Bioactive Polyketides and Benzene Derivatives from Two Mangrove Sediment-Derived Fungi in the Beibu Gulf

To discover bioactive natural products from mangrove sediment-derived microbes, a chemical investigation of the two Beibu Gulf-derived fungi strains, Talaromyces sp. SCSIO 41050 and Penicillium sp. SCSIO 41411, led to the isolation of 23 natural products. Five of them were identified as new ones, including two polyketide derivatives with unusual acid anhydride moieties named cordyanhydride A ethyl ester (1) and maleicanhydridane (4), and three hydroxyphenylacetic acid derivatives named stachylines H–J (10–12). Their structures were determined by detailed nuclear magnetic resonance (NMR) and mass spectroscopic (MS) analyses, while the absolute configurations were established by theoretical electronic circular dichroism (ECD) calculation. A variety of bioactive screens revealed three polyketide derivatives (1–3) with obvious antifungal activities, and 4 displayed moderate cytotoxicity against cell lines A549 and WPMY-1. Compounds 1 and 6 at 10 μM exhibited obvious inhibition against phosphodiesterase 4 (PDE4) with inhibitory ratios of 49.7% and 39.6%, respectively, while 5, 10, and 11 showed the potential of inhibiting acetylcholinesterase (AChE) by an enzyme activity test, as well as in silico docking analysis.


