Fusarins G–L with Inhibition of NO in RAW264.7 from Marine-Derived Fungus Fusarium solani 7227

Six new fusarin derivatives, fusarins G–L (1–6), together with five known compounds (5–11) were isolated from the marine-derived fungus Fusarium solani 7227. The structures of the new compounds were elucidated by means of comprehensive spectroscopic methods (1D and 2D NMR, HRESIMS, ECD, and ORC) and X-ray crystallography. Compounds 5–11 exhibited potent anti-inflammatory activity by inhibiting the production of NO in RAW264.7 cells activated by lipopolysaccharide, with IC50 values ranging from 3.6 to 32.2 μM. The structure–activity relationships of the fusarins are discussed herein.


Introduction
Fusarium species are widely distributed in plants and soils worldwide. They have been proven to be an important source of plant pathogens in agriculture [1,2] and are also excellent sources of bioactive natural products. Among these, fusarin-related compounds are representative of Fusarium natural products, including epolactaene [3]; fusarins A, C, D, and F [4]; L-755,807 [5]; and NG-393 and NG-391 [6] (Figure 1), which possess a γ-lactam nucleus with a polyunsaturated side chain. Fusarium species have also received widespread attention in terms of both total chemical synthesis and biosynthesis [7,8]. For example, lucilactaene has been considered as a cell cycle inhibitor in p53-transfected cancer cells, which could arrest cell-cycle progression in a p53-independent manner in cells possessing a temperature-sensitive p53 protein, isolated from a strain of Fusarium species [9]. L-755,807 was reported as a bradykinin binding inhibitor, displaying bradykinin B2 receptor antagonist activity with an IC 50 of 71 µM [5]. NG-391 has displayed the impairment of nucleic acid formation, while translation remains unaffected, in K-562 human cancer cells [10].

