During the last decade, marine-derived fungi have proven to be a prolific source of structurally novel and biologically active natural compounds, which gained considerable attention [1, 2]. Especially the fungus isolated from mangrove could produce structurally novel and bioactive compounds, such as antiosteoporotic citrofulvicin [3], anti-inflammatory chrysogenester [4], antibacterial brocapyrrozin A [5], and antivirus simpterpenoid A [6]. In our search for new bioactive natural products from mangrove-derived fungi in the South China Sea, we have found many bioactive compounds, including cytotoxic indole diterpenes, chlorinated xanthones, anthraquinone derivatives, dihydroisocoumarins and isocoumarins [7,8,9,10,11]. In our ongoing search for new bioactive compounds from mangrove-derived fungus, an endophytic fungus Cladosporium sp. JS1-2 obtained from the mangrove Ceriops tagal, was selected for further research because its EtOAc extract showed insecticidal activity against newly hatched larvae of Helicoverpa armigera Hubner. One new pentenoic acid derivative 1,1’-dioxine-2,2’-dipropionic acid (1) and a new natural product 2-acetate-3,5,6-trimethylpyrazine (2) and six known compounds (3–8) were isolated from the EtOAc extract of the fungus (Fig. 1). In this report, we described the isolation, structure elucidation, antimicrobial and insecticidal activities of all compounds.

Fig. 1
figure 1

The structure of compounds 1–8

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The fungus Cladosporium sp. JS1-2 was isolated from the mangrove plant C. tagal, collected from Dongzhaigang of Hainan Province in China in July, 2016. The strain was deposited in the Key Lab of Tropical Medicinal Resource Chemistry of Ministry of Education, College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou, P.R. China. The fungus was identified according to its morphological characteristics and a molecular biological protocol by 18S rRNA amplification and sequencing of the ITS region. The sequence data have been submitted to GenBank, with an accession number MK234874, and the fungal strain was identified as Cladosporium sp.

The fungus Cladosporium sp. JS1-2 was cultured in 200 ml of potato dextrose broth at 30 °C on a rotary shaker (120 rpm) for 5 days to prepare the seed culture. Large-scale fermentation was carried out in 200-Erlenmeyer flasks (1000 ml), each containing potato dextrose broth (8 g), and purified water (200 ml), which were soaked overnight before autoclaving at 121 °C for 20 min. These Erlenmeyer flasks were added to 3 ml seed broth after cooling to room temperature, and maintained at room temperature for 30 days in stationary phase.

The fungal cultures was extracted with EtOAc (3 × 10 L, 24 h each), which were filtered through cheesecloth. The extracts were concentrated in vacuo to yield an oily residue (25.3 g), which was subjected to silica gel CC (petroleum ether, EtOAc v/v, gradient 100:0–0:100) to generate six fractions (Fr. A−Fr. E). Fr. C was chromatographed on a silica gel column by stepwise-gradient elution using petroleum ether/ethyl acetate from 100: 0 to 0: 100. The fractions were further purified with preparative HPLC with an isocratic solvent system of 0.1% formic acid in 30% acetonitrile-water at a flow rate of 2 ml min−1 to obtain compounds 1 (7 mg) and 4 (3.1 mg). Fr. D was further purified by Sephadex LH20 column (CHCl3/MeOH v/v, 1: 1) and eluted with preparative HPLC with an isocratic solvent system of 0.1% formic acid in 40% acetonitrile-water at a flow rate of 2 ml min−1 to obtain compounds 2 (3.4 mg) and 6 (3.7 mg). Like the above method, we obtained 3 (4.0 mg) and 7 (4.4 mg) from Fr. B, and acquired 5 (3.3 mg) and 8 (3.9 mg) from Fr. E.

Compound 1 was obtained as colorless crystals and had a molecular formula of C10H12O6 as determined by the HR-ESI-MS at m/z 251.0529 [M + Na]+ (calcd. for 251.0526), indicating five degrees of unsaturation. The 13C NMR and DEPT 135 spectra data (Table 1) exhibited 5 carbon signals, including one carbonyl carbon (δC 173.3), one double bond (δC 153.0 and 143.3) and two methylene carbons (δC 32.2 and 29.1). Since one carbonyl group and one double bond accounted for two out of five degrees of unsaturation, implying that 1 was a dimer, and the remaining one degree of unsaturation were assumed for the presence of one ring system in 1. The 1H NMR spectrum of 1 (Table 1) in DMSO-d6 showed one downfield proton signal at δH 8.45 (1 H, s), two methylene proton signals at δH 2.95 (2 H, t, J = 7.6 Hz), and 2.67 (2 H, t, J = 7.6 Hz). The 1H-1H COSY correlation of H-4 and H-5, together with the HMBC correlations (Fig. 2) from H-3 to C-2; from H-4 to C-2, C-3, C-5, and C-6; from H-5 to C-2, C-4, and C-6, indicated that 1 comprised two (Z)- 5-hydroxypent-4-enoic acid units and it was a symmetrical dimer. The structure of 1 was also determined by X-ray diffraction analyses (Fig. 3). Hence, compound 1 was determined as 1,1’-dioxine-2,2’-dipropionic acid.

