Two New Picoline-Derived Meroterpenoids with Anti-Acetylcholinesterase Activity from Ascidian-Derived Fungus Amphichorda felina

Amphichoterpenoids D (1) and E (2), two new picoline-derived meroterpenoids with a rare 6/6/6 tricyclic pyrano[3,2-c]pyridinyl-γ-pyranone scaffold, were isolated from the ascidian-derived fungus Amphichorda felina SYSU-MS7908. Their structures, including the absolute configurations, were established by extensive spectroscopic methods (1D and 2D NMR and high-resolution mass spectrometry) and ECD calculations. Compounds 1 and 2 showed anti-acetylcholinesterase (anti-AChE) activities with IC50 values of 12.5 μM and 11.6 μM, respectively. The binding interactions between 1, 2, and AChE were investigated using molecular docking analyses.


Results and Discussion
Compound 1 was isolated as a white powder. Its molecular formula was established as C 16  3) belonging to the pyridine ring (ring A), the remaining three sp 2 carbons were classified as one carbonyl group (δ C 196.1) and an olefin group (δ C 168.2, 101.6). The 1D NMR data and molecular formula indicated that 1 is an amphichoterpenoid type meroterpenoid with a tricyclic ring system [12]. Further analyses of the 1 H-1 H COSY and HMBC spectra resulted in the identification of the planar structure of 1. The 1 H-1 H COSY spectrum indicated the presence of two independent spin systems, H 2 -11/H-10/H 3 -12 and H 2 -1 /H-2 ( Figure 3). The HMBC cross-peaks from H-2 to C-3, C-4, and C-6, and from H-5 to C-3, and their chemical shifts, can establish the core fragment of the pyridine ring (unit A). Subsequently, the substructure of the γ-pyranone ring (unit B), located at C-6 of unit A, was determined by analyzing the 1 H-1 H COSY of H 2 -11/H-10/H 3 -12, and the key HMBC correlations from H-5 to C-7; from H-8 to C-6, C-7, and C-10; from H-11 to C-7; from H 3 -12 to C-9, C-10, and C-11 ( Figure 3). The 1 H-1 H COSY of H 2 -1 /H-2 and the HMBC correlations from H 2 -1 to C-3, C-4, C-5, C-2 , and C-3 ; from H 3 -5 to C-2 , C-3 and C-4 , H 3 -4 to C-2 , C-3 and C-5 along with the required one degree of unsaturation, revealed a dimethyl-substituted pyran ring (unit C) fused with unit A. Therefore, the resulting planar structure of 1 was established ( Figure 3). Further analyses of the 1 H-1 H COSY and HMBC spectra resulted in the of the planar structure of 1. The 1 H-1 H COSY spectrum indicated the presenc pendent spin systems, H2-11/H-10/H3-12 and H2-1′/H-2′ ( Figure 3). The HMB from H-2 to C-3, C-4, and C-6, and from H-5 to C-3, and their chemical shifts the core fragment of the pyridine ring (unit A). Subsequently, the substru pyranone ring (unit B), located at C-6 of unit A, was determined by analyz COSY of H2-11/H-10/H3-12, and the key HMBC correlations from H-5 to C-7; 6, C-7, and C-10; from H-11 to C-7; from H3-12 to C-9, C-10, and C-11 (Figure COSY of H2-1′/H-2′ and the HMBC correlations from H2-1′ to C-3, C-4, C-5, from H3-5′ to C-2′, C-3′ and C-4′, H3-4′ to C-2′, C-3′ and C-5′ along with the degree of unsaturation, revealed a dimethyl-substituted pyran ring (unit C) f A. Therefore, the resulting planar structure of 1 was established ( Figure 3).   Compound 2 was obtained as a white solid. The molecular formula was assigned as C 16 (Table 1) revealed that 2 possesses an identical planar structure to 1, confirmed by the extensive 2D NMR spectroscopic analysis ( Figure 3). Based on compounds 1 and 2 purified from the same fraction with different optical rotations, a minor variation (±0.03) of δ C (C-10, C-11, and C-12), their NOESY spectrum analysis ( Figure S23), and opposite Cotton effects at 320 nm in their experimental ECD spectra (Figure 4), it is speculated that they were 10-epimers.
The acetylcholinesterase (AChE) inhibitor is the primary drug target fo heimer's disease [10,32,33]. The huperzine A, physostigmine, berberine, a drugs (galanthamine and rivastigmine) were representative natural prod AchE reversible inhibitors with significant activity [34][35][36]. Here, the AChE tivities of compounds 1 and 2 were evaluated by Ellman's method and using as the positive control [12,37]. Compounds 1 and 2 exhibited AChE inhib with IC50 values of 12.5 μM and 11.6 μM, respectively, significantly less a positive control, rivastigmine (IC50, 3.9 μM). About 38% of the naturally-der (55 molecules) were considered potential AChE inhibitors with an IC50 ≤ 1 Here, we have added a new class of natural meroterpenoid alkaloids for AC Besides, because compounds 1, 2, and (+)/(−)-amphichoterpenoids A (3, 4) have the same planar structure but quite different anti-AChE activities in vi Compounds 1 and 2 are a pair of epimers exhibiting the same planar structure with only 2 chiral centers at C-10 and C-2 , suggesting four possible configurations (10S, 2 R), (10R, 2 S), (10R, 2 R) and (10S, 2 S). Thus, the absolute configurations were determined by calculating their theoretical ECD and comparing them to the experimental curves and cotton effect values. The theoretical ECD spectrums were constructed by the timedependent density functional theory (TDDFT) method at the B3LYP/6-311G** level in methanol. The predicted ECD curves of (10S, 2 R) −1 and (10R, 2 R) −2 were matched well with the experimental ones ( Figure 4). Furthermore, the experimental ECD curve of 1 was close to that of (10S, 2 R) −amphichoterpenoid B previously reported by Jiang M. et al. using X-ray diffraction, which supported the absolute configurations of 1 as 10S, 2 R ( Figure S24) [12]. So, the absolute configurations of 1 and 2 were determined as 10S, 2 R, and 10R, 2 R, respectively. Consequently, the structures of 1 and 2 were established, as shown in Figure 2, and were named amphichoterpenoids D and E.
