Pestalotiopols E–J, Six New Polyketide Derivatives from a Marine Derived Fungus Pestalotiopsis sp. SWMU-WZ04-1

Chemical epigenetic cultivation of the sponge-derived fungus Pestalotiopsis sp. SWMU-WZ04-1 contributed to the identification of twelve polyketide derivatives, including six new pestalotiopols E–J (1–6) and six known analogues (7–12). Their gross structures were deduced from 1D/2D NMR and HRESIMS spectroscopic data, and their absolute configurations were further established by circular dichroism (CD) Cotton effects and the modified Mosher’s method. In the bioassay, the cytotoxic and antibacterial activities of all compounds were evaluated. Chlorinated benzophenone derivatives 7 and 8 exhibited inhibitory effects on Staphylococcus aureus and Bacillus subtilis, with MIC values varying from 3.0 to 50 μg/mL. In addition, these two compounds were cytotoxic to four types of human cancer cells, with IC50 values of 16.2~83.6 μM. The result showed that compound 7 had the probability of being developed into a lead drug with antibacterial ability.


Introduction
Marine fungi provide a prospective source for the discovery of novel drug leads [1].Traditional methods used in the investigation of marine fungi mainly focus on sample collection, the cultivation of fermentation broth, and mycelium.However, it is becoming difficult to discover novel structurally and biologically active metabolites using these traditional methods, but the development of genetic technology can assist [2].Research findings demonstrate that a large portion of gene clusters are silenced under standard fermentation conditions [3][4][5].To explore these potential secondary metabolites, activation of silent biosynthetic gene clusters has become an important strategy.At present, the silent gene clusters have been activated through multiple strategies, such as the OSMAC strategy [6][7][8][9][10], epigenetic modification [6,11,12], and genome mining.Of these, the chemical epigenetic strategy has proved to be an effective strategy for enhancing cryptic secondary metabolites [13].
Mar. Drugs 2024, 22, x 2 of 12 [19], were obtained from sponge-derived fungi Pestalotiopsis sp. 1 H and 13 C assignments of the pestalotiopols E-J (1-6) were accomplished, the absolute configurations of the compounds were determined by CD techniques, and furthermore, the biological activities of the separated organic molecules were assessed.

