Cytosporones with Anti-Inflammatory Activities from the Mangrove Endophytic Fungus Phomopsis sp. QYM-13

Six previously undescribed cytosporone derivatives (phomotones A-E (1–5) and phomotone F (13)), two new spiro-alkanol phombistenes A-B (14–15), and seven known analogs (6–12) were isolated from the mangrove endophytic fungus Phomopsis sp. QYM-13. The structures of these compounds were elucidated using spectroscopic data analysis, electronic circular dichroism (ECD), and 13C NMR calculations. Compound 14 features an unprecedented 1,6-dioxaspiro[4.5]decane ring system. All isolates were evaluated for their inhibitory effect on nitric oxide (NO) in LPS-induced RAW264.7 cells. The results showed that compounds 1, 6, 8, and 11 exhibited potent bioactivities by comparing with positive control. Then, compound 1 displayed the anti-inflammatory effect by inhibiting the MAPK/NF-κB signaling pathways. Molecular docking further revealed the possible mechanism of compound 1 interaction with ERK protein.

Mangrove endophytic fungi have attracted the attention of many researchers due to their ability to produce structurally novel and remarkably bioactive secondary metabolites [7].To date, thousands of new metabolites have been isolated from mangrove endophytic fungi [8].As part of our ongoing search for bioactive compounds from mangrove endophytic fungi, the strain Phomopsis sp.QYM-13, isolated from healthy leaves of Kandelia candel, was investigated.We recently reported twelve new cytochalasins, phomopchalasins D-O, including brominated and iodinated cytochalasins, with significant cytotoxicity from this fungus [9].Subsequently, another eight new cytosporones phomotones A-E (1-5), phomotone F (13), and phombistenes A-B (14-15) (Figure 1) were obtained in our further research work.In bioassays, compounds 1, 6, 8, and 11 exhibited significant anti-inflammatory activities.Herein, the isolation, structure elucidation, biological assays, molecular docking, and structure-activity relationship of these isolated compounds are described.

