Anti-inflammatory Polyketides from the Marine-Derived Fungus Eutypella scoparia

Three new polyketides, eutyketides A and B (1 and 2) and cytosporin X (3), along with four known compounds (4–7), were obtained from the marine-derived fungus Eutypella scoparia. The planar structures of 1 and 2 were elucidated by extensive HRMS and 1D and 2D NMR analyses. Their relative configurations of C-13 and C-14 were determined with chemical conversions by introducing an acetonylidene group. The absolute configurations of 1–3 were determined by comparing their experimental electronic circular dichroism (ECD) data with their computed ECD results. All of the isolated compounds were tested for their anti-inflammatory activities on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophages. Compounds 5 and 6 showed stronger anti-inflammatory activities than the other compounds, with the inhibition of 49.0% and 54.9% at a concentration of 50.0 µg/mL, respectively.


Introduction
Eutypella species, which are one genus of the ubiquitous fungi, are widely distributed in many extreme environments, including Antarctica, tropical forests, and marine organisms [1][2][3]. Chemical investigations of Eutypella species have resulted in diverse metabolites, including γ-lactones, benzopyrans, cysporins, terpenoids, and nitrogen-containing compounds [4,5]. Among them, many bioactive secondary metabolites were obtained, such as antibacterial scoparasin B [5], cytotoxic phenochalasin B [6], and antitumor diaporthein B [7]. The Eutypella genus has become an attractive target for discovering leading compounds due to its remarkable biological activity and novel complex structures. In recent years, a lot of work has been carried out on the isolation, total synthesis, pharmacological research, and drug development for the genus Eutypella [8][9][10].
As part of our ongoing investigation of bioactive natural products from marine-derived fungi [11][12][13][14][15][16], the strain Eutypella scoparia HBU-91 attracted our attention because the EtOAc extract of the culture showed anti-inflammatory activity. As a result, the new eutyketides A and B (1 and 2) and cytosporin X (3), together with four known compounds (4-7) (Figure 1), were obtained by using silica gel and LH-20 column chromatography and semipreparative HPLC. Structurally, compounds 1 and 2 were a pair of epimers with vic-diol unit on their side chain, while 3 exhibited a skeleton characterized by a polyketide moiety and a terpenoid part. All of the isolated compounds were tested for their anti-inflammatory activities. Herein, we report their isolation, structure elucidation, and biological activities.

