Bioactive Alkaloids from the Mangrove-Derived Fungus Nigrospora oryzae SYSU-MS0024

Chemical investigation of marine fungus Nigrospora oryzae SYSU-MS0024 cultured on solid-rice medium led to the isolation of three new alkaloids, including a pair of epimers, nigrosporines A (1) and B (2), and a pair of enantiomers, (+)-nigrosporine C (+)-3, and (−)-nigrosporine C (−)-3, together with eight known compounds (4–11). Their structures were elucidated based on extensive mass spectrometry (MS) and 1D/2D nuclear magnetic resonance (NMR) spectroscopic analyses and compared with data in the literature. The absolute configurations of compounds 1–3 were determined by a combination of electronic circular dichroism (ECD) calculations, Mosher’s method, and X-ray single-crystal diffraction technique using Cu Kα radiation. In bioassays, compound 2 exhibited moderate inhibition on NO accumulation induced by lipopolysaccharide (LPS) on BV-2 cells in a dose-dependent manner at 20, 50, and 100 μmol/L and without cytotoxicity in a concentration of 100.0 μmol/L. Moreover, compound 2 also showed moderate acetylcholinesterase (AChE) inhibitory activities with IC50 values of 103.7 μmol/L. Compound 5 exhibited moderate antioxidant activity with EC50 values of 167.0 μmol/L.

The mangrove-derived fungus Nigrospora oryzae SYSU-MS0024 was isolated from sediment, which was collected from the Zhuhai Mangrove Special Protected Area, Guangdong Province, China.Chemical investigation of its EtOAc extract led to the discovery of three new alkaloids, including a pair of epimers, nigrosporines A (1) and B (2), and a pair of enantiomers, (+)-nigrosporine C and (−)-nigrosporine C (3), together with eight known compounds (4-11) (Figure 1).Herein, the details of the isolation, structural elucidation, and bioactivities of compounds 1-11 are reported.
The mangrove-derived fungus Nigrospora oryzae SYSU-MS0024 was isolated from sediment, which was collected from the Zhuhai Mangrove Special Protected Area, Guangdong Province, China.Chemical investigation of its EtOAc extract led to the discovery of three new alkaloids, including a pair of epimers, nigrosporines A (1) and B (2), and a pair of enantiomers, (+)-nigrosporine C and (−)-nigrosporine C (3), together with eight known compounds (4-11) (Figure 1).Herein, the details of the isolation, structural elucidation, and bioactivities of compounds 1-11 are reported.

Results and Discussion
The EtOAc extract of mangrove sediment-derived fungus Nigrospora oryzae SYSU-MS0024 was performed on repeated silica gel and Sephadex LH-20 column chromatography, followed by semi-preparative HPLC to afford three new alkaloids, nigrosporines A-C (1-3), and eight known compounds 4-11.

Results and Discussion
The EtOAc extract of mangrove sediment-derived fungus Nigrospora oryzae SYSU-MS0024 was performed on repeated silica gel and Sephadex LH-20 column chromatography, followed by semi-preparative HPLC to afford three new alkaloids, nigrosporines A-C (1-3), and eight known compounds 4-11.

General Experimental Procedures
Optical rotations were carried out on an MCP-200 polarimeter (Anton Paar, Graz, Austria) with MeOH as solvent at 25 °C.UV spectra were acquired on a Blue Star A spectrophotometer.IR data were performed on a Fourier transformation infrared spectrometer coupled with an EQUINOX 55 infrared microscope (Bruker, Fällanden, Switzerland).Here, 1D and 2D NMR spectra were tested on a Bruker Avance 400 MHz spectrometer (Bruker, Fällanden, Switzerland) using TMS as an internal standard.HR-ESIMS and ES-IMS data were recorded on an LTQ-Orbitrap LC-MS spectrometer (Thermo Corporation, Waltham, MA, USA) and an ACQUITY QDA (Waters Corporation, Milford, MA, USA),

General Experimental Procedures
Optical rotations were carried out on an MCP-200 polarimeter (Anton Paar, Graz, Austria) with MeOH as solvent at 25 • C. UV spectra were acquired on a Blue Star A spectrophotometer.IR data were performed on a Fourier transformation infrared spectrometer coupled with an EQUINOX 55 infrared microscope (Bruker, Fällanden, Switzerland).

Fungal Material
The fungal strain Nigrospora oryzae SYSU-MS0024 was isolated from sediment, which was collected from the Zhuhai Mangrove Special Protected Area, Guangdong Province, China, in December 2021.This strain was identified by the sequence analysis of the rDNA ITS (internal transcribed spacer) region [29].The sequence data of the fungal strain have been deposited at GenBank with accession no.PP422138.A BLAST search result suggested that the sequence was most similar (99.81%) to the sequence of Nigrospora oryzae SYSU-MS0024 (compared to MZ151376.1).The strain was preserved at the School of Marine Sciences, Sun Yat-Sen University.

Extraction and Isolation
The strain Nigrospora oryzae SYSU-MS0024 was grown on a solid-rice medium in a 1000 mL culture flask containing 50 g of rice and 50 mL of 3% artificial seawater after sterilization.A total of 120 flasks of fungal incubation were cultivated at room temperature for 30 days.The solid fermented substrate was extracted with MeOH four times to obtain a crude extract, then dissolved in H 2 O, and continuously extracted four times with EtOAc solvent.The EtOAc extract (30 g) was subjected to a silica gel column eluting with linear gradient petroleum ether/EtOAc (from 8:2 to 0:1) to obtain six fractions (A-F).Fr

X-ray Crystallographic Analysis
Compounds 1 and 3 were obtained as colorless crystals using the vapor diffusion method.The single crystal X-ray diffraction data were recorded on a Rigaku Oxford Diffraction with Cu-Kα radiation (λ = 1.54178A).The structures were solved by direct methods (SHELXS-97 and Olex2-1.2) and refined using full-matrix least-square difference Fourier techniques [31].Crystallographic data for 1 and 3 have been deposited with the Cambridge Crystallographic Data Centre.Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: 44-(0)1223-336033, or e-mail: deposit@ccdc.cam.ac.uk).

Calculation of the ECD Spectra
The Merck molecular force field (MMFF) and density functional theory (DFT) calculations were performed using Spartan'14 software from Wavefunction Inc. (Irvine, CA, USA) and the Gaussian 09 program, respectively [32].Conformers were subjected to DFT geometry optimizations at the B3LYP/def2-SVP level with the solvation model PCM for methanol.

12 Figure 6 .
Figure 6.X-ray crystallographic analysis and molecular packing properties of 3.