The acetylcholinesterase (AChE) inhibitor is the primary drug target for treating Alzheimer's disease [10,32,33]. The huperzine A, physostigmine, berberine, and marketed drugs (galanthamine and rivastigmine) were representative natural products derived-AchE reversible inhibitors with significant activity [34][35][36]. Here, the AChE inhibitory activities of compounds 1 and 2 were evaluated by Ellman's method and using rivastigmine as the positive control [12,37]. Compounds 1 and 2 exhibited AChE inhibitory activity with IC 50 values of 12.5 µM and 11.6 µM, respectively, significantly less active than the positive control, rivastigmine (IC 50 , 3.9 µM). About 38% of the naturally-derived alkaloids (55 molecules) were considered potential AChE inhibitors with an IC 50 ≤ 10 µM [34,35]. Here, we have added a new class of natural meroterpenoid alkaloids for AChE inhibitors. Besides, because compounds 1, 2, and (+)/(−)-amphichoterpenoids A (3, 4) ( Figure 2) [12] have the same planar structure but quite different anti-AChE activities in vitro (Table S3), molecular docking analysis was performed to investigate the mechanism of the inhibitory effects of amphichoterpenoids on the AChE enzyme (PDB ID: 1QTI). The results ( Figure 5, Table 2) suggested that compounds 1-4 matched well in the protein-binding pocket of AChE protein, but different interactions with AChE were found. Generally, the low binding energy indicats that the active compound is easily bound to the protein. The binding energy of the AChE enzyme and 1 was −9.3 kcal mol −1 , with three hydrogen bonds and two interacting residues, Arg289 and Phe288, which was similar to that of 2 ( Figure 4, Table 2). However, the binding affinity between 3 and the AChE enzyme was −7.9 kcal mol −1 , with two hydrogen bonds and two interaction residues, Leu305 and Glu306, that was higher than that of 1, 2, and lower than that of 4 (binding energy: −6.8 kcal mol −1 , without hydrogen bond) ( Figure 5, Table 2). It is well known that there are four subsites in the inhibitor-binding gorge-like pocket of AChE, including the catalytic active site (CAS, including Ser200, His440, and G1u327), the peripheral anionic site (PAS, including Tyr70, Tyr121, Trp279, and Asp72), the hydrophobic site (or choline-binding site, including Trp84, Glu199, Phe330, and Tyr442), and the acyl pocket (including Phe288 and Phe290). The binding site between the compounds 1, 2 and AChE is not CAS but other active sites (Tyr121 and Trp279 residues in the PAS, Phe330 residue in the hydrophobic site, and Phe288 and Phe290 residues in the acyl pocket) that play an important role in the enzyme activity, while compound 3 can interact with some amino acid residues at the substrate binding site (not CAS or PAS) in the pocket of AChE to form hydrogen bonds and hydrophobic interactions, which partially inhibit AChE activity. These results further supported the different acetylcholine inhibitory activities of 1−4 in vitro.
Molecules 2022, 27, x FOR PEER REVIEW 5 of 10 two interacting residues, Arg289 and Phe288, which was similar to that of 2 ( Figure 4, Table 2). However, the binding affinity between 3 and the AChE enzyme was −7.9 kcal mol −1 , with two hydrogen bonds and two interaction residues, Leu305 and Glu306, that was higher than that of 1, 2, and lower than that of 4 (binding energy: −6.8 kcal mol −1 , without hydrogen bond) ( Figure 5, Table 2). It is well known that there are four subsites in the inhibitor-binding gorge-like pocket of AChE, including the catalytic active site (CAS, including Ser200, His440, and G1u327), the peripheral anionic site (PAS, including Tyr70, Tyr121, Trp279, and Asp72), the hydrophobic site (or choline-binding site, including Trp84, Glu199, Phe330, and Tyr442), and the acyl pocket (including Phe288 and Phe290). The binding site between the compounds 1, 2 and AChE is not CAS but other active sites (Tyr121 and Trp279 residues in the PAS, Phe330 residue in the hydrophobic site, and Phe288 and Phe290 residues in the acyl pocket) that play an important role in the enzyme activity, while compound 3 can interact with some amino acid residues at the substrate binding site (not CAS or PAS) in the pocket of AChE to form hydrogen bonds and hydrophobic interactions, which partially inhibit AChE activity. These results further supported the different acetylcholine inhibitory activities of 1−4 in vitro.