Results
Compound 1 was isolated as a white solid with the molecular formula of C 18 H 23 O 7 based on the negative HRESIMS data m/z 351.14534 [M-H] − .The 1 H NMR spectrum of 1 showed the presence of two aromatic protons (δ H 6.20 (d, J = 2.2 Hz), 6.26 (d, J = 2.2 Hz)) and one methyl group (δ H 1.24 (t, J = 7.1 Hz)).The 13 C NMR (Table 1) and HSQC spectra (Figure S3) of 1 revealed the coexistence of eighteen carbons attributable to one methyl, eight methylenes (one oxygenated), two methines, and seven unprotonated carbons (three carbonyl carbons and four olefinic carbons).These data suggested 1 to be a cytosporone class, and the 1 H and 13 C NMR data of 1 were similar to those of 12 [10].The main difference involved the absence of the oxygenated methylene in 12 and the appearance of carboxyl carbon in 1, which might mean the terminal hydroxyl group in 12 was oxidized to a carboxyl group in 1.The deduction was further confirmed with the spin system of H 2 -10/H 2 -11/H 2 -12/H 2 -13/H 2 -14/H 2 -15 in the 1 H-1 H COSY spectrum (Figure 2), as well as the HMBC correlations (Figure 2) from H 2 -10 to C-9 and from H 2 -15 to C-16.Thus, the structure of 1 was assigned as shown in Figure 1.
For compound 14, a yellow solid, the molecular formula C14H22O4 with four degrees of unsaturation was established using HRESIMS.The 1 H NMR spectrum (Table 3) showed two methyl groups at δH 1.34 (d, J = 6.2 Hz) and 1.30 (d, J = 6.4 Hz), and three olefin protons at δH 5.73 (ddd, J = 4.1, 10.7, 14.7 Hz), δH 5.07 (d, J = 10.7 Hz), and δH 5.30 (d, J = 17.1 Hz).The 13 C NMR and HSQC spectra of 14 revealed the existence of fourteen carbons attributable to two methyls, five methylenes (one olefinic carbon), five methines (one olefinic carbon), and two unprotonated carbons.From an analysis of the 1 H-1 H COSY spectrum, four independent coupling fragments could be inferred (Figure 2).Furthermore, the HMBC correlations from H-1, H-3, H-6, and H2-13 to C-4 and from H-3 and H-5 to C-7 indicated the furan ring and pyranoid ring were fused at positions C-3/C-4, and one group of terminal olefin was located at C-4.Then, the HMBC correlations from H-8 and H-10 to C-7 and the chemical shift at C-4 (δC 108.5) enabled the construction of a 1,6-dioxaspiro[4.5]decanering system.Therefore, the planar structure of 14 was tentatively assigned.Compound 2 was isolated as a colorless oil with the molecular formula of C 16 H 19 O 7 based on the negative HRESIMS data m/z 323.11392 [M-H] − .Detailed analysis of the NMR data (Table 1) indicated that 2 was structurally similar to 1, with the difference being the absence of two methylenes.It was supported by its 1 H-1 H COSY cross-peaks of H 2 -10/H 2 -11/H 2 -12/H 2 -13 and HMBC correlations from H 2 -10 to C-9 and from H 2 -13 to C-14.Thus, the structure of 2 was established.
Compound 3 was isolated as a colorless oil with the molecular formula of C 14 H 15 O 7 based on the negative HRESIMS data m/z 295.08227 [M-H] − .The 1 H and 13 C NMR data of 3 (Table 1) were similar to those of 2, except for the absence of an ethyl group (δ C 60.4, 13.1) in 3.Then, the HMBC correlations (Figure 2) further confirmed the deduction above.
Compound 4 was isolated as a colorless oil with the molecular formula of C 14 H 15 O 7 based on the negative HRESIMS data m/z 295.08240 [M-H] − .A comparison of the NMR data of 4 (Table 2) with those of 2 revealed that their structures were similar, with the only difference being the absence of two methylenes.The 1 H-1 H COSY cross-peaks of H 2 -10/H 2 -11 and HMBC correlations from H 2 -10 to C-9 and from H 2 -11 to C-12 further confirmed the structure.Compound 5 was isolated as a colorless oil with the molecular formula of C 16 H 21 O 6 based on the positive HRESIMS data m/z 309.1333 [M + H] + .The 1 H and 13 C NMR data of 5 (Table 2) were similar to those of 6 [10], except for the absence of an ethyl group (δ C 61.5, 14.2) in 5.The deduction was further confirmed by 1 H-1 H COSY and HMBC correlations (Figure 2).
For compound 13, a yellow solid, the molecular formula C 17 H 22 O 3 with seven degrees of unsaturation was established using HRESIMS.The NMR (Table 2) information of 13 was similar to 10, with the main difference being the presence of one methyl group (δ H 2.64), one olefin proton (δ H 5.95), and two olefin carbons (δ C 167.3, 110.5) in 13, and the disappearance of the carboxyl group at C-1 in 10.The HMBC correlations from H 3 -2 to C-3, C-4, and C-8, from H-10 to C-9, C-11, and C-12, together with the spin system of H 2 -12/H 2 -13/ H 2 -14/H 2 -15/H 2 -16/H 2 -17/H 3 -18 in the 1 H-1 H COSY spectrum (Figure 2) further confirmed the deduction.Thus, the structure of 13 was verified.