Structural Elucidation
Eutyketide A (1) was obtained as a pale yellow oil. The molecular formula of 1 was determined to be C 18   ]. The 1 H and 13 C NMR data revealed that 1 shares the same carbon framework as graphostrin I, a polyketide obtained from the Atlantic hydrothermal fungus Graphostroma sp. MCCC 3A00421 [17]. The main differences between them were the presence of a methyl at C-6 and a vic-diol [−OHCH−CHOH−] substructure at C-13/14 in 1 instead of the group [−CH 2 −CH 2 −] and the absence of the hydroxy group at C-18 in graphostrin I. The above differences were confirmed by the COSY cross-peaks of H-12/H-13/H-14/H 2 -15 and H 2 -17/H 2 -18/H 3 -19 and the HMBC correlations from H-8 to C-1, C-5, and C-6, from H-13 to C-11 and C-15, and from H-14 to C-12 and C-16, respectively ( Figure 2). In addition, trans geometries at C-9−C-10 and C-11−C-12 double bonds were assigned by the large coupling constants (J 9,10 = 15.0 Hz and J 11,12 = 15.0 Hz) [17]. By detailed analysis of its 2D NMR spectra, the planar structure of 1 was assigned.
Eutyketide B (2) was also obtained as a pale yellow oil. It exhibited the same molecular formula as 1, C 18 H 26 O 5 , according to the pseudomolecular ion at m/z 345.1669 [M + Na] + in the HRESIMS spectrum. Detailed analysis of the 1 H and 13 C NMR spectra of 2 (Table 1) revealed that its 1D NMR data were similar to those of 1. The differences were attributable to the signals [δ H 4.05 (m, H-13) and 3.51 (m, H-14); δ C 138.7 (C-12), 74.4 (C-13), and 33.2 (C-15) in 2 vs. δ H 4.23 (dd, J = 6.0, 4.2 Hz, H-13) and 3.72 (m, H-14); δ C 137.5 (C-12), 75.2 (C-13), and 32.2 (C-15) in 1], indicating that the structural differences between them should be located in this part of the structure (C-13 and C-14). Thus, it was deduced that 1 and 2 were either C-13 or C-14 epimers.    The calculation of the solution conformers is the most time-demanding part of th ECD calculation in conformationally flexible molecules and may be aided by simplifyin the input geometry to reduce the number of conformers and save computational time [18 For example, alkyl side chains and unsaturated side chains with isolated chromophore The calculation of the solution conformers is the most time-demanding part of the ECD calculation in conformationally flexible molecules and may be aided by simplifying the input geometry to reduce the number of conformers and save computational time [18]. For example, alkyl side chains and unsaturated side chains with isolated chromophores in an achiral environment could be simplified by truncation [18]. The absolute configuration of the hydroxyl group at C-13 was affected by the conjugate system, which can be determined by ECD calculation [19]. For 1 and 2, the absolute configurations of C-13 and C-14 were determined by comparing their experimental electronic circular dichroism (ECD) results with the computed results of their simplified model compounds. The group of C-15 to C-19 was a saturated alkyl side chain with no chromophore and had a negligible effect on the ECD spectrum. Thus, the C-15 to C-19 alkyl substituent was truncated to a methyl group as model compound 1b (Figure 4). Molecules of (13S,14S)-1b, (13R,14R)-1b, (13R,14S)-1b, and (13S,14R)-1b were chosen for ECD calculations, which were carried out at the B3LYP/6-311+G(d,p) level in MeOH using the PCM model. The predicted ECD spectrum of (13R,14R)-1b matched well with the experimental ECD curve of 1, and the predicted ECD spectrum of (13R,14S)-1b was in good agreement with the experimental ECD data of 2 ( Figure 4). Therefore, the absolute configurations of 1 and 2 could be defined as 13R,14R and 13R,14S, respectively. The calculation of the solution conformers is the most time-demanding part of the ECD calculation in conformationally flexible molecules and may be aided by simplifying the input geometry to reduce the number of conformers and save computational time [18]. For example, alkyl side chains and unsaturated side chains with isolated chromophores in an achiral environment could be simplified by truncation [18]. The absolute configuration of the hydroxyl group at C-13 was affected by the conjugate system, which can be determined by ECD calculation [19]. For 1 and 2, the absolute configurations of C-13 and C-14 were determined by comparing their experimental electronic circular dichroism (ECD) results with the computed results of their simplified model compounds. The group of C-15 to C-19 was a saturated alkyl side chain with no chromophore and had a negligible effect on the ECD spectrum. Thus, the C-15 to C-19 alkyl substituent was truncated to a methyl group as model compound 1b (Figure 4). Molecules of (13S,14S)-1b, (13R,14R)-1b, (13R,14S)-1b, and (13S,14R)-1b were chosen for ECD calculations, which were carried out at the B3LYP/6-311+G(d,p) level in MeOH using the PCM model. The predicted ECD spectrum of (13R,14R)-1b matched well with the experimental ECD curve of 1, and the predicted ECD spectrum of (13R,14S)-1b was in good agreement with the experimental ECD data of 2 ( Figure 4). Therefore, the absolute configurations of 1 and 2 could be defined as 13R,14R and 13R,14S, respectively. To the best of our knowledge, compounds 1 and 2 are very similar to prosolanapyrones and their congeners [20]. They share the same pyranone framework with long alkyl side chains. In addition to the conjugate double bonds, compounds 1 and 2 also contain a vic-diol unit on their side chains, while prosolanapyrones just possess double bonds on their side chains.  To the best of our knowledge, compounds 1 and 2 are very similar to prosolanapyrones and their congeners [20]. They share the same pyranone framework with long alkyl side chains. In addition to the conjugate double bonds, compounds 1 and 2 also contain a vic-diol unit on their side chains, while prosolanapyrones just possess double bonds on their side chains.  Table 2). The NMR data revealed that 3 belongs to the family of hexahydrobenzopyrane Mar. Drugs 2022, 20, 486 5 of 10 skeletons and is characterized by a polyketide moiety and a terpenoid part (the red part in Figure 5) with a tricyclic structure containing a hexahydrobenzopyrane moiety fused with an oxirane ring [1]. Careful comparison of the NMR data of 3 with those of the known hexahydrobenzopyrane cytosporin D (4) indicated that the structure of 3 is closely related to 4. The notable difference between them lay in the presence of two methylene signals  2). The NMR data revealed that 3 belongs to the family of hexahydrobenzopyrane skele tons and is characterized by a polyketide moiety and a terpenoid part (the red part in Figure 5) with a tricyclic structure containing a hexahydrobenzopyrane moiety fused with an oxirane ring [1]. Careful comparison of the NMR data of 3 with those of the known hexahydrobenzopyrane cytosporin D (4) indicated that the structure of 3 is closely related    The relative configuration of 3 was determined by analysis of the NOESY data ( Figure 6). The NOESY correlations of H-3/H 3 -12, H 3 -11/H-10, H-10/H-7, H-10/H-4β, and H-4α/H-6 indicated that H-3 and H-6 were situated on the same side of the molecule with an α-orientation, while C-5, C-6, H-7, and H-10 were accordingly assigned to be β-configured. In addition, the observed NOEs are consistent with the structure and relative configuration of 4.

