Ergosteroid derivatives from an algicolous strain of Aspergillus ustus

One new ergosteroid derivative, isocyathisterol (1), and eight known compounds (2–9) were isolated from the culture of an algicolous strain (cf-42) of Aspergillus ustus obtained from the fresh tissue of marine green alga Codium fragile. The structure and absolute configuration of 1 were unequivocally identified by using NMR and mass spectroscopic methods as well as quantum chemical calculations. Compound 1 exhibited weak antibacterial activity.


Results and discussion
Compound 1 was obtained as colourless crystals. A molecular formula of C 28 H 42 O 2 was assigned by analysis of HR-EI-MS (m/z 410.3184 [M] þ , calcd for C 28 H 42 O 2 , 410.3185). The IR absorption bands at 3394 and 1655 cm 21 corresponded to the presence of a hydroxyl group and a conjugated carbonyl group. The 1 H NMR spectrum (Table S1) exhibited two methyl singlets, four methyl doublets, and one singlet and two doublets as well as two double doublets ascribed to five olefinic protons. The 13 C NMR spectrum (Table S1) indicated 28 resonances, which were classified into 6 methyls, 6 methylenes, 11 methines and 5 quaternary carbons on the basis of DEPT and HSQC experiments. A detailed NMR data comparison with those reported for cyathisterol revealed the close similarity of them (Kawahara et al. 1994). The 1 H-1 H COSY and HMBC correlations further confirmed the same planar structure of 1 and cyathisterol (Kawahara et al. 1994).
The relative configuration of 1 was determined by coupling constants and NOE correlations. Me-18 and Me-19 were located on the same face according to their NOE correlations with H-11b, which was axial and opposite to H-9 based on the large coupling constant between them. The axial orientation of H-9, H-14 and H-17 was also established by the analysis of coupling constants, and the same orientation of H-14 and H-17 was further supported by their correlation in the NOESY spectrum. The configurations of side-chain moiety (C-20 to C-25) were deduced to be in agreement with those of 2 -9 through the biogenic consideration, which were supported by the identical NMR data of 1 and cyathisterol. In addition, H-7 exhibited NOE correlations with H-15a and H-15b, which established the b-orientation of OH-8 by analysis of the stereo structure produced via the Dreiding force field in MarvinSketch (2012). Collectively, the abovementioned data evidenced the structure of 1 to be ergosta-4,6,22-trien-8b-ol-3-one, trivially named isocyathisterol. It was deduced to be a C-9 and/or C-14 isomer of cyathisterol according to their different NMR data around C-8 (Kawahara et al. 1994). In addition, the specific optical rotation (½a 20 D þ 61.3) of 1 differed from that (½a 25 D þ 133) of cyathisterol (Kawahara et al. 1994), which further supported the different configurations of these two steroids.
Compound 1 was tested for biological activities against several target organisms including bacteria, fungi and brine shrimp ( Table 1). The results demonstrated that it exhibited weak antibacterial activity against Escherichia coli and Staphylococcus aureus (inhibitory diameters of 6.7 and 5.7 mm, respectively) at 30 mg/disc, and no antifungal activity against Colletotrichum lagenarium and Fusarium oxysporum as well as toxicity against Artemia salina was detected.

Computational details
A conformational search for compound 1 was performed via the Dreiding force field in MarvinSketch (2012), and the geometries were further optimised at the B3LYP/6-31G(d) level in methanol without vibrational imaginary frequencies. The predominant conformer was subjected to the theoretical calculations of ECD spectrum at the B3LYP/6-31G(d) level in methanol using the TD-DFT method, which was drawn via SpecDic software with sigma ¼ 0.35 and UV shift ¼ 10 nm (Bruhn et al. 2011). All the above-mentioned calculations were performed with the integral equation formalism-polarisable continuum model as implemented in Gaussian 09 (Frisch et al. 2010).

Bioassays
Antibacterial activity against E. coli and S. aureus, antifungal activity against phytopathogenic C. lagenarium or F. oxysporum, and toxicity against brine shrimp (A. salina) of compound 1 were tested as described previously (Miao et al. 2012) with chloramphenicol, amphotericin B and K 2 Cr 2 O 7 as positive controls, respectively. The bioassay results are shown in Table 1.

Conclusion
Nine ergosteroid derivatives (1 -9), including a new one (1) with the weak antibacterial activity, were isolated and identified from an alga-endophytic strain (cf-42) of A. ustus, which further added the molecular diversity and evidenced the metabolic talent of this fungus.

Supplementary material
Supplementary details relating to this paper are available online, alongside experimental details, 1D/2D NMR, IR, and mass spectra as well as Cartesian coordinates.