Setosphlides A–D, New Isocoumarin Derivatives from the Entomogenous Fungus Setosphaeria rostrate LGWB-10

Investigation of the entomogenous fungus Setosphaeria rostrate LGWB-10 from Harmonia axyridis led to the isolation of four new isocoumarin derivatives, setosphlides A–D (1–4), and four known analogues (5–8). Their planar structures and the relative configurations were elucidated by comprehensive spectroscopic methods. The absolute configurations of isocoumarin nucleus for 1–4 were elucidated by their ECD spectra. The C-10 relative configurations for the pair of C-10 epimers (1 and 2) were established by comparing the magnitude of the computed 13C NMR chemical shifts (Δδcalcd.) with the experimental 13C NMR values (Δδexp.) for the epimers. All of the isolated compounds (1–8) were evaluated for their cytotoxicities against four human tumor cell lines MCF-7, MGC-803, HeLa, and Huh-7.


Introduction
Symbiosic microorganisms from insects, which are wellknown as a rich source of bioactive natural products, have attracted widespread attention [1][2][3]. Especially, due to the special environmental conditions, the bioactive natural products from symbiotic microorganisms as a rich source of various compounds with complex structures and excellent activities, may reshape the experts' views on the drug ability of natural products [4]. Among them, isocoumarin derivatives have been isolated as antifungal, insecticidal, and phytotoxic In Honor of Professor Jun Zhou.

Results and Discussion
The relative configurations of C-3 and C-4 in 1-3 were determined by their NOESY correlations. For compounds 1 and 2, NOE correlations from H-3 to H-4 were observed.  While, the NOESY correlations between H-4 and H 2 -9 were present for 3. In order to assign the absolute configurations of C-3 and C-4 in 1-3, electronic circular dichroism (ECD) was carried out for them. The absolute configurations of the C-3 methine carbon in 1-3 were deduced by the application of the circular dichroism (CD) exciton chirality method. Further more, According to the earlier references [7,8], the negative ECD Cotton effect for 1-3 around 275 nm (Fig. 3) indicated the 3R,4R, 3R,4R, and 3S,4R configurations for 1, 2, and 3, respecitively.
However, it was difficult to determine the absolute configuration of C-10 in 1-3 due to the high conformational flexibility of the chains in them. Especially, the experimental ECD spectra of 1-3 were almost identical, indicating that the ECD method had limitations in the assignment of the C-10 absolute configurations for them. Recently, computational methods for atomic chemical shift calculations have been developed and used for the relative configuration identifications of complex natural compounds [9][10][11]. Compounds 1 and 2 are a pair of epimers with more than one stereogenic carbon. The carbons near C-10 in 1 should have different chemical shifts from those of the corresponding carbons in 2. Thus, the configurations at C-10 of 1 and 2 could be established by comparing the magnitude of the computed chemical shifts (Δδ calcd. ) for two epimers of (10R)epimer and (10S)-epimer [12]. The relative errors (Δδ calcd. ) between the computed 13 C chemical shifts of (10R)-epimer and (10S)-epimer, and the relative errors (Δδ exp. ) between experimental 13 C NMR data of 1 and 2 were summarized in Table 3. Based on the relative error magnitudes (Δδ calcd. and Δδ exp. ), the configurations of C-10 for 1 and 2 were suggested to be R and S, respectively. However, the absolute configuration at C-10 in 4 was undetermined since only one of its C-10 epimer was not obtained.
Setosphlide D (4) was also isolated as a colourless oil with the molecular formula C 15 H 18 O 7 determined by HRESIMS. The NMR spectra (Tables 1 and 2) of 4 showed a high similarity to those of 1-3. The most significant difference in the 1 H NMR spectra was the presence of an additional methine signal at δ H (3.43, m) in 4. Furthermore, the key HMBC from H-9 to C-4 indicated that C-4 and C-9 were connected via an oxygen bridge, forming the third ring, a furan C-ring. The relative configuration of C-3, C-4, and C-10 of 4 was determined by NOESY experiment, which showed NOE correlations from H-10 to H-3 and H-4. ECD spectrum suggested the 3R,4R,10R configuration of 4 (Fig. 3).

General Experimental Procedures
OR and UV data were acquired on Perkin-Elmer 341 and 241 spectrophotometers, respectively. ECD spectra were measured using a JASCO J-715 spectrometer. 1D and 2D NMR data were recorded on a Bruker AM-600 spectrometer. HRESIMS spectra were recorded on a Bruker apex-ultra 7.0T spectrometer. HPLC was carried out on a Waters 600-2489 with a YMC column (YMC-Pack ODS-A, 250 × 10 mm). Column chromatography (CC) were conducted over silica gel (200-300 mesh) and Sephadex LH-20 gel (25-100 μm). TLC were conducted with silica gel GF 254 plates.

Isolation of the Fungal Material
The fungal strain Setosphaeria rostrate LGWB-10 was isolated from the Harmonia axyridis collected in Baoding, Hebei Province, China. The voucher specimen of the fungus

Computational Section
The molecules of (10R)-epimer (1) and (10S)-epimer (2) was constructed and used for conformational searches using the MMFF94S force field by using BARISTA software. A total of 45 stable conformers for 1 and 48 stable conformers for 2 with relative energy within a 10.0 kcal/ mol energy window were obtained and optimized at the gas-phase B3LYP/6-311 + G(d) level using the Gaussian 09 package. MPW1PW91 theory at the basis set of B3LYP/6-311 + G(d,p) in the gas phase was applied for 13 C NMR calculation for (10R)-epimer (1) and (10S)-epimer (2).