1 Introduction

Alternaria fungi are one of the important biological resources with great potentials, from which a rich source of novel structures with a variety of bioactivities were discovered successively [1,2,3]. They mainly included nitrogen-containing metabolites [4], diterpenoids [5], meroterpenoids [6], and polyketides [7, 8]. And these metabolites exhibited a broad range of biological activities, such as anti-inflammatory [9], phytotoxic [10], cytotoxic [11], acetylcholinesterase inhibitory [12], and antimicrobial activities [13], which attracted, and still attract increasing attentions from the chemists and pharmacologists.

During the chemical investigation of structurally interesting and biologically active constituents from fungus A. brassicicola, our group have identified a variety of novel structures. Notably, brassicicene N represented the first fusicoccane-derived diterpenoid with a tetracyclic skeleton bearing an oxabicyclo[4.3.1]nonane unit [14]; alterbrassicene A possessed an unprecedented 5/9/4-fused carbocyclic skeleton, exhibiting potent IKKβ inhibitory effect [9]; alterbrassicicene A, featuring a newly transformed monocyclic carbon skeleton, showed strong agonistic action upon PPAR-γ [15]; alterbrassinoids A–D represented the first class of fusicoccane-derived diterpenoid dimers [16]. Based on our previous work, we amplified and fermented this potential fungus again. As a result, three new compounds (1–3) along with three known alkaloids (4–6) were afforded in the present phytochemical study on the fungus A. brassicicola. Herein, this paper aims to report the isolation, structural elucidation, and the cytotoxic activity of these isolated compounds (Fig. 1).

Fig. 1
figure 1

Chemical structures of compounds 1–6

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2 Results and discussion

Compound 1 was isolated as a colorless needle crystal, and its molecular formula was confirmed as C15H21NO4 based on the HRESIMS data at m/z 302.1378 (Additional file 1). According to the characteristic signals of the 1D NMR data (Table 1) showing H-2′/H-6′ (δH 7.15, d J = 8.5 Hz) and H-3′/H-5′ (δH 6.74, d, J = 8.5 Hz); and C-1′ (δC 133.2), C-2′/C-6′ (δC 128.9), C-3′/C-5′ (δC 116.3), and C-4′ (δC 158.0), the para-substituted benzene ring can be deduced. The spin coupling system (Fig. 2) of H3-7 (δH 0.9)/H2-6 (δH 1.31)/H2-5 (δH 1.53)/H2-4 (δH 4.02) suggested the existence of a butyl fragment. The spin coupling system (Fig. 2) of H-1 (δH 5.25)/H2-2 (δH 2.79), along with the HMBC correlations (Fig. 2) of H2-4 and H-1 with C-3 (δC 172.4) and of H-2′ with C-1 (δC 51.2), suggested that the butyl moiety was connected to the 3-(4-hydroxyphenyl)propanoic acid group. In addition, the HMBC correlations from H3-2″ (δH 1.92) and H-1 to C-1″ (δC 172.2) were observed, which confirmed an acetamido motif attached to the C-1. Thus, the planar structure of 1 was corroborated (Fig. 1). The absolute configuration of 1 was confirmed as 1R using the single-crystal X-ray diffraction analysis [Flack parameter = 0.01(5), CCDC 2049878] (Fig. 3). Therefore, the structure of 1 was determined and named as alteralkaloid A.

Table 1 1H NMR and 13C NMR data (δ in ppm, J in Hz) for 1–3
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Fig. 2
figure 2

Key 1H–1H COSY and HMBC correlations of compounds 1–3

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Fig. 3
figure 3

X-Ray crystallographic structure of 1

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Compound 2, obtained as a colorless oil, showed an [M + Na]+ peak at m/z 238.1473 in the HRESIMS analysis, matching a molecular formula of C11H21NO3, which was indicative of two degrees of unsaturation. Based on its 1D NMR data (Table 1), a total of 11 carbons were observed, from which two methyl groups (δC 14.0 and 22.5), seven methylene groups (δC 20.2, 23.4, 29.8, 31.8, 34.6, 40.0, and 65.3), and two carbonyls (δC 173.2 and 175.3) were included. The characteristic signals [spin coupling system: H2-6 (δH 4.07)/H2-7 (δH 1.61)/H2-8 (δH 1.40)/H3-9 (δH 0.95)] of the butyl moiety were also observed in the 1H–1H COSY spectrum of 2 (Fig. 2). In addition, the spin coupling system of H2-1 (δH 3.17)/H2-2 (δH 1.52)/H2-3 (δH 1.69, 1.55)/H2-4 (δH 2.34) along with the HMBC correlations (Fig. 2) from H2-3 and H2-6 to C-5 (δC 175.3) and from H3-2′ (δH 1.92) and H2-1 to C-1′ (δC 173.2) were clearly observed, which defined the structure of 2 as butyl 5-acetamidopentanoate and this compound was named as alteralkaloid B (Additional file 1).

Compound 3 was obtained as a colorless oil. Its HRESIMS data at m/z 294.1689 indicated that the molecular formula was C14H25NO4, suggesting three degrees of unsaturation. The 1D NMR (Table 1) and HSQC data showed 14 carbon signals, which was similar to that of 2, including two methyl groups (δC 14.0 and 18.5), eight methylene groups (δC 20.2, 25.9, 31.8, 32.4, 39.3, 44.5, 60.8, and 65.4), two carbonyls (δC 175.0 and 169.7), and two olefinic carbons (δC 120.9 and 151.6). The structure contained one double bond and two carbonyl groups, suggesting that 3 was a chain compound. The 1H–1H COSY correlations (Fig. 2) of H2-1 (δH 3.23)/H2-2 (δH 1.80)/H2-3 (δH 2.36) and H2-5 (δH 4.08)/H2-6 (δH 1.62)/H2-7 (δH 1.40)/H3-8 (δH 0.95), together with the HMBC correlations (Fig. 2) from H2-2 and H2-5 to C-4 (δC 175.0), suggested the existence of a butyl butyrate fragment (A unit). In addition, two spin coupling systems of H2-1″ (δH 2.32)/H2-2″ (δH 3.70) and H3-4′ (δH 2.12)/H-3′ (δH 5.71) in the 1H–1H COSY spectrum of 3 (Fig. 2) and the key HMBC correlations (Fig. 2) of H3-4′ and H2-1″ with C-3′ (δC 120.9) and C-2′ (δC 151.6), indicated the existence of a 2-(2-hydroxyethyl)but-2-enamide fragment (B unit). The deduction that C-1 of A unit was connected to the nitrogen atom of B unit was supported by the HMBC correlation from H2-1 to C-1′ (δC 169.7). To sum up, the structure of 3 was confirmed and named as alteralkaloid C.

Three known compounds were confirmed as lobechine (4) [17], 4-[2-formyl-5-(methoxymethyl)-1H-pyrrol-1-yl]butanoic acid (5) [18], and 2-furanmethanol-(5′→11)-1,3-cyclopentadiene-[5,4-c]-1H-cinnoline (6) [19] by comparing their NMR data with the literatures.

Compounds 1–6 were tested for the cytotoxicity against six cell lines, including five human cancer cell lines (HepG2, Hep3B, HT-29, HeLa, and OCVAR) and the normal liver cell LO2. Unfortunately, no compound showed obvious cytotoxicity against the above cell lines (IC50 > 40 µM) (Additional file 1).