7]. In our continuous search for bioactive secondary metabolites from marine fungi, a large-scale culture of a marine-derived fungus, identified as Aspergillus niger LS11 in the rice medium led to the isolation of three new compounds, named asperitaconic acids A–C (1–3). In this paper, we presented the isolation, structure characterization, and biological activities of asperitaconic acids A–C.

The producing strain LS11 was isolated from a marine sponge Haliclona sp. collected from the Linshui, Hainan Province, China, and identified as A. niger based on the ITS sequencing. The fungal strain was first grown on potato dextrose agar plate (PDA, 8.0 g of potato extract, 20 g of glucose, 35 g of sea salt, and 20 g of agar in 1 L of distilled water) for 3 days. Agar plugs were inoculated into 250 mL Erlenmeyer flasks containing 100 mL of PDB (8.0 g of potato extract, 20 g of glucose, and 35 g of sea salt in 1 L of distilled water) as seed culture at 28 °C in a shaker (150 rpm) for 3 days. A scaled-up fermentation was conducted on solid rice cultures in 1 L Erlenmeyer flasks (20 flasks; 160 mL of distilled water and 120 g of rice; autoclave). Each flask was inoculated with 10 mL of the seed culture and fermented at 28 °C without shaking for 30 days. The fungal culture was then extracted three times with EtOAc at room temperature. The resulting EtOAc extract was evaporated to dryness under reduced pressure to yield a crude extract (10.5 g). The extract was fractionated by ODS MPLC (30–100% methanol in water, flow rate 20 mL/min, 120 min) to obtain five subfractions (1−5). Fr. 3 was finally purified by reversed-phase HPLC using 40% MeCN/H2O as eluent to afford compounds 1 (2 mg) and 2 (3 mg). Fr. 4 was separated by reversed-phase HPLC, eluting with MeCN/H2O to give 3 (3 mg) (Fig. 1).

Fig. 1
figure 1

Chemical structures of compounds 1–3

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Asperitaconic acid A (1) was isolated as a colorless oil. Its molecular formula was deduced as C12H20O5 based on the HRESIMS data at m/z 262.1648 [M + NH4]+, indicating 3° of unsaturation. The 13C NMR in combination with the HSQC spectrum of 1 showed 12 carbon signals for two carbonyl groups at δC 173.8 (C-1) and 170.6 (C-4), two olefinic groups at δC 137.9 (C-3) and 128.9 (C-11), including one exo-methylene group, one methoxyl group at δC 52.1 (C-12), five methylene groups at δC 32.5 (C-9), 31.3 (C-5), 28.9 (C-7), 27.3 (C-6), and 25.4 (C-8), one oxymethylene group at δC 62.9 (C-10), along with one methine group at δC 46.2 (C-2) (Table 1). 1H-NMR signals at δH 3.65 (t, J = 7.6 Hz, 2H), 1.57 (m, 2H), 1.34 (m, 2H) × 3, 1.91 (m, 1H), and 1.69 (m, 1H), each integrating for two protons indicated the existence of the 1-hexanol side chain. The detail NMR data suggested that 1 shared the same itaconic acid core structure as hexylitaconic acid, a known itaconic acid derivative obtained from the fungus Aspergilus niger [8], except that the presence of a hydroxyl group and a methoxyl group. The complete structure of 1 was subsequently accomplished by the 1H–1H COSY and HMBC spectra. The main difference was the hexyl side chain substituted by a hydroxyl group at C-10 in 1, which was confirmed by the COSY cross-peaks of H2-9/H2-10 and H-5/H-6 and the HMBC correlations of H2-9 with C-7 and C-8 and H2-5 with C-7, respectively (Fig. 2). The location of the methoxyl group was further corroborated to be at C-12 by key HMBC cross-peak of H3-12/C-1. The absolute configuration at C-2 was unambiguously assigned as S based on the value of the optical rotation [α]22D + 2.7 (c 0.50, MeOH) for 1 and the R-(−)/S-(+) relationship [9]. Consequently, the structure of 1 was determined to be as shown in Fig. 1 and named asperitaconic acid A.

Table 1 1H (600 MHz) and 13C (150 MHz) NMR spectroscopic data of 1–3 (CDCl3)
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Fig. 2
figure 2

Key 1H–1H COSY and HMBC correlations of asperitaconic acids A–C (1–3)

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Asperitaconic acid B (2) was assigned to be a molecular formula of C13H20O6 with 4° of unsaturation by the HRESIMS at m/z 290.1585 [M + NH4]+. A direct comparison of the 1H and 13C NMR data of 2 (Table 1) with those of 1 revealed that they share the similar structure, except that 2 has one more acetyl group at C-10 and one less methoxy group, which was further confirmed by the HMBC correlations of H3-12 (δH 2.05, s)/C-11 (δC 171.8) and H2-10 (δH 4.05, t, J = 6.7 Hz)/C-11 (Fig. 1). Finally, considering the optical rotation [α]22D + 7.70 (c 2.50, MeOH), the absolute configuration at C-2 of 2 was established as S. Therefore, the structure of 2 was established as depicted in Fig. 1.

Asperitaconic acid C (3) was obtained as a colorless oil that presented a positive HRESIMS ion at m/z 246.1336 [M + NH4]+, corresponding to the molecular formula C11H16O5 and 4° of unsaturation. The 1H and 13C NMR spectral data of 3 (Table 1) were closely related to those of 1, indicating that 3 was a structural analog of 1, with the exception of the absence of resonances for the hydroxyl and methoxy moiety and the appearance of resonance for the ketone group in 3, which was supported by its molecular formula and resonance at δC 209.5 (C-9). This conclusion was further supported by the HMBC correlations from H3-10 (δH 2.14) and H2-7 (δH 1.60) to C-9. The optical rotation data [α]22D + 2.8 (c 0.90, MeOH) observed for 3 defined the absolute configuration to be S as shown in Fig. 1 according to the R-(−)/S-(+) relationship [9].

Antimicrobial activities of compounds 1−3 were evaluated against clinically isolated S. aureus and Escherichia coli strains using broth microdilution in 96-well microplates [10]. Chloramphenicol was used as positive control. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of the test compound completely inhibiting visible growth of the test microorganisms. Compounds 1−3 had inhibitory effect on S. aureus with MIC values of 16, 32, and 32 μg/mL, respectively (chloramphenicol, MIC value of 4 μg/mL). However, compounds 1−3 were inactive against E. coli at the concentration of 64 μg/mL (chloramphenicol, MIC value of 2 μg/mL). Asperitaconic acids A–C (1−3) were assayed for cytotoxicity against HepG2 and HeLa cancer cell lines by the CCK-8 (Cell Counting Kit-8) method [11] using doxorubicin as positive control. However, compounds 1−3 were found to be noncytotoxic at the 100 μM concentration on both human cancer cell lines.