*3.6. Molecular Docking Study*

Virtual docking is implemented in the AutoDock tool of AutoDock4.2.6 software [33]. This is a common docking method that allows the ligand to have sufficient flexibility and maintain the rigidity of the target protein. The X-ray crystal structure of iNOS (PDB ID: 3E6T) [27] was obtained from the RCSB protein database (PDB) database. Before docking simulation, PyMOL was used to delete the original ligand and water molecules from the crystal structure, and the protein was saved in PDB format (receptor.pdb). The compound structure was drawn using ChemDraw 2D software, which was converted into three-dimensional (3D) structure by ChemDraw 3D software, and then stored as a file in PDB format. Furthermore, the molecular structure was optimized by Gaussian software. AutoDock tools converted both protein and ligand into PDBQT format for subsequent docking. Focusing on the protein, the parameters of the grid box were set to 126 × 126 × 126 points and the Lamarckian genetic algorithm was used to link the algorithm with 100 GA operations. Finally, PyMOL was used to visualize and analyze the results.

#### **4. Conclusions**

In conclusion, chemical investigation of the mangrove endophytic fungus BJR-P2 resulted in the isolation and identification of four new compounds (**1**–**4**), with four known analogs (**5**–**8**). Their structures were elucidated by extensive spectroscopic methods and quantum chemical calculations. The anti-inflammatory activity evaluation was carried out by screening their inhibition activity on NO production. The results showed compound **2** exhibited significant inhibitory activity with an IC50 value of 12 μM. This study may provide a new chemical lead candidate for the discovery of anti-inflammatory agents.

**Supplementary Materials:** The HRESIMS and NMR spectrum are available online at: https://www. mdpi.com/article/10.3390/md20090583/s1. Figure S1. HRESIMS of compound **1**. Figure S2. 1H NMR spectrum of compound **1**. Figure S3. 13C NMR spectrum of compound **1**. Figure S4. HMQC spectrum of compound **1**. Figure S5. HMBC spectrum of compound **1**. Figure S6. 1H, 1H-COSY spectrum of compound **1**. Figure S7. NOESY spectrum of compound **1**. Figure S8. HRESIMS of compound **2**. Figure S9. 1H NMR spectrum of compound **2**. Figure S10.13C NMR spectrum of compound **2**. Figure S11. HMQC spectrum of compound **2**. Figure S12. HMBC spectrum of compound **2**. Figure S13. 1H, 1H-COSY spectrum of compound **2**. Figure S14. NOESY spectrum of compound **2**. Figure S15. HR-ESI-MS spectrum of compound **3**. Figure S16. 1H NMR spectrum of compound **3**. Figure S17. 13C NMR spectrum of compound **3**. Figure S18. HMQC spectrum of compound **3**. Figure S19. HMBC spectrum of compound **3**. Figure S20. 1H, 1H-COSY spectrum of compound **3**. Figure S21. NOESY spectrum of compound **3**. Figure S22. HR-ESI-MS spectrum of compound **4**. Figure S23. 1H NMR spectrum of compound **4**. Figure S24. 13C NMR spectrum of compound **4**. Figure S25. HMQC spectrum of compound **4**. Figure S26. HMBC spectrum of compound **4**. Figure S27. 1H, 1H-COSY spectrum of compound **4**. Figure S28. NOESY spectrum of compound **4**. Figure S29. Experimental (Exp.) and calculated (Cal.) 1H and 13C chemical shift values of **3** and its possible isomers. Figure S30. DP4+ analysis of **3**. Figure S31. Experimental (Exp.) and calculated (Cal.) 1H and 13C chemical shift values of **4** and its possible isomers. Figure S32. DP4+ analysis of **4**. Figure S33. Proposed biosynthetic pathways for compounds **1**–**4**.

**Author Contributions:** C.C. and G.Y. performed the experiments for the isolation, structure elucidation, and biological evaluation and prepared the manuscript; J.T., J.L., W.L., L.W. contributed to part work of fermentation, extraction, structure characterization of all the compounds; Y.L. supervised the research work and revised the manuscript. Conceptualization, Y.L.; methodology, C.C. and G.Y.; software, C.C. and G.Y.; validation, Y.L.; formal analysis, C.C., G.Y., J.T.; investigation, C.C., G.Y., J.L., W.L., L.W.; resources, Y.L.; data curation, C.C., G.Y., J.T.; writing—original draft preparation, C.C. and G.Y.; writing—review and editing, Y.L. and C.C.; supervision, Y.L.; project administration, Y.L.; funding acquisition, Y.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China, grant number 41876153; Guangdong Marine Economy Development Special Project (No. GDNRC [2022]35).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** The authors gratefully acknowledge grant from the National Natural Science Foundation of China (No. 41876153) and Guangdong Marine Economy Development Special Project (No. GDNRC [2022]35).

**Conflicts of Interest:** The authors declare no conflict of interest.