Numerous inflammatory targets including iNOS, COX-1, COX-2, ICAM, IL-5, IL-17, JAK1, JAK2, SIRT2, and TNF-α were investigated through virtual screening for all compounds.The results indicated that iNOS showed stronger binding affinity with 1-13 than other targets (Table S2).Thereafter, all compounds were evaluated for their inhibitory activities against LPS-induced nitric oxide (NO) production in RAW 264.7 mouse macrophages.At non-cytotoxic concentrations, the results showed (Table 4) that compounds 1, 6, 8, and 11 exhibited significant anti-inflammatory activities with IC 50 values of 10.0, 12.0, 13.4, and 11.5 µM, respectively.Compounds 2, 12, and 13 showed potent anti-inflammatory activities compared with the positive control ( L -NMMA: 32.8 µM).Thereafter, the preliminary structure-activity relationship was discussed.The compounds 2, 6, 8, and 11 displayed higher anti-inflammatory activity than 3, 5, 7, and 10, which indicated that the acetyl group at C-1 is beneficial for activity.The carboxyl group in the side chain at C-16 may contribute to the activity by comparing 1 with 6, 8, 9, 11, and 12.Moreover, the number of carbons in the side chain at C-8 may has not affected the anti-inflammatory activity by comparing the IC 50 values of 1-12.Cytosporones were reported to have anti-inflammatory activity.To explore the antiinflammatory mechanism of these compounds, the inhibitory effects of inflammationrelated iNOS and COX-2 for new compound 1 were measured using western blot.As a result, the protein expression of iNOS and COX-2 were apparently down-regulated after treatment of 1 with different concentrations (20.0, 10.0, and 5.0 µM) in a dose-dependent manner and dose-independent manner, respectively (Figure 6).These results were consistent with the project of target docking.Cytosporones were reported to have anti-inflammatory activity.To explore the antiinflammatory mechanism of these compounds, the inhibitory effects of inflammation-related iNOS and COX-2 for new compound 1 were measured using western blot.As a result, the protein expression of iNOS and COX-2 were apparently down-regulated after treatment of 1 with different concentrations (20.0, 10.0, and 5.0 µM) in a dose-dependent manner and dose-independent manner, respectively (Figure 6).These results were consistent with the project of target docking.Mitogen-activated protein kinases (MAPK) and nuclear factor-κB (NF-κB) pathway were known to regulate the inflammatory response by modulating multiple pro-inflammatory cytokines in macrophages.So as to elucidate the mechanism of action by which compound 1 inhibited the level of NO, the MAPK and NF-κB signaling pathway of 1 was further investigated.As expected, the phosphorylation levels of p38, JNK, and ERK were significantly up-regulated after the treatment of RAW264.7 cells with LPS in the MAPK signaling pathway.Meanwhile, the phosphorylation levels of p38, JNK, and ERK were reduced after pretreatment of RAW264.7 cells with different concentrations of compound 1.In addition, the western blotting methods have detected the expression of phosphorylation p65 in the NF-κB signaling pathway.The results implied that pretreatment with compound 1 obviously decreased the levels of phosphorylation p65 (Figure 7).Taken together, compound 1 displayed the anti-inflammatory effect by inhibiting the MAPK/NF-κB signaling pathways.Mitogen-activated protein kinases (MAPK) and nuclear factor-κB (NF-κB) pathway were known to regulate the inflammatory response by modulating multiple pro-inflammatory cytokines in macrophages.So as to elucidate the mechanism of action by which compound 1 inhibited the level of NO, the MAPK and NF-κB signaling pathway of 1 was further investigated.As expected, the phosphorylation levels of p38, JNK, and ERK were significantly up-regulated after the treatment of RAW264.7 cells with LPS in the MAPK signaling pathway.Meanwhile, the phosphorylation levels of p38, JNK, and ERK were reduced after pretreatment of RAW264.7 cells with different concentrations of compound 1.In addition, the western blotting methods have detected the expression of phosphorylation p65 in the NF-κB signaling pathway.The results implied that pretreatment with compound 1 obviously decreased the levels of phosphorylation p65 (Figure 7).Taken together, compound 1 displayed the anti-inflammatory effect by inhibiting the MAPK/NF-κB signaling pathways.In the MAPK signaling pathway, the levels of phosphorylation ERK were remarkably diminished compared with p38 and JNK.To further expound the role of phosphorylation ERK in compound 1 effects, molecular docking analysis was performed to investigate an inside interaction between compound 1 and ERK protein (PDB:5v60).As shown in Figure 8, the results indicated that 1 binds deeply in the active site pocket between Lys114, Tyr113, Asp111, Ser153, Glu33, Met38, and Ala35.Four hydrogen bonds are formed between the hydroxyl group at C-5 and C-7, and carbonyl at C-9 of 1 with Met108, Asp106, Gln105, and Cys166, respectively.In addition, multiple van der Waals-interacting residues such as Leu107, Ile31, Asn154, Lys54, Asp167, and Gly32 are generated between the ligand and the receptor protein, as well as π-π stacking interaction with Ala52, Val39, and Leu156.Thus, compound 1 could effectively activate the ERK signaling.In the MAPK signaling pathway, the levels of phosphorylation ERK were remarkably diminished compared with p38 and JNK.To further expound the role of phosphorylation ERK in compound 1 effects, molecular docking analysis was performed to investigate an inside interaction between compound 1 and ERK protein (PDB:5v60).As shown in Figure 8, the results indicated that 1 binds deeply in the active site pocket between Lys114, Tyr113, Asp111, Ser153, Glu33, Met38, and Ala35.Four hydrogen bonds are formed between the hydroxyl group at C-5 and C-7, and carbonyl at C-9 of 1 with Met108, Asp106, Gln105, and Cys166, respectively.In addition, multiple van der Waals-interacting residues such as Leu107, Ile31, Asn154, Lys54, Asp167, and Gly32 are generated between the ligand and the receptor protein, as well as π-π stacking interaction with Ala52, Val39, and Leu156.Thus, compound 1 could effectively activate the ERK signaling.