Introduction
The mangrove wetland ecosystems, located at tropical and subtropical intertidal estuarine zones, possess rich biodiversity and include an enormous diversity of microorganisms [1]. From these, a vast range of fungi species in mangrove sediment play a vital role in biogeochemical cycles to sustain the mangrove wetland ecosystems [2]. Mangrove sediment-derived fungi are widely considered to be a pivotal and prolific reservoir of structurally unique and biologically active secondary metabolites with promising medicinal, agricultural, or industrial applications [2,3].
The genus Talaromyces is widely distributed in marine environments, soil, plants, and foods. The extreme living conditions have led the fungi to develop more specific metabolic patterns, and marine-derived Talaromyces spp. can produce a number of structurally diverse substances with a wide range of bioactivities, such as anti-inflammatory meroterpenoids, thioester containing benzoate derivatives that exhibit α-glucosidase inhibitory activity, and oxaphenalenone dimers with broad antibacterial activity [4].
Penicillium species are among the most widespread fungal organisms on earth and contains more than 350 species. Many Penicillium species can produce plentiful secondary metabolites, such as alkaloids [5], polyketides [6] and terpenoids [7], that can ascribe specific structural characteristics and significant biological activities.
Compound 4 was isolated as a yellow oil. Its molecular formula was established as C 13 (Table 1) with 1 revealed that 4 shared part of the structure of 1, with one acid anhydride moiety. By further analysis of the chemical shift, the coupling constant and the molecular formula, 4 was determined as shown in Figure 1. The structure had been reported as a synthetic product in a patent without NMR data and trivial name [11]. Herein, it was discovered as a new natural product and named maleicanhydridane (4).
Compounds bearing acid anhydride moieties are rare in nature. Cordyanhydrides A and B, bearing two and three acid anhydride moieties, were originally described from the insect pathogen fungus Cordyceps pseudomilitaris [12] and Amazonian endophytic Talaromyces fungi [10], with the absolute configuration unsolved. To the best of our knowledge, this study is the first example to obtain cordyanhydride derivatives from marinederived microbes. In addition, 4 should be a precursor compound for the biosynthesis of 1-3.
Compound 5 has been reported as a fungal metabolite [13] and was isolated as an epimer at C-7 of 7-epiaustdiol (6) [14] with almost identical NMR data. The absolute configurations of 7-epiaustdiol (6) and 8-O-methylepiaustdiol (7) were determined as shown in Figure 1 (Tables S1 and S2), which led to the determination of the 7R,8S absolute configuration of 5 ( Figure 3). Thus, 5 was assigned as (7R, 8S)-austdiol (5).  (Table 2) (Table 2) of 10 displayed 14 carbon resonances consistent with four non-protonated carbons (including three sp 2 carbons and a carbonyl), five sp 2 methine groups, three methylene groups, and two methyl groups (one of them oxygenated). The 1 H-1 H COSY spectrum ( Figure 2) indicated the presence of three independent spin systems of H-2/H-3, H-5/H-6, and H 2 -10/H-11. Comparison of its NMR data with those of stachyline G from Mortierella sp. revealed closed similarities except for the presence of an additional oxygenated methyl signal [15]. Extensive analysis of its HMBC spectrum revealed key signals from H 3 -9 to C-8, indicating that the methoxy was linked to C-8 and formed a methyl ester group. The geometric configuration of the ∆ 10,11 double bond was determined to be in Z configuration by the NOESY correlations ( Figure 2) of H 2 -13 with H 2 -10, and H 3 -14 and H-11. The gross structure was constructed as shown in Figure 1 and named stachyline H (10).  Compound 11 was isolated as a brown oil, and its molecular formula was designated as C 14  Comparison of NMR spectroscopic data of 11 with 10 indicated that they shared the same planar structures, supported by the HMBC and COSY correlations ( Figure 2). Upon, detailed interpretation of its 13 C NMR data, we found that the C-13 resonance was shielded from δ C 21.0 in 10 to δ C 13.8 in 11, while the C-14 resonance was deshielded from δ C 59.8 in 10 to δ C 65.4 in 11. The above obvious differences in the chemical shifts at C-13 and C-14 hinted that the geometric configuration of the ∆ 10,11 double bond is different in both compounds. The NOESY spectrum ( Figure 2) of 11 revealed key signals of H 3 -14 with H 2 -10, and H 2 -13 and H-11, indicating the double bond ∆ 10,11 was in E configuration. Accordingly, it was elucidated and named stachyline I (11).  Table 2) (Table 2) of 12 displayed 14 carbon resonances consistent with four non-protonated carbons (including one sp 3 carbon, two sp 2 carbons and a carbonyl), five sp 2 methine groups (including one oxygenated and four sp 2 hybridized), two methylene groups (one of them oxygenated), and three methyl groups (one of them oxygenated). Analysis of its NMR data revealed that the structure of 12 closely resembled that of 10. The difference was the replacement of signals for the 4-hydroxy-2-en-3-methylbutoxy unit substituted at the C-4 position in 10 with those for the 2,3-dihydroxy-3-methylbutoxy moiety in 12. The 1 H-1 H COSY correlation of H 2 -10/H-11 and the HMBC correlations of H 2 -10 with C-4 and C-11, H-11 with C-12, and H 3 -13/H 3 -14 with C-11 and C-12 support the above deduction. In addition, the planar structure of 12 was similar to the known compound stachyline E except for the presence of an additional oxygenated methyl [15]. The recorded optical rotation for 12 was [α] 25 D −4 (c 0.1, CH 3 OH), which has the same angle as that of stachyline E, [α] 23 D −6 (c 0.1, CH 3 OH), suggesting that the configuration of C-11 in 12 is the same as that of stachyline E. Consequently, the structure of 12 was determined and assigned stachyline J (12). Compounds 10-12 may be separation artifacts.
In order to further understand the interaction between the compounds and AChE protein, so as to improve the activity by structure optimization in the future, docking studies were carried out for 5, 10, and 11 in the active site of AChE (PDB: 1UT6) to gain insights into their molecular interactions. As a result, these ligands were favorably accommodated within the binding cleft with analogous anchoring conformations, exhibiting binding free energies (designated as S value) spanning from −8.7 to −8.4 kcal/mol. Compounds 5, 10, and 11 interacted with the AChE active site mainly through hydrogen bonds, π-π stacking contacts, and hydrophobic interactions (Figure 4). Compound 5 formed hydrogen bonds with amino acid residues TYR121 and SER122 within the target protein at distances of 3.6 Å and 3.0 Å, respectively. It also exhibited π-π stacking contacts with TRP84 and PHE330, as well as hydrophobic interactions with TRP84, PHE330, ILE439, and TYR442. Compound 10 established hydrogen bonds with TRP84, GLY118, GLY119, SER200, TRP432, and HIS440 at distances of 2.9 Å, 3.5Å, 3.0Å, 2.8Å, 3.2Å, and 3.9Å, respectively. It also showed π-π stacking contacts with TRP84 and PHE330, in addition to hydrophobic interactions with TRP84, TYR121, PHE290, PHE330, PHE331, ILE439, and TYR442. Compound 11 formed hydrogen bonds with TRP84, SER122, GLY123, and TRP43 at distances of 2.9 Å, 3.5Å, 3.3Å, and 3.3Å, respectively. It also exhibited π-π stacking contacts with TRP84 and PHE330, as well as hydrophobic interactions with ASP72, TRP84, TYR121, PHE330, ILE439, and TYR442. The binding of these compounds to the enzyme was stabilized through these interactions.

General Experimental Procedures
The UV spectrum was recorded on a Shimadzu UV-2600 PC spectrometer (Shimadzu, Beijing, China). The IR spectrum was obtained using an IR Affinity-1 spectrometer (Shimadzu). Optical rotations were determined with an Anton Paar MPC 500 polarimeter. HRESIMS spectra were recorded with a Bruker maXis Q-TOF mass spectrometer. The NMR spectra were recorded on a Bruker Avance-500 spectrometer (Bruker BioSpin International AG, Fällanden, Switzerland), and chemical shifts were recorded as δ-values. Semipreparative high-performance liquid chromatography (HPLC) was performed on the Hitachi Primaide with a DAD detector, using an ODS column (YMC-pack ODS-A, 10 × 250 mm, 5 µm). Thin-layer chromatography analysis (TLC) and column chromatography (CC) were carried out on plates precoated with silica gel GF254 (10-40 µm) and over silica gel

Fungal Material
The fungal strains SCSIO 41050 and SCSIO 41411 were isolated from a mangrove sediment sample, collected from Gaoqiao mangrove wetland (21.

