New anti-inflammatory guaianes from the Atlantic hydrotherm-derived fungus Graphostroma sp. MCCC 3A00421

Nine new guaianes (graphostromanes A–I, 1–9) were isolated from the deep-sea-derived fungus Graphostroma sp. MCCC 3A00421, along with four known ones (10–13). The relative configurations were established mainly by detailed analysis of the NMR and HRESIMS data, while the absolute configurations were assigned using the X-ray crystallography and modified Mosher’s method. All isolates were evaluated for their inhibitory effects against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 macrophages. Graphostromanes F (6) showed remarkable inhibitory effect with an IC50 value of 14.2 μM, which was even stronger than that of aminoguanidine, a positive control with an IC50 value of 23.4 μM.

Compound 4 was established the molecular formula C 15 H 24 O 2 on the basis of its HRESIMS. The 1 H and 13 C NMR spectra were very similar to those of 3 except that a ketone group (δ C 220.6) rather than a hydroxy unit was located at C-3. The assumption was corroborated by the HMBC relationships from 15-Me (δ H 1.08) to the carbonyl carbon. Therefore, 4 was assigned as (1R,4S,5S,7R,10R)-3-oxo-11(13)-en-10-hydroxyguaiene, and named graphostromane D.
Compound 5 exhibited the same molecular formula as that of 1 according to its positive HRESIMS at m/z 261.1833 [M + Na] + . Close comparison of its 1 H and 13 C NMR spectra to those of 1 revealed a general similarity except that the hydroxy unit should be attached to C-2 instead of C-3 in 5. This was confirmed by the HMBC correlations of 15-Me to the methylene unit at δ C 43.1 of the C-3 position. In the NOESY spectrum, cross-peaks from H-9a (δ H 1.85) to Me-14 (δ H 1.33) and H-2 revealed H-2 was in β orientation (Fig. 4). By the modified Mosher's method, C-2 was determined as S-configuration (Fig. 6). Accordingly, 5 was established to be (1S,2S,4S,5S,7R,10R)-11(13)-en-2,10-dihydroxyguaiene, and named graphostromane E.
All isolates were evaluated for their anti-inflammatory activities against LPS-induced NO production in RAW264.7 macrophages (Table 3). Compound 6 exhibited remarkable anti-inflammatory activity with an IC 50 value of 14.2 μM, which was stronger than that of the positive control, aminoguanidine, with an IC 50 of 23.4 μM. In addition, 4, 9, and 13 showed weak anti-inflammatory activities with IC 50 values of 72.9, 79.1, and 88.2 μM, respectively.
In conclusion, chemical investigation on the deep-sea-derived fungus Graphostroma sp. MCCC 3A00421 led to the isolation of 9 new (graphostromanes A-I, 1-9) and 4 known (10-13) guaianes. They are first examples of guaiane sesquiterpenoids reported from the marine-derived fungi. Additionally, 6 showed potent anti-inflammatory activity against LPS-induced NO production in RAW264.7 macrophages, indicating its potential usage for anti-inflammatory drugs.

Materials and Methods
General Experimental Procedures. An automatic polarimeter Rudolph IV Autopol was used for recording optical rotation data at 25 °C. A Xevo G2 Q-TOF mass spectrometer was used for measuring HRESIMS. A Bruker Avance 400 MHz NMR spectrometer was used for measuring 1 H, 13 C, HSQC, COSY, HMBC, and NOESY spectra. Chemical shifts (δ) were expressed in ppm referring to the solvent peaks, and coupling constants are in Hz. A Bruker D8 Advance X-ray single-crystal diffractometer was used for measuring X-ray data with Cu Kα radiation. Column chromatography (CC) were performed on Sephadex LH-20 (18- 3 (3a, 3b, 3c, and 3d) at mPW1PW91/6-311 + G(2d,p) level in pyridine-d 5 .       Table 3. Anti-inflammatory activities of compounds 1-13 against LPS-stimulated NO production by in RAW 264.7 macrophages. a Positive control.