Results
Fusarin G (1) was isolated as a white solid, and 6.4 mg was obtained from 197.9 g of crude extract. HRESIMS gave a molecular formula of C 9 H 11 O 4 , implying four degrees of unsaturation. Based on analysis of the 1 H NMR data of 1 (Table 1), there were two olefinic proton signals, δ H 7.08 (1H, qd, J = 7.2, 1.1 Hz) and 7.7 (1H, m); two methyls, δ H 1.78 (3H, d, J = 1.4 Hz) and 1.76 (3H, d, J = 1.4 Hz); and one methoxy, H-8 δ H 3.76 (3H, s). The planar structure of 1 was identified through 1 H-1 H COSY and HMBC analysis ( Figure 3). The core moiety of compound 1, a hexadienoic acid with a 5-substituted methyl, was established by means of HMBC correlations from H-4 to C-2, C-5, and C-6; H-9 to C-4, C-5, and C-6; and H-1 to C-2 and C-3; together with 1 H-1 H COSY data from H-1 and H-2. A methyl ester was linked to C-3, which was deduced from the HMBC correlation from H-8, H-4 and H-2 to C-7. The structure was further confirmed using X-ray crystallography. The X-ray structure ( Figure 4) clearly showed that the geometric configurations were both E; two methyl groups on olefinic carbon were located on the same side but not on the same face with a small angle. The chemical shift of H-2 (δ H 7.08) and H-4 (δ H 7.36) were downfield shifts, due to the de-shielding effect of carbonyl C-7 (δ C 166.7). This suggests that H-2 and H-4 are located on the same side as carbonyl C-7 in liquid.   Compound 2 was isolated as a white solid, and 7.3 mg was obtained from 197.9 g of crude extract. HRESIMS provided a molecular formula of C9H14O4. Analysis of the 1 H NMR data (Table 1) indicated that the planar structure of 2 was similar to that of 1. This was further supported by HMBC correlations ( Figure S15) from H-2 to C-3, C-4, and C-7    Compound 2 was isolated as a white solid, and 7.3 mg was obtained from 197.9 g of crude extract. HRESIMS provided a molecular formula of C9H14O4. Analysis of the 1 H NMR data (Table 1) indicated that the planar structure of 2 was similar to that of 1. This Compound 2 was isolated as a white solid, and 7.3 mg was obtained from 197.9 g of crude extract. HRESIMS provided a molecular formula of C 9 H 14 O 4 . Analysis of the 1 H NMR data (Table 1) indicated that the planar structure of 2 was similar to that of 1. This was further supported by HMBC correlations ( Figure S15) from H-2 to C-3, C-4, and C-7 and H-4 to C-5, C-6, and C-9, which also revealed the methyl C-9 to be connected to C-5. HMBC correlations also identified a methyl ester at C-3. Based on these data, the structure of 2 was determined as shown in Figure 2. To further identify the absolute configurations of compound 2, we compared the calculated optical rotation of (5S)-2 (+127.5) with the experimental data (+3.7), which indicated that the absolute configuration of 2 was (5S)-2. Compound 2 was named fusarin H.
Compound 3 was obtained as a yellow oil, and 9.8 mg was obtained from 197.9 g of crude extract. The molecular formula of C 16 H 20 O 4 was deduced from its HRESIMS data. Compared with the NMR data ( Table 2) of 2 and 3, compound 3 shares a methyl butenoate fragment. The structural differences were apparent in the 1 H-1 H COSY NMR data ( Figure  S25) of 3. These data revealed a spin system comprised of five olefinic hydrogens, H-6, H-7, H-8, H-9, and H-10, indicating that an unsaturated chain existed. HMBC ( Figure S24) correlations from H-16 to C-10, C-11, and C-12 and from H-15 to C-6 and C-5 suggested that 3 had a dimethylundecatetraenoic acid chain linked to C-5. At the same time, HMBC correlations from H-4 to C-5 and C-13 were observed. This suggested that C-4 connected with the methyl butyrate moiety at C-3. Therefore, the structure of compound 3 was identified and named fusarin I. Compound 4 was isolated as a yellow amorphous solid, and 5.1 mg was obtained from 197.9 g of crude extract. The molecular formula was assigned as C 15 H 20 O 4 based on HRESIMS data. Compared to the NMR data of 3 (Table 2), 4 showed similar spectroscopic features to 3, which had an unsaturated chain and methyl butyrate moiety. The main difference in compound 4 was the absence of one olefinic proton and carboxyl acid, and the presence of an additional keto carbonyl and a secondary oxygenated carbon. HMBC correlations from H-12 to C-11, C-10, and C-9 revealed that keto carbonyl was located on C-11. In addition, a secondary alcohol was attached to C-4, determined on the basis of the chemical shift of C-4 (δ C 71.8, δ H 5.06) and the HMBC correlation of H-15 and H-1 with C-4. Hence, the planar structure of compound 4 was deduced, as shown in Figure 5. To further identify the absolute configurations of compound 4, the calculated optical rotation of (4S)-4 (+226.63) was compared with the experimental data (+25.2), which indicated the absolute configuration of 4 was S. Finally, compound 4 was named fusarin J.  