Fermentation and Extraction
The fungal strains were cultured in 200 mL seed medium (15 g malt extract, 10g sea salt, 1 L H 2 O) in 500 mL Erlenmeyer flasks at 28 • C for 3 days on a rotary shaker (180 rpm). Large-scale fermentations of SCSIO 41050 and SCSIO 41411 were incubated statically at 25 • C for 30 days using a rice medium (200 g rice, 2.5% sea salt, 230 mL H 2 O) in the 1 L flask (×60 and ×45, respectively). The fermented culture was extracted three times with EtOAc, yielding a reddish extract (130 g) and a brown extract (53.2 g), respectively.

Spectroscopic Data of New Compounds
Cordyanhydride A ethyl ester (1)

ECD Calculation of 5
Conformational analyses were carried out via Monte Carlo searching by means of Spartan'14 software (v1.1.4, Wavefunction, Irvine, CA, USA) using a Molecular Merck force field. The results showed ten lowest energy conformers within an energy window of 14 Kcal/mol. Then, these conformers were further re-optimized by the TD-DFT method at the B3LYP/6-31G(d) level in methanol using the Gaussian 16 program (A.03, Guassian, Pittsburgh, PA, USA) [27]. ECD calculations were further carried out at the B3LYP/6-311+G (d, p) level in methanol by adopting 50 excited states. The ECD spectra were generated based on Boltzmann distribution theory by SpecDis (1.70.1, SpecDis, Berlin, Germany) under a half band width of 0.3 eV and shifted by −25 nm to facilitate comparison to the experimental data.

Cytotoxicity Bioassay
Cytotoxicities against PC-3 (human prostate cancer cell line), 22Rv1 (human prostate cancer cell line), WPMY-1 (human prostatic stromal myofibroblast cell line), and A549 (human lung cancer cell), purchased from Shanghai Cell Bank, Chinese Academy of Sciences, were evaluated. Cell viability was analyzed by 3-(4,5)-dimethylthiahiazo (-z-yl)-3,5-diphenytetrazoliumromide (MTT) assay as previously described [29]. In brief, cells were seeded in a 96-well plate at a density of 5 × 10 3 per well overnight and treated with compounds for the required time. OD 570 values were detected using a Hybrid Multi-Mode Reader (Synergy H1, BioTek, Santa Clara, CA, USA). The experiment was independently repeated three times.

NF-κB Bioassay
The suppression of LPS-induced NF-κB activation in RAW264.7 cells was assessed using a luciferase reporter gene assay as detailed previously [30].

Enzyme Inhibitory Activities Assay
The protocols for expression, purification, and enzymatic assays of PDE4D2 were similar to those we described previously [26]. The inhibitory activity of AChE was assessed in vitro following a modified Ellman method [31].

Molecular Docking
The molecular docking simulation was implemented by utilizing the software AutoDock Tools (ADT 1.5.6) [32]. The crystal structure of AChE from Tetronarce californica (PDB ID: 1UT6) [33] was acquired from the Protein Data Bank (http://www.rcsb.org, accessed on 21 April 2005). The structures of ligands were generated in ChemBioOffice 18.0 (PerkinElmer Informatics, Waltham, MA, USA), followed by an MM2 calculation to minimize the conformation energy. The size of the grid box was 2.3 × 62.7 × 55.6, centered at x: 31.1, y: 27.7, z: 50.7. The other docking parameters, settings, and calculations were default, and the docking results were analyzed using the software PyMOL 2.4.0 (Schrödinger, New York, NY, USA).

Conclusions
In conclusion, the chemical investigation of the two mangrove-sediment fungal stains Talaromyces sp. SCSIO 41050 and Penicillium sp. SCSIO 41411 afforded 23 different compounds. Among these, cordyanhydride A ethyl ester (1) and stachylines H-J (10-12) were identified as new compounds, and maleicanhydridane (4) as a new natural product. Although some natural maleic anhydrides have been reported from Talaromyces species [34,35], the discovery of three cordyanhydride derivatives (1-3) with obvious antifungal activities was impressive. Other active natural products were also revealed, such as 4 with moderate cytotoxicity against cell lines A549 and WPMY-1, 1 and 6 with PDE4 inhibitory activities, and 5, 10, and 11 with potential for inhibiting AChE. The obtained results highlight the immense potential of the mangrove wetland ecosystem to yield novel natural products as well as bioactive compounds.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.