Introduction

The rapid development of drugs resistance in human diseases such as cancers and invasive fungal infections calls for a class of structurally unique lead drug compounds and beneficial therapeutic agents with new pharmacological mechanisms1. Marine endophytic fungi may play an important role in the discovery of unique secondary metabolites with chemical and biological properties unlike those found in terrestrial ones in recent years2. Bicyclic polyketides, a class of compounds with a 4,6-dimethylhepta-2,4,6-trienoic acid substructure and decalin derivative motif from acetyl Co-A and malonyl Co-A interdispersed with units of methylmalonyl Co-A, are moderate inhibitors of angiogenesis, and inhibit proliferation of filamentous fungi and plant pathogens3. Bicyclic polyketides had been discovered from terrestrial fungal metabolites, such as the antarones from Penicillium antarcticum4, embellistarin from Embellisia chlamydospora5, and hamigerone from Hamigera avellenea6. To discover bioactive compounds from marine fungi, a new bicyclic polyketide curvulaide A (1) (Fig.1) was isolated from the culture of the marine fungus Curvularia sp. IFB-Z10.

Fig. 1
figure 1

The chemical structure of 1

Full size image

Curvularia sp. IFB-Z10 was isolated from the gut flora of fish (white croaker Argyrosomus argentatus); it produces novel alkaloids named curvulamine7 and curindolizine8, when cultured in shake flask in the Czapek’s medium. These compounds exhibited significant antibacterial and anti-inflammatory activities. In this study, we aimed to explore the “new fate” of polyketide analogs from the metabolites of Curvularia sp. IFB-Z10 through solid-state fermentation9. A new bicyclic polyketide, curvulaide A (1), together with its known congener preussilide D3 were produced by solid-state cultivated Curvularia sp. IFB-Z10. Curvulaide A (1) was evaluated for antitumor and anti-anaerobic bacteria activities, and showed moderate cytotoxicity against human hepatoma cell lines BEL7402/5-Fu with IC50 value of 12.46 μM, and exhibited moderate anti-anaerobic bacteria activity against periodontal pathogen Porphyromonas gingivalis with an MIC value of 62.5 μM. Hence, we reported the isolation, structural elucidation, and biological activity evaluation of 1.

Curvulaide A (1) was isolated as a pale-yellow gum, and its molecular formula was determined to be C25H30O4 (11 degrees of unsaturation) based on the HRESIMS ion at m/z 417.2035 [M+Na]+ (calcd. 417.2036). In the 1H NMR spectra (Table 1), six methyl groups including four singlet methyl groups (δH 2.08, 2.21, 2.09, 1.55, each 3H), two doublet methyl groups (δH 1.23, d, J = 6.5 Hz, 6H) were observed. It also displayed a pair of characteristic trans-coupled olefinic protons (δH 7.41, d, J = 15.5 Hz; 5.94, d, J = 15.5 Hz), a doublet olefinic proton (δH 3.82, d, J = 4.0 Hz), and two specific singlet olefinic protons (δH 7.07, 6.47). The 13C NMR spectrum (Table 1) showed two carbonyl carbons (δC 217.9, 208.8), a conjugated carbonyl carbon (δC 170.9), and twelve alkenic carbons. Thus, the aforementioned data suggested that compound 1 was a bicyclic polyketide like preussilide C3.

