Cytotoxic pimarane-type diterpenes from the marine sediment-derived fungus Eutypella sp. FS46

Abstract Two new pimarane-type diterpenes, scopararanes H-I (1–2), along with five known ones (3–7) were isolated from the culture broth of a marine sediment-derived fungus Eutypella sp. FS46, which was obtained from the South China Sea. Their structures were established by extensive spectroscopic analysis. All of them were evaluated for their cytotoxic activities against MCF-7, NCI-H460 and SF-268 tumour cell lines. Scopararane I (2) showed moderate inhibitory activities.


Introduction
The genus Eutypella is well known for producing biologically meaningful secondary metabolites, such as polyketides, benzopyrans, cytosporins, terpenoids, cytochalasins, and cyclic dipeptides (Pongcharoen et al. 2006;Ciavatta et al. 2008;Isaka et al. 2009). In continuation of our efforts to search for structurally diverse and biologically significant metabolites from marine fungi Sun, Li, Tao, Chen et al. 2012;Sun et al. 2013), the culture broth of Eutypella sp. FS46 was found to show potent cytotoxic activity against human breast adenocarcinoma cell line (MCF-7) with an inhibition rate of 68.4% . Based on the speculation and inspection of novel chemical constituents in this species, a further extensive chemical investigation of the culture broth led to the discovery of two new pimarane-type diterpenes, scopararanes H-I (1-2) (Figure 1), as well as five known ones including scopararane C (3) (Sun, Li, Tao, Chen et al. 2012), libertellenone A (4) (oh et al. 2005), 11-deoxydiaporthein A (5) (Yoshida et al. 2007), diaporthein A (6) (Dettrakul et al. 2003), and diaporthein B (7) (Dettrakul et al. 2003). Herein, we described the isolation, structure elucidation, and cytotoxicity of these isolated diterpenes.

Results and discussion
Compound 1 was obtained as yellowish oil. Its molecular formula was established as C 20 H 28 o 4 with seven indices of hydrogen deficiency based on the sodium molecular ion [M + Na] + determined at m/z 355.1883 (C 20 H 28 o 4 Na, calcd 355.1885) in the high-resolution electrospray ionisation mass spectroscopy (HreSIMS). The Ir absorptions of compound 1 at 3410 and 1652 cm −1 showed the presence of a hydroxyl and carbonyl groups. The 13 C NMr data (Table  S1) , s, H-17) indicated four methyl groups. The presence of 20 carbons in 1 together with four singlet methyls and a number of methylene characteristic protons implied that 1 possessed a diterpenoid skeleton. The 1 H-1 H CoSY spectrum of 1 revealed the presence of following fragments: H-1/H-2, H-11/H-12, and H-15/H-16. Moreover, the HMBC correlations from both H 3 -18 and H 3 -19 to C-3, C-4, C-5; H 3 -18 to C-19; H 3 -19 to C-18; H 3 -20 to C-1, C-5, C-9 and C-10; H-17 to C-12, C-13, C-14 and C-15; H-16 to C-13; H-14 to C-7, C-9, C-12 and C-15; H-11 to C-10; H-6 to C-7 and C-10; H-5 to C-7 and C-9; and H-1 to C-3 were unambiguously assigned the connections between rings A-C in scopararane H (1). Therefore, the planar structure for 1 was confirmed as depicted.
The relative configuration of 1 ought to be identical to that of libertellenone A (4) according to proton coupling constants and NoeSY spectrum result. The Noe correlations between H 3 -19 and H-5, H-5 and H-11α suggested that these protons were cofacial and arbitrarily assigned as α-orientation. Moreover, the coupling constants of 15.2,13.1,3.6 Hz) and Noe correlations between H-17/H 3 -20 demonstrated that C-17, C-19 and C-20 were in axial configuration on the top face of the molecule. The hydroxyl group at C-9 was established as α-orientation according to the Noe correlation between H-11β and H 3 -20. However, the stereochemistry at C-6 and C-7 of 1 could not be established based upon available spectral data because of the overlap of H-6 and H-7. Finally, the structure of 1 was identified as shown and given the trivial name scopararane H.
Compound 2 was obtained as white solid. The molecular formula was established to be C 26 H 36 o 7 with nine indices of hydrogen deficiency, based on the sodium molecular ion [M + Na] + determined at m/z 483.2335 (C 26 H 36 o 7 Na, calcd 483.2359) in the high-resolution electrospray ionisation mass spectroscopy (HreSIMS). The Ir spectrum of 2 showed the presence of a hydroxyl group (3456 cm −1 ) and two carboxyl groups (1734, 1660 cm −1 ). The 1 H NMr spectrum of 2 was similar to that of 1, indicating that 2 was also a pimarane diterpene. Comparing with the previous literature, 2 was much similar to libertellenone H (Lu et al. 2014), exhibiting a distinct upfield proton at 0.69 ppm strongly indicative of the presence of a cyclopropane ring. Besides, compound 2 exhibited two more carbonyl signals at δ C (171.9, C-1′) and δ C (178.1, C-1″). Meanwhile, the sharp singlet methyl signal at δ H 1.99 (3H, s, H-1′) suggested the presence of an acetyl group, and the signals at 1.12 (3H, d, J = 7.0 Hz, H-3″) and 1.13 (3H, d, J = 7.0 Hz, H-4″), 2.51 (1H, m, H-2″) revealed an additional isopropyl group. 1 H-1 H CoSY spectrum of 2 revealed the H-1 to H-2 partial structure and also correlations between H-11/H-12, H-15/H-16, and H-3″/H-2″/H-4″. The HMBC correlations from H-3 to C-1′ and H 2 -18 to C-1″ indicated the acetyl and isobutyryl groups were located at the C-1′ and C-1″ respectively. Moreover, the connections between rings A-C in scopararane I (2) could also be unambiguously assigned by the HMBC correlations. Therefore, the planar structure for 2 was confirmed.
By the comparison of proton-coupling constants and NoeSY correlations, the relative configuration of 2 was deduced to be identical with those of libertellenone A (4) and scopararane H (1). Thus, the structure of 2 was identified as shown and given the trivial name scopararane I.
All of the seven pimarane diterpenes were preliminarily evaluated for their cytotoxicities against MCF-7, NCI-H460 and SF-268 cell lines with cisplatin as the positive control. The new compound, scopararane I (2), showed moderate cytotoxic activities with IC 50 values of 83.91, 13.59, and 25.31 μg mL −1 against the three cell lines, respectively. The known compound 7 was previously reported with potent cytotoxic activities against the selected tumour cell lines by our group (Sun, Li, Tao, Chen et al. 2012).