Compound 5 was obtained as a yellow amorphous substance, and 1.7 mg was obtained from 197.9 g of crude extract. The molecular formula was assigned as C26H26O5 based on HRESIMS data. The 1D NMR data (Table 3) indicated that compounds 5 and 3 had common fragments, except for the change in the carboxyl group C-12 (δC 196.94), replaced with a γ-pranone unit, which was supported by the 1 H-1 H COSY ( Figure 6) of H-13, H-14 and H-16, H-17, with two spin systems and HMBC correlations ( Figure 6) from H-14 to C-12, C-13, and C-15; H-16 to C-15; and H-17 to C-13. To further identify the absolute configurations of compound 5, the calculated optical rotation of (13S)-5 (−309.39) was compared with the experimental data (+1.8), which indicated the absolute configuration of 5 was R. Compound 5 was named fusarin K. Compound 5 was obtained as a yellow amorphous substance, and 1.7 mg was obtained from 197.9 g of crude extract. The molecular formula was assigned as C 26 H 26 O 5 based on HRESIMS data. The 1D NMR data (Table 3) indicated that compounds 5 and 3 had common fragments, except for the change in the carboxyl group C-12 (δ C 196.94), replaced with a γ-pranone unit, which was supported by the 1 H-1 H COSY ( Figure 6) of H-13, H-14 and H-16, H-17, with two spin systems and HMBC correlations ( Figure 6) from H-14 to C-12, C-13, and C-15; H-16 to C-15; and H-17 to C-13. To further identify the absolute configurations of compound 5, the calculated optical rotation of (13S)-5 (−309.39) was compared with the experimental data (+1.8), which indicated the absolute configuration of 5 was R. Compound 5 was named fusarin K.  Fusarin L (6) was obtained as a yellow amorphous substance, and 11.3 mg was obtained from 197.9 g of crude extract. HRESIMS analysis gave a molecular formula of C21H24NO6 with 10 degrees of unsaturation. Based on the analysis of 1D and 2D NMR data (Table 3), compound 6 belongs to a fusarin derivative and has a similar structure to that of 5 in the comparison of their NMR data. The HMBC correlations from methyl proton H-17 to C-15 and C-14 and from H-14 to C-13 and C-16 indicated an epoxy-γ-lactam ring with a methyl at C-15, which was linked to keto carbonyl C-11 in compound 6, which replaced a pyrone moiety in compound 5. Hence, the planar structure of 6 was confirmed. The theoretical ECD spectra (Figure 7) of compound 6 were calculated by means of a quantum chemical method at the [b3lyp/6-31+g(d,p)] level for a pair of configurations-13R, 14R, and 15S and 13R, 14R, and 15R. The computed configurations of 13R, 14R, and 15S were well in agreement with the experimental results. Furthermore, the computed Fusarin L (6) was obtained as a yellow amorphous substance, and 11.3 mg was obtained from 197.9 g of crude extract. HRESIMS analysis gave a molecular formula of C 21 H 24 NO 6 with 10 degrees of unsaturation. Based on the analysis of 1D and 2D NMR data (Table 3), compound 6 belongs to a fusarin derivative and has a similar structure to that of 5 in the comparison of their NMR data. The HMBC correlations from methyl proton H-17 to C-15 and C-14 and from H-14 to C-13 and C-16 indicated an epoxy-γ-lactam ring with a methyl at C-15, which was linked to keto carbonyl C-11 in compound 6, which replaced a pyrone moiety in compound 5. Hence, the planar structure of 6 was confirmed. The theoretical ECD spectra (Figure 7) of compound 6 were calculated by means of a quantum chemical method at the [b3lyp/6-31+g(d,p)] level for a pair of configurations-13R, 14R, and 15S and 13R, 14R, and 15R. The computed configurations of 13R, 14R, and 15S were well in agreement with the experimental results. Furthermore, the computed optical rotation of (13R, 14R, 15S)-6 with +351.83 was consistent with the experimental value (+44.95). Thus, combined with ECD, ORC, and the literature [13], the absolute configuration of 6 was proposed, with stereochemical assignments of 13R, 14R, and 15S.
Mar. Drugs 2021, 19, x 7 of 11 optical rotation of (13R, 14R, 15S)-6 with +351.83 was consistent with the experimental value (+44.95). Thus, combined with ECD, ORC, and the literature [13], the absolute configuration of 6 was proposed, with stereochemical assignments of 13R, 14R, and 15S. All the compounds were evaluated for their inhibition of nitric oxide (NO) production in RAW264.7 cells activated by lipopolysaccharide (LPS) using the Griess assay with indomethacin as a positive control (Table 4). Compounds 9, 10, and 11 displayed strong All the compounds were evaluated for their inhibition of nitric oxide (NO) production in RAW264.7 cells activated by lipopolysaccharide (LPS) using the Griess assay with indomethacin as a positive control (Table 4). Compounds 9, 10, and 11 displayed strong inhibition of nitric oxide (NO) production, with IC 50 values of 7.6, 3.6, and 8.4 µM, respectively, whereas compounds 2, 3, 4, and 5 showed moderate anti-inflammatory activity, with IC 50 values of 22.3, 13.1, 13.9, and 21.9 µM. In comparing the anti-inflammatory activity of fusarins G and H, 2 showed much stronger activity than that of 1, indicating that the double bond (C4-C5) of 1 is a disadvantage in terms of anti-inflammatory activity. Compound 5 exhibited stronger anti-inflammatory effects than those of 6, indicating that the pyrone moiety made a more positive contribution to the anti-inflammatory activity than the epoxy-γ-lactam ring. Table 4. Inhibitory activity of all compounds 1-11 against lipopolysaccharide (LPS)-induced NO production in the murine macrophage cell line (RAW 264.7 cells).