Acetonide Formation of 1 and 2
A mixture of 1 (1.0 mg), 2,2-dimethoxypropane (2.0 mL), and p-TsOH (0.2 mg) was stirred at room temperature for 0.5 h. Saturated aqueous NaHCO 3 (6.0 mL) was then added, and the reaction mixture was extracted with EtOAc (24 mL × 3). The organic solvents were removed with a high-vacuum pump, and the crude mixture was subjected to preparative HPLC to obtain acetonide product 1a (0.96 mg). Acetonide product 2a (0.91 mg) was obtained from 2 under the same conditions as 1a. Compound

Computational Section
A conformational search for the molecules was carried out using the MMFF94S force field and Compute VOA software, with relative energies ranging from 0−10.0 kcal/mol energy, respectively. The conformers were optimized at the B3LYP/6-31G(d)//B3LYP/6-311+G(d) levels with Gaussian 09 software [24]. Then, stable conformers with relative energy within a 2.5 kcal/mol energy window were chosen for ECD calculations at the B3LYP/6-311+G(d,p) level in methanol using the PCM model, with a total of 60 excited states. A standard deviation of 0.3 eV was used for ECD simulations. Boltzmann statistics were applied for the final simulations of the ECD spectra by the software SpecDis 1.64 [25].

Cell Culture and Viability Assay
Compounds 1−7 were first tested for their cytotoxic effects on RAW264.7 cells at 3.13, 6.25, 12.5, 25.0, and 50.0 µg/mL. Murine monocytic RAW264.7 macrophages were cultivated at 37 • C with 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM), which was added with 10% (v/v) fetal bovine serum (FBS) as well as 1% (v/v) penicillin/streptomycin. RAW264.7 cells were grown in 96-well plates and then incubated with the tested compounds for 24 h. A solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) at a concentration of 5.0 mg/mL was substituted for the culture medium. After incubation at 37 • C for 4 h, the MTT solution was removed, and DMSO was chosen for the dissolution of the formazan crystals. The absorbance was measured at 540 nm with a microplate reader [26]. Compounds 3−7 exhibited no toxicity at a concentration of 50.0 µg/mL, 1 showed no toxicity at a concentration of 25.0 µg/mL, and 2 displayed no toxicity at a concentration of 6.25 µg/mL.

Inhibition of NO Production Assay
The Griess assay was applied to evaluate the production of NO through the level of nitrite (NO 2 ) in the medium [27]. RAW264.7 cells were inoculated into 96-well plates, and then LPS at a concentration of 1.0 µg/mL was added to induce inflammation. The tested compounds at different concentrations were added to the above mixture. The Griess reaction was used for the quantification of NO production in the supernatant. The absorbance was measured at 540 nm with a microplate reader. All of the experiments were carried out in triplicate.

Conclusions
In summary, three new polyketides (1−3), together with four known compounds (4−7), were isolated from the marine-derived fungus Eutypella scoparia. Chemical conver-Mar. Drugs 2022, 20, 486 9 of 10 sions and TDDFT ECD calculations were used to determine the absolute configurations of 1−3. Compounds 1, 5, and 6 exhibited certain anti-inflammatory activities on nitric oxide (NO) production in RAW264.7 cells induced by lipopolysaccharide (LPS). Our findings will contribute to the diversity of these fungal metabolites.