Table 1 The 1H NMR (600 MHz) and 13C NMR (150 MHz) of 1 in CD3OD
Full size table

The 1H NMR data analyses of 1 showed much similarity with those of preussilide C, except for the loss of two specific methine protons (δH 1.76, d, J = 12.5 Hz; 3.21, d, J = 10.5 Hz) in preussilide C, and the presence of two additional alkenic carbons (δC 136.3, 136.9) in 1. The HMBC correlations from H-18 (δH 7.41, d, J = 15.5 Hz) to C-20 (δC 170.9) and C-16 (δC 142.5), from H-16 (δH 3.82, d, J = 4.0 Hz) to C-14 (δC 132.0) and C-18 (δC 151.6), from Me-25 (δH 3.09, s) to C-16, C-17 (δC 142.5), and C-18 implied a 4,6-dimethylhepta-2,4,6-trienoic acid motif (substructure A) in the structure of 1. Two specific doublet methyl groups and one singlet methyl group in 1H NMR and the HMBC correlations from H-9 (δH 2.43, m) to Me-22 (δH 1.23, d, J = 6.5 Hz), H-11 (δH 3.48, m) to Me-23, H2-8 (δH 2.95, m) to C-7 (δC 136.3), C-12 (δC 136.9), Me-22 and Me-23 to C-10 (δC 217.9) and H-11 to C-12 and C-7 indicated a decalin derivative motif (substructure B) in 1 like in preussilide C. The HMBC correlation between H-14 and C-13 suggested the connection point between substructure A and substructure B located at C-13. The singlet methyl (δH 2.08) and two germinal coupled protons (δH 3.82, d, J = 18.0 Hz; 3.88, d, J = 18.0 Hz) showed HMBC correlations to the ketone carbonyl carbon (δC 208.8), and this indicated a methyl ketone unit in 1 located at C-4. Thus, the planar structure of 1 was determined as shown in Fig. 2.

Fig. 2
figure 2

Selective HMBC and ROESY correlations of 1

Full size image

The relative configuration of 1 was determined based on vicinal coupling constants and ROESY correlations. The coupling constant J18,19 = 15.5 Hz indicated an E-configuration of this double bond. The trisubstituted double bonds Δ14,15 and Δ16,17 were assigned as E-configurations based on the ROESY correlations between H-16 and H-14, H-18 as well as the ROESY correlations between Me-25 and Me-24, H-19. The ROESY correlations between the H-9 and H-11 indicated Me-22 and Me-23 were co-facial, and H-11 and H-9 were on the same face of the decalin motif. Thus, the relative configuration of the decalin moiety was determined as shown in Fig. 1, which was consistent with the configuration of the revised preussilide A10.

Unfortunately, the curvulaide A (1) failed to crystallize in different solvent. To determine the absolute configuration of curvulaide (1), the theoretical calculation of ECD spectra was performed with the Gaussian 09 program package, which was conducted in MeOH using time-dependent density functional theory at B3LYP/6–311G** (Fig. 3), and led us to determine the absolute configuration of 1 (9S, 11R) by comparing experimental and calculated ECD spectra.

Fig. 3
figure 3

Experimental and calculated ECD spectra of 1

Full size image

The anti-anaerobic bacteria activity was evaluated against periodontal pathogen Porphyromonas gingivalis by broth microdilution method in a 96-well plate11. Compound 1 exhibited moderate activity with the MIC value of 62.5 μM (tinidazole was added as the positive control with MIC value of 7.8 μM). The cytotoxic activity of 1 was assessed against human hepatoma cell line BEL7402 and the drug-resistant cell line BEL7402/5-Fu12, and showed effective cytotoxicity with IC50 values of 19.85 μM and 12.46 μM, respectively13 [the 5-Fu was used as the positive control (BEL7402, IC50: 14 μM; BEL7402/5-FU, IC50: 1630 μM)].

In conclusion, a new bicyclic polyketide, curvulaide A (1), with a 4,6-dimenthyhepta-2,4,6-trienoic acid substructure and decalin derivative motif, was produced by solid-state fermentation with Curvularia sp. IFB-Z10 using rice as substrate. Compound 1 showed moderate anti-anaerobic bacteria activity and cytotoxic activities. Therefore, the isolation of the new bicyclic polyketide 1 suggested that more unique bioactive compounds can be isolated from Curvularia sp. IFB-Z10, and these novel compounds could serve as lead compounds for new drug discovery.

Curvulaide A (1): pale-yellow resin, [α]23D + 10.3 (c, 0.1, MeOH); UV (MeOH) λmax (log ε) 207 nm (4.56) 280 nm (4.02); 1H and 13C NMR (CD3OD, 600 MHz), see Table 1; negative ESIMS m/z 393.2 [M − H]−; positive ESIMS m/z 417.2 [M + Na]+; HRESIMS m/z 417.2035 [M + Na]+ (calcd for C25H30O4Na, 417.2036).