Table 1 1H NMR (400 MHz) and 13C NMR (100 MHz) data of compounds 1 and 2
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Fig. 2
figure 2

1H-1H COSY (in blue lines) and HMBC (in red lines) correlations of 1–2

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Fig. 3
figure 3

X-ray structureof Compound 1

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Compound 2 was isolated as yellow powder and gave a HR-ESI-MS ion peak at m/z 195.1133 [M + H]+ (calcd for 195.1128), corresponding to a molecular formula of C10H14N2O2 with five degrees of unsaturation. The 13C NMR and DEPT 135 spectra (Table 1) data exhibited 10 carbon signals, including one ester carbonyl carbon (δC 170.1), two double bonds (δC 150.8, 148.5, 148.4, and 144.6), one oxygenated methylene carbon (δC 64.4), and four methyl carbons (δC 21.2, 21.0, 20.5, and 20.0). The 1H NMR spectrum of 2 (Table 1) showed one methylene proton signal at δH 5.12 (2 H, s, H-6’), four methyl protons at δH 2.44 (3 H, s, H-2’), 2.43 (3 H, s, H-5’), 2.42 (3 H, s, H-3’), and 2.06 (3 H, s, H-8’). The HMBC correlations (Fig. 2) from H-8’ to C-7’; from H-6’ to C-5, C-6, and C-7’; from H-2’ to C-2; from H-3’ to C-3; from H-5’ to C-5, primary ascertained the structure of compound 2.

According to the NMR spectra of compound 2, we were uncertain the location of C-2 (δC 148.4) and C-3 (δC 150.8). To further verify the structure, a calculation of the 13C NMR chemical shifts of structure 2 at the B3LY/6-31 G (d)//B3LYP/6-31 G (d, p) level with the PCM model in DMSO was obtained [12], and the calculated chemical shifts agreed well with the experimental data (Fig. 4), of which the Fig. 4 showed a correlation coefficient (R2) of 0.9972, indicating that the calculation of the 13C NMR chemical shifts of structure 2 was suitable. Finally 2 was determined as 2,3,5-trimethyl-6-methylacetate-pyrazine. Interestingly, compound 2 was firstly reported as a synthetic intermediate for the synthesis of piperlongumine-ligustrazine hybrids [13] and 2 was isolated from a natural source for the first time

Fig. 4
figure 4

Regression analysis of experimental versus calculated 13C NMR chemical shifts of compound 2 [at the B3LYP/6-31G(d)//B3LYP/6-31G(d, p)level]

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The structures of known compounds 3–8 were identified by comparison of their 1H/13C NMR spectra with those in the literature as vermistatin (3) [14], citrinin H1 (4) [15], secalonic acid D (5) [16], ladosporol E (6) [17], cladosporol C (7) [18], and cytochalasin D (8) [19].

All compounds were evaluated for their antibacterial activities against five terrestrial pathogenic bacteria, including S. aureus (ATCC 27154), Staphylococcus albus (ATCC 8799), B. cereus (ATCC 11778), Escherichia coli (ATCC 25922), and Micrococcus luteus (ATCC 10240) by the microplate assay method [20]. The result (Table 2) showed that 1, 2, 4, 6 and 7 showed antibacterial activities against S. aureus with the MIC values of 25.0, 12.5, 6.25, 1.56 and 6.25 μg ml−1, respectively. Ciprofloxacin was used as positive control with the MIC value of 0.39 μg ml−1.

Table 2 Antibacterial activity for compounds 1–8
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Compounds 1–8 were also evaluated for growth inhibition activity against newly hatched larvae of H. armigera Hubner [21]. Compounds 1–4 and 6 showed growth inhibition activities against newly hatched larvae of H. armigera Hubner with the IC50 values of 150, 100, 150, 100 and 150 μg ml−1, respectively. Azadirachtin was used as positive control with the IC50 value of 25 μg ml−1.

Compound 1: colorless crystals. mp. 108–110 °C; IR (KBr) νmax 3536, 1750, 1315 cm−1; 1H and 13C NMR see Table 1; HR-ESI-MS m/z 251.0529 [M + Na]+ (calcd. for C10H12O6Na+, 251.0526).

Compound 2: yellow powder. IR (KBr) νmax 1598, 1445, 873 cm−1; 1H and 13C NMR see Table 1; HR-ESI-MS m/z 195.1133 [M + H]+ (calcd. for C10H15N2O2+, 195.1128).

Crystal data for 1: C10H12O6, Mr = 600.60, monoclinic, a = 5.6567 (3) Å, b = 13.2069 (5) Å, c = 8.0189 (5) Å, α = 90°, β = 90°, γ = 90°, V = 523.64 (5) Å3, space group P212121, Z = 2, Dx = 1.447 mg/mm3, μ (Cu Kα) = 0.816 mm−1, and F (000) = 240. Independent reflections: 924 (Rint = 0.0619). The final R1 values were 0.0566, wR2 = 0.1185 (I > 2σ (I)).

In summary, we isolated and characterized one new pentenoic acid derivative 1 and a new natural product 2, along with other six known compounds (3-8) from the fungus Cladosporium sp. JS1-2. Compounds 1 and 2 showed insecticidal activities against H. armigera Hubner and antimicrobial activities against S. aureus.