New Carboxamides and a New Polyketide from the Sponge-Derived Fungus Arthrinium sp. SCSIO 41421

New carboxamides, (±)-vochysiamide C (1) and (+)-vochysiamide B (2), and a new polyketide, 4S,3aS,9aR-3a,9a-deoxy-3a hydroxy-1-dehydroxyarthrinone (3), were isolated and identified from the sponge-derived fungus Arthrinium sp. SCSIO 41421, together with other fifteen known natural products (4–18). Their structures including absolute configurations were determined by detailed NMR, MS spectroscopic analyses, calculated electronic circular dichroism (ECD), as well as quantum-chemical NMR calculations. Preliminary bioactivity screening and molecular docking analysis revealed that several natural products exhibited obvious enzyme inhibitory activities against acetylcholinesterase (AChE), such as 2,3,6,8-tetrahydroxy-1-methylxanthone (4) with an inhibitory rate 86% at 50 μg/mL.


Introduction
Marine sponge derived fungi featured with easy culture and efficient productivity have been proven to be a prolific source of structurally diverse and novelty secondary metabolites [1]. Plenty of new secondary metabolites of sponge-derived fungi have been discovered with striking bioactive properties such as antifungal [2], antiviral [3], antioxidant [4], and cytotoxic [5] properties; meanwhile, their structures are diverse including polyketones, terpenes, and alkaloids [6,7].
Arthrinium sp. distributing in both terrestrial and marine habitats and comprising more than 32 species [8] produce a vast array of secondary metabolites, especially for xanthones, peptides, and terpenes [9], which showed a variety of activities covering antibacterial [9], cytotoxic [10], and antitumoral [11] activities.

Results and Discussion
Compound 1 was obtained as a colourless oil, and had the molecular formula C 10 H 15 NO 4 (four degrees of unsaturation) as determined by its HRESIMS data, which showed a protonated ion peak at m/z 214.1077 ([M + H] + ). The 1 H and 13 C NMR data (Table 1) showed one aromatic/olefinic methine (δ C/H 124.9/6.53, C-3), two methyls (δ C/H 20.6/1.15, C-11; 20.6/1.15, C-12), two methylenes (δ C/H 58.3/3.56, C-8; 58.3/3.56, C-9), two methines (δ C/H 25.1/2.71, C-10; 56.0/3.99, C-7), two carbonyls (δ C 171.4, C-2; δ C 171.2, C-5), and one hydroxy (δ H 4.79). Based on the detailed analysis of its HMBC spectrum (Figure 2), which exhibited correlations from H-3 to C-4 and C-2, from H-8 to C-7, from H-9 to C-7, and from H-10 to C11, C12, C-3, C-4, and C-5, a ring containing carboxamide was deduced. The configuration of ∆ 3 was established as Z through NOESY correlations observed between the isopropyl group and H-3 ( Figure 2). Although 1 has one stereogenic carbon (C-7), its optical rotation was close to zero and the inapparent Cotton effect in the electronic circular dichroism (ECD) spectra suggested that 1 was not enantiomerically pure. Unfortunately, with many attempts for various chiral columns and mobile phase systems, 1 could not be successfully separated and was named as a racemic (±)-vochysiamide C.  Compound 2 was obtained as a white solid and was determined to ha molecular formula C11H17NO4 from the HRESIMS data, a protonated ion peak 228.1233 ([M + H] + ). Analysis of the 1 H and 13 C NMR data (Table 1) of 2 with tho displayed almost superimposable structural relationships. The remarkable dist was the replacement of a hydrogen at C-9 in 1 with a methyl (δC/H 20.9/1.19, C-1  Compound 2 was obtained as a white solid and was determined to have the molecular formula C 11 H 17 NO 4 from the HRESIMS data, a protonated ion peak at m/z 228.1233 ([M + H] + ). Analysis of the 1 H and 13 C NMR data (Table 1) of 2 with those of 1 displayed almost superimposable structural relationships. The remarkable distinction was the replacement of a hydrogen at C-9 in 1 with a methyl (δ C/H 20.9/1.19, C-10) in 2. The observed HMBC ( Figure 2) correlations from H-10 to C-7 and from H-8 to C-9 further verified this deduction. The configuration of ∆ 3 was the same as that assigned for 1 based on the NOESY correlations ( Figure 2). Moreover, H-7/H-9 were established to adopt the opposite orientation due to the NOESY correlation between H-7/H-10.
All the isolated compounds were assessed for their enzyme inhibitory activities against acetylcholinesterase (AChE) . Compounds 3, 4, 6, 8, 11, and 15 exhibited obvious inhibition against AChE with an inhibitory rate more than 80% at 50 µg/mL, comparative to the positive control tacrine with an inhibitory rate 83.7% at 50 µg/mL ( Table 2). Among them, 2,3,6,8-tetrahydroxy-1-methylxanthone (4) showed the strongest activity relatively, with an inhibitory rate 86% at 50 µg/mL. Subsequently, molecular docking analysis was conducted to investigate the binding modes between active compounds and AChE. Compounds 2-12, 15, and 17 appeared to interact with AChE protein (PDB ID: 4EY7) perfectly with the docking scores from −6.213 to −9.383 (Table 2) (the positive ligand E20 with the docking score −12.482, and tacrine with the docking score −9.965). As shown in Figure 4, phenolic hydroxy groups of 4 (the docking score −9.383) formed four hydrogen bonds with the active site residues GLU 202, TYR 133, GLY 120, and ASP 74. Additionally, the aromatic ring of 4 formed a π-π stacking interaction with TRP 86.