General experimental procedures
optical rotation was measured on an Anton Paar MCP-500 spectropolarimeter (Anton Paar, Graz, Austria). The Ir spectrum was recorded on an Ir Affinity-1 spectrophotometer in cm −1 (Shimadzu, Kyoto, Japan). uV spectrum was measured on a SHIMADZu uV-2600 uV-visible spectrophotometer (Shimadzu, Kyoto, Japan). 1D and 2D NMr spectra were recorded on a Bruker Avance-500 spectrometer with TMS as internal standard, δ in ppm, J in Hz (Bruker, Fällanden, Switzerland). HreIMS was measured on a Thermo MAT95XP high-resolution mass spectrometer and eIMS on a Thermo DSQ eI mass spectrometer (Thermo Scientific, MA, uSA). All solvents were analytical grade (Guangzhou Chemical Plant, Guangzhou, China). Silica gel (200-300 mesh) was used for column chromatography, and precoated silica gel GF 254 plates (Qingdao Marine Chemical Inc, Qingdao, China) were used for TLC spotting. C 18 reversedphase silica gel (40-63 μm, Merck, Darmstadt, German), and Sephadex LH-20 gel (Pharmacia Fine Chemical Co Ltd, New Jersey, Sweden) were also used for column chromatography (CC). TLC spots were visualised under uV light and by dipping into 10% H 2 So 4 in alcohol followed by heating.

Fungal material and identification
The strain FS46 was isolated from a marine sediment sample, which was collected at the depth of 292 m in the South China Sea (21°42.289′ N, 117°18.262′ e), in December, 2009. The strain was identified by sequence analysis of rDNA ITS (internal transcribed spacer) region. The sequence of ITS region of the marine fungus Eutypella sp. FS46 has been submitted to GenBank (Accession No. Ku192989). By using BLAST (nucleotide sequence comparison program) to search the GenBank database, Eutypella sp. FS46 has 99.6% similarity to Eutypella sp. e9901c (Accession No. JN637945). The strain is preserved at the Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Institute of Microbiology.

Fermentation, extraction and isolation
Eutypella sp. FS46 was cultured in potato dextrose broth (potato 20%, glucose 2%, K 2 HPo 4 0.3%, MgSo 4 ·7H 2 o 0.15%, vitamin B 10 mg L −1 ), which was prepared in 50% (v/v) sea water instead of distilled water. The fungus Eutypella sp. FS46 was maintained on 50% (v/v) sea water potato dextrose agar medium at 28 °C for 5 days, and then, three pieces (0.5 × 0.5 cm 2 ) of mycelial agar plugs were inoculated into 20 × 500 mL erlenmeyer flasks, each containing 250 mL 50% (v/v) sea water potato dextrose broth. After four days of incubation at 28 °C on a rotary shaker at 130 r.p.m, 25 mL seed cultures was aseptically transferred into each of a total of 200 flasks (1000 mL) containing 500 mL of 50% (v/v) sea water potato dextrose broth. The liquid cultivation that followed was kept for seven days at 28 °C and 130 r.p.m on a rotary shaker.
The culture (100 L) was centrifuged to give the broth and mycelia. The broth was exhaustively extracted with etoAc for four times, and then, the etoAc layers were combined and evaporated under reduced pressure at temperature not exceeding 40 °C to yield a dark brown gum (28.9 g). The dark brown gum was subjected to silica gel column chromatography (200-300 mesh) with a gradient system of increasing polarity (petroleum ether/etoAc/ MeoH), to afford 38 fractions (Fr. 1-38).

Conclusion
The chromatographic separation of the culture broth of a marine sediment-derived fungus Eutypella sp. FS46 yielded seven compounds. Their structures were determined by using spectroscopic methods as two new pimarane-type diterpenes, scopararanes H-I (1-2), and five known ones (3-7). The cytotoxic activities of all isolated compounds were evaluated. The new compound, scopararane I (2), showed different levels of inhibitory activities with IC 50 values ranging from 13.59 to 83.91 μg mL −1 .

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
Financial support for this research was provided by the National Natural Science Foundation of China