Fungal Material, Fermentation and Isolation
The strain Phomopsis sp.QYM-13 was described as previously reported [9].Briefly, the mycelia of the fungus were inoculated into a 250 mL potato dextrose medium for 5 days to prepare the seed culture.Thereafter, the spore suspension was transferred into liquid mediums each containing 300 mL (3 g of potato extract, glucose 20 g/L, artificial sea salts 20 g/L, 100 × 1 L Erlenmeyer flasks) for 30 days at 25 • C.Then, the extracts (21.2 g) were obtained from the mycelia and broth as the methods previously described.It was subjected to silica gel column chromatography (CC, 200 mesh silica) eluting with petroleum ether/EtOAc (9:1 to 1:9) to yield 6 fractions (Fr.A-F).Fr.A was fractionated with Sephadex LH-20 CC (CH 2 Cl 2 /MeOH v/v, 1:1) to yield 2 fractions (Fr.A1-A2).Fr.A1 was further separated using silica gel CC (CH

ECD and NMR Calculation Methods
The ECD calculation was performed as described previously [7,14].The conformers were subjected to geometric optimization at the level of B3LYP/6-31+G in the liquid phase.Thereafter, the optimized conformers were calculated using the TD-DFT method at the RB3LYP/6-311G (14) and PBEPBE/LAN12DZ (15) levels, respectively.
Typically, the Merck molecular force field in Spartan's 10 software was used for the conformational analysis of compound 14.Conformers with populations exceeding 5% according to the Boltzmann distribution were optimized using the B3LYP/6-311+G (d, p) level in the polarizable continuum model (PCM) with methanol as the solvent.Subsequently, NMR calculations were performed using the gauge invariant atomic orbital (GIAO) method at the mPW1PW91-SCRF/6-311+G (d, p) level with PCM in methanol (Gaussian 09).Finally, the shielding constants were averaged using Boltzmann distribution theory for each stereoisomer, and their experimental and calculated data were analyzed using DP4+ probability.
Funding: We are grateful for the financial support from the National Natural Science Foundation of China (U20A2001, 42276114), the Key Scientific Research Project in Colleges and the Universities of Anhui Province (2022AH050706), and the Natural Science Foundation of Anhui Province (2308085QH302).
Informed Consent Statement: Not applicable.

Figure 4 .
Figure 4. Comparisons of calculated and experimental 13 C NMR data of 14.

Figure 5 .
Figure 5. Experimental and calculated ECD spectra of 14 and 15.

Figure 4 .
Figure 4. Comparisons of calculated and experimental 13 C NMR data of 14.

Figure 4 .
Figure 4. Comparisons of calculated and experimental 13 C NMR data of 14.

Figure 5 .
Figure 5. Experimental and calculated ECD spectra of 14 and 15.

Figure 5 .
Figure 5. Experimental and calculated ECD spectra of 14 and 15.

Figure 7 .
Figure 7. Influences of compound 1 on the MAPK and NF-κB pathway detected with western blotting.(A) The expression levels of p-JNK, p-ERK, p-P38, and GAPDH detected with Western blotting.(B) The proportion of p-JNK, p-ERK, and p-P38 to GAPDH content.(C) The expression levels of p-P65 and β-actin detected with western blotting.(D) The proportion of p-P65 to β-actin content.Data rendered are the mean ± SD, n = 3.In comparison to the control, *** p < 0.001.In comparison to the LPS, # p < 0.05, ## p < 0.01, ### p < 0.001.

Figure 7 .
Figure 7. Influences of compound 1 on the MAPK and NF-κB pathway detected with western blotting.(A) The expression levels of p-JNK, p-ERK, p-P38, and GAPDH detected with Western blotting.(B) The proportion of p-JNK, p-ERK, and p-P38 to GAPDH content.(C) The expression levels of p-P65 and β-actin detected with western blotting.(D) The proportion of p-P65 to β-actin content.Data rendered are the mean ± SD, n = 3.In comparison to the control, *** p < 0.001.In comparison to the LPS, # p < 0.05, ## p < 0.01, ### p < 0.001.

-5 and 13. 4 a 5 a 13 b No δ C δ H (J in Hz) δ C δ H (J in Hz) δ C δ H (J in Hz)
Measured in MeOD-d 4 , b measured in DMSO-d 6 . a
a positive control.
a positive control.