Fungal Strain
The Arthrinium sp. SCSIO 41421 was isolated from a spongia sample collected from Weizhou Island, Guangxi, China, in October 2020. The strain was stored on Muller Hinton broth (MB) agar (malt extract 15 g, artificial sea salt 24 g, and agar 18 g) slants at 4 • C, and a voucher specimen was deposited in the CAS Key Laboratory of Tropical Marine Bioresources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China. It was identified as Arthrinium sp. by analysis of its ITS region of the rDNA as described in the Supporting Information (GenBank database accession no. OP022423).

Fermentation, Extraction and Isolation
The strain was cultured on MB agar plates at 25 • C for 7 days. The seed medium (malt extract 15 g, artificial sea salt 24 g in 1.0 L of tap distilled H 2 O, pH 7.4-7.8) in 1000 mL Erlenmeyer flasks (300 mL per flask) was inoculated with strain SCSIO 41421 and incubated at 25 • C for 3 days on a rotating shaker (180 rpm). Then, seed medium was inoculated into a 1000 mL × 80 Erlenmeyer flasks containing solid rice medium (200 g of rice, 6 g artificial sea salt, and 250 mL tap distilled H 2 O in each flask). After cultivation at 25 • C for 30 days, each culture broth was extracted with an equal volume of ethyl acetate three times and broken with an ultrasonic treatment apparatus for 10 min. The organic extract was then concentrated under vacuum to afford the crude extract (62.8 g). The

Measurement of AChE Inhibition Activity
AChE inhibitory activities of 1-18 were assessed according to the spectrophotometric method with slight modification. Tacrine was used as a positive control with the IC 50 value of 0.068 µM [30].

Molecular Docking
The Schrödinger 2017-1 suite (Schrödinger Inc., New York, NY, USA) was employed to perform the docking analysis. The crystal structure of AChE (PDB code: 4EY7) [32] obtained from the Protein Data Bank was used as a starting model with all of the waters and the N-linked glycosylated saccharides removed, and was constructed following the Protein Prepare Wizard workflow in Maestro 11-1. The prepared ligands were then flexibly docked into the receptor using the induced-fit module with the default parameters. The figures were generated using PyMol molecular graphics software (Schrödinger 2017-1, Schrödinger Inc., New York, NY, USA).

NMR Computational Methods
In general, conformational analyses were carried out via random searching in Spartan'14 software and Gaussian 09 software using the MMFF94 force field with an energy cutoff of 2.5 kcal/mol for compounds 2 and 3. The generated conformers were reoptimized using DFT method at the B3LYP/6-31G (d, p) level by the Gaussian 09 program. Subsequently, NMR shielding constants in PCM chloroform or DMSO were computed using the atomic orbital (GIAO) method at the B3LYP/6-31G (d, p) level in Gaussian 09. Boltzmann weights in chloroform or DMSO were computed through Molclus [33]. Shielding constants were used to perform DP4 + probability analysis [13].

ECD Computational Methods
The Molecular Merck force field (MMFF) and density functional theory (DFT)/TDDFT calculations of 2 and 3 were performed with the Spartan'14 and Gaussian 09 software, respectively, using default grids and convergence criteria. A MMFF conformational search generated low energy conformers with a Boltzmann population of over 5%, which were subjected to geometry optimization using the DFT method at the B3LYP/6-311G* level in MeOH using the IEFPCM model. The overall theoretical calculation of the ECD was conducted in MeOH using TDDFT at the B3LYP/6-311G* level for the stable conformers of 2 and 3. The ECD spectra of the different conformers were generated using the Multiwfn [34] with a half-bandwidth of 0.2-0.4 eV, according to the Boltzmann-calculated contribution of each conformer after the UV correction.