General Experimental Procedures
Optical rotations were carried out on an MCP 200 (Anton Paar, Graz, Austria) polarimeter. UV spectra were measured at a Lambda 950 UV-Vis-NIR spectrophotometer

Fungal Material
The strain was identified as Amphichorda felina (syn. Beauveria felina) SYSU-MS7908 based on the rDNA ITS sequence (GenBank NO. MT786206) [12]. The strain was preserved at Guangdong Microbial Culture Collection Center (GDMCC NO. 61059) and the School of Marine Sciences, Sun Yat-Sen University.

Extraction and Isolation
The strain A. felina SYSU-MS7908 was grown on Yeast extract Peptone Dextrose Agar at 26 • C. Then it was cut into pieces and cultivated on rice medium (40 mL rice, 40 mL water with 3% artificial sea salt, and 0.3% peptone) in 200 flasks for 28 days at room temperature. The solid fermented substrate was extracted exhaustively with MeOH three times to obtain a crude extract, then suspended in water and continuously extracted three times with EtOAc. The EtOAc extract (170 g) was fractionated to CC on silica gel (200-300 mesh) and was eluted with petroleum ether/EtOAc of increasing polarity (from 9:1 to 0:10) to obtain six fractions (A-F). Fr

Calculation of the ECD Spectra
Molecular Merck force field (MMFF) and TDDFT ECD calculations were performed with Spartan'14 software package (Wavefunction Inc., Irvine, CA, USA) and Gaussian 09 program package, respectively, using default grids and convergence criteria. MMFF conformational search generated low-energy conformers within a 10 kcal/mol energy window were subjected to geometry optimization using the DFT method at the B3LYP/6-31 G(d, p) in gas. Frequency calculations were run at the same condition to estimate their relative thermal free energies (∆G) at 298.15 K. Energies of the low-energy conformers with Boltzmann distribution over 1% in MeOH were re-calculated at the B3LYP/6-311G** level. Solvent effects were taken into account by using an IEF-PCM model. The TDDFT calculations were performed in MeOH using the B3LYP/6-311G** level for all conformers. Rotatory strengths for a total of 20-50 excited states were calculated. The ECD spectra were produced by the programs SpecDis 1.6 (University of Würzburg, Würzburg, Germany) and OriginPro 8.5 (OriginLab, Ltd., Northampton, MA, USA) using a Gaussian band shape from dipole-length dipolar and rotational strengths with 0.30 eV exponential half-width. The equilibrium population of every conformer at 298.15 K was calculated from its relative free energies using Boltzmann statistics. The calculated spectra of 1 and 2 were generated from the low-energy conformers according to the Boltzmann distribution of each conformer in the MeOH solution. All calculations were performed by Tianhe-2 in National Super Computer Center in Guangzhou.

Anti-Acetylcholinesterase Activity
The acetylcholinesterase (AChE) inhibitory activity of compounds 1 and 2 was evaluated by the modified Ellman's method with rivastigmine as a positive control [12,37]. All experiments were performed in triplicate. The detailed experiment was shown in supporting information.

Molecular Docking
The binding interaction between compounds 1-4 and AChE enzyme at the active site was investigated by molecular docking. Their 3D structures were optimized to establish the lowest energy state and saved in mol.2 file format by chem3D 16.0 software. The protein crystallographic structure of the AChE (PDB ID: 1QTI) was selected from the RCSB Protein Data Bank with 2.5 Å resolution [38]. Molecular docking was conducted using Autodocktools-1.5.6 and PyMOL-2.3.4. Autodock Vina-1.2 [39] was used to study the interaction. PyMOL-2.3.4 and LigPlot + were applied to analyze the result of the binding mode.