General Experimental Procedures
One-dimensional and two-dimensional NMR spectra were acquired in CDCl 3 with a Bruker Avance 400 MHz NMR spectrometer (Fällanden, Switzerland). HR-ESI-MS data were measured using a maXis Q-TOF mass spectrometer in positive ion mode (Bruker, Fällanden, Switzerland). UV and CD spectra were acquired using a JASCO J-810 circular dichroism spectrometer (JASCO International Co. Ltd., Hachioji, Tokyo, Japan). X-ray diffraction intensity data were recorded on an XtalLAB PRO single-crystal diffractometer, using Cu-Kα radiation (Rigaku, Japan). The data were corrected for absorption, using

Fungal Isolation and Fermentation
Fusarium solani 7227 was isolated from a seawater sample collected in February 2018 from the South China Sea. The fungus was identified as Fusarium solani by morphological observation and analysis of the internal transcribed spacer (ITS) regions of its rDNA, the sequence data of which were deposited in GenBank with the accession number MN922526. The strain was grown on PDA plates at 28 • C for 5 days; then it was cut into small pieces and inoculated into 1.5 L of PDA medium as a seed liquid at 28 • C for 2 days in a shaker; 1400, 1436, 1589, 1660, 1716, 1754, 2854, 2925, 2950, 3473 cm -1 ( Figure S45); 1 H NMR (CDCl 3 , 400 MHz) and 13 C NMR (CDCl 3 , 100 MHz), see  Figure S54); 1 H NMR (CDCl 3 , 400 MHz) and 13 C NMR (CDCl 3 , 100 MHz), see Table 3; HRESIMS m/z 386.16132 [M − H] − (calcd. for C 21 H 24 NO 6 , 387.16091).

Computational Detail
In optical rotation and ECD calculations, conformational analysis of compounds was performed using Spartan'14 software (Wavefunction Inc., Irvine, CA, USA) in the MMFF force field and the b3lyp/6-31+g(d,p) level was used to optimize the conformers, using this approach with the solvent model for MeOH [15,16]. Conformers with Boltzmann distributions over 5% were selected for ECD and OR calculations in methanol at the b3lyp/6-31+g(d,p) level. The ECD and OR spectra were generated using the programs SpecDis 1.71 (University of Würzburg, Würzburg, Germany) and OriginPro 2021 (OriginLab, Ltd., Northampton, MA, USA) based on dipole-length rotational strengths by applying Gaussian band shapes with sigma = 0.30 ev [17,18]. The optical rotations were calculated using time-dependent density functional theory. The b3lyp/6-31+g(d,p) level was employed in methanol for calculating optical rotations at A = 589.3 nm. All the calculations were performed using Tianhe-2 in the National Super Computer Center in Guangzhou.

Crystal Structure Analysis
Colorless crystals of compound 1 were obtained from a solvent of chloroform. Crystal data were acquired using the hemisphere technique on a Rigaku Oxford Diffraction diffractometer with graphite-monochromated Cu-Kα (radiation λ = 1.54178 Å). The structure was solved by means of direct methods using SHELXT; refinement was carried out by full-matrix least squares on F2, using the SHELXT program suite on Olex2 Launcher [19,20].

Anti-Inflammatory Activity
Evaluation of their inhibition of nitric oxide (NO) production in RAW264.7 cells activated by lipopolysaccharide (LPS) was carried out using the Griess assay with indomethacin as a positive control, according to a previous report [21]. RAW264.7 cells were purchased from the cell bank of the Chinese Academy of Sciences.

Conclusions
In this study, a chemical investigation on the secondary metabolites of Fusarium solani 7227, isolated from seawater in the South China Sea, led to the isolation of compounds 1-11. Among the isolates, compounds 1 and 2 were found to be possible precursor compounds and compounds 3-6 were a series of new fusarin-related compounds with a polyunsaturated side chain. Together with the known compounds 7-11, all compounds were evaluated for their inhibition of NO production in RAW264.7 cells activated by LPS. Compounds 2-5 and 7-11 displayed potent anti-inflammatory activity, with IC 50 values ranging from 3.6 to 32.2 µM. A preliminary structure-activity relationship analysis indicated that the anti-inflammatory activities depend on the double bonds between C-4 and C-5 for compounds 1 and 2 and on the substitution group of the polyunsaturated chain for compounds 3-11.