Results
Compound 1 appeared as a white solid, and its molecular formula was C 19 H 26 O 6, whose quasi-molecular ion peak was located at m/z 373.1635 [M + Na] + by HRESIMS.The 1 H NMR spectrum (Table 1) showed two aromatic proton signals [δ H 6.60 (d, J = 8.1 Hz, H-4), 6.87 (d, J = 8.1 Hz, H-5)], which belong to a 1,2,3,4-tetrasubstituent benzene, and one olefinic proton signal [δ H 5.57 (brt, J = 7.2 Hz, H-2')].The 13 C NMR spectrum of compound 1 showed 19 carbon resonances (Table 1), involving three CH 3 , four CH 2 , three sp 2 -CH carbons, and five not-containing proton carbons.One carbonyl carbon was proposed based on resonances at δ C 172.8, and five oxygenated carbon signals at δ C 71.0, 71.0, 71.7, 73.3, 82.3.The 1 H NMR data was initially analyzed, and it was found that 1 had similar structural characteristics to heterocornol L [20].The major difference was that a carbonyl signal was missing and an oxygenated methine was present in 1.This was proved by the HMBC correlations between H-10 (δ H 3.71) and C-11 (δ C 71.7), C-9 (δ C 39.1), and C-12 (δ C 18.9).The location of the vicinal diol side chain was confirmed by HMBC correlations from H-8 (δ H 5.53) to C-7 (δ C 143.4.), C-6 (δ C 125.9), C-10 (δ C 73.3), C-1 (δ C 71.0), and 1 H-1 H COSY correlations of H-8/H-9/H-10/H-11/H-12 (Figure 2).Furthermore, HMBC correlations found H-1'(δ H 3.27) to C-5' (δ C 71.0), C-2' (δ C 128.8), C-3' (δ C 132.3), and C-7 (δ C 143.4), together with 1 H-1 H COSY correlations located connections from H-1'/H-2' suggested the existence of 5'-O-acetyl isoamylene group located at C-6 in 1 (Figure 2).Therefore, the planar structure of 1 was established.To establish its relative configuration, the NOESY spectrum of 1 was performed; unfortunately, the NOESY spectrum of 1 was not useful for the assignment of the relative configuration at C-10 and C-11.According to the previously reported [21][22][23][24], in 3rythron vicinal diols (J > 4.0 Hz), the value of the coupling constant of the methine hydrogens was larger than 4 Hz, while in threo ones (J < 2.0 Hz), it is smaller than 2 Hz.On this account, consistent with the known pyriculin A and B [25] (J = 3.7/3.8Hz), heterocornol I [20], indicating that the configurations of C-10 and C-11 in 1 (J = 4.7 Hz) were suggested as erythro.The absolute configuration of 1 (8S,10R,11S) was elucidated from the observed positive Cotton effect at 210 nm and positive Cotton effect at 313 nm in the CD and Mo2(AcO)4-induced CD spectra of 1, respectively (Figure 3) [19].Compound 2 was purified as a white solid with the molecular formula C19H26O6, determined from the HRESIMS data.The NMR data of 2 were very similar to those of 1, To establish its relative configuration, the NOESY spectrum of 1 was performed; unfortunately, the NOESY spectrum of 1 was not useful for the assignment of the relative configuration at C-10 and C-11.According to the previously reported [21][22][23][24], in rythron vicinal diols (J > 4.0 Hz), the value of the coupling constant of the methine hydrogens was larger than 4 Hz, while in threo ones (J < 2.0 Hz), it is smaller than 2 Hz.On this account, consistent with the known pyriculin A and B [25] (J = 3.7/3.8Hz), heterocornol I [20], indicating that the configurations of C-10 and C-11 in 1 (J = 4.7 Hz) were suggested as erythro.The absolute configuration of 1 (8S,10R,11S) was elucidated from the observed positive Cotton effect at 210 nm and positive Cotton effect at 313 nm in the CD and Mo 2 (AcO) 4 -induced CD spectra of 1, respectively (Figure 3) [19].Compound 2 was purified as a white solid with the molecular formula C 19 H 26 O 6 , determined from the HRESIMS data.The NMR data of 2 were very similar to those of 1, which manifests that both of them had the same planar structures.Compared with compound 1, the configuration at C-10 and C-11 in the vicinal diol side chain was discrepant, which was proved by the chemical shifts of C-8 (∆δ C 1.1 ppm), C-9 (∆δ C −1.8 ppm), C-10 (∆δ C 1.1 ppm), and C-11 (∆δ C −1.8 ppm).According to the chemical shift of C-10 (δ C 74.4) and C-11 (δ C 69.9) [26], and the coupling constant between H-10 and H-11 (1.6 Hz), the configuration of C-10 and C-11 was inferred to be threo.We speculated that 2 had the same configuration at C-8 (8S) as 1 according to their closely similar CD spectrum.The absolute configuration of C-10,11-diols in 2 was determined by Mo 2 (AcO) 4 -induced CD (Figure 3).
In order to further confirm the absolute configuration of 2, the modified Mos method was performed (2a and 2b) [27][28][29][30] (Figures S36 and S37).biogenetic considerations, we suggested that the absolute configuration of 4 was the 8S, 10R configuration.These assignments were further confirmed by the CD spectrum of 4 (Figure 3).Thus, we determined the structure of 4 and named it pestalotiopol H. Compounds 5 and 6 were acquired as a mixture in a nearly 1:1 ratio, which was established by their HRESIMS and the 13  The absolute configurations of C-8 and C-11 in 5 and 6 were assigned 8R, 10R, and 8S, 10R, respectively, which were consistent with other analogs from the marine fungus Pestalotiopsis vaccinii [31].Considering that two strains belong to the same genus, Pestalotiopsis, we proposed that they arose from the same biosynthetic pathway and shared the chiral center.
In the present study, polyketide derivatives 1-12 were evaluated for their cytotoxic activities via MTT assay (Table 3).The cytotoxicity results demonstrated that compound 7 was mildly cytotoxic to HepG2, with IC 50 values of 16.2 µM; the IC 50 values of 8, which was weakly cytotoxic to human cancer cells, ranged from 34.8 to 63.1 µM; unfortunately, compounds 1-6, at concentrations of 100 µM, did not show cytotoxicity against these tumor cell lines.Bacteriostatic effects 1-12 were performed (Table 3).Compounds 7 and 8 showed antibacterial effects on Staphylococcus aureus and Bacillus subtilis, with MIC values varying from 3 to 50 mg/mL.No obvious bioactivities were found in the other compounds at 100 µM or 100 mg/mL.The results showed that there was considerable potential to develop compound 7 as a lead drug with antimicrobial activity.
In order to further verify the activity of compounds 7 and 8 against HepG2, the anti-apoptotic protein Bcl-2 (PDB ID: 4LVT) was used as a target for molecular docking.Compounds 7 and 8 bound to ASN9, TRP192, ILE186, TRP141, PHE195, TRP141, LEU198, TYR199, and GLN187 residues and formed hydrogen bonds and non-polar interactions, respectively.Noticeably, the docking results showed that compounds 7 and 8 were well matched in the docking body of 4LVT and had good interaction with 4LVT.The binding free energies of systems were negative (−5.11 kcal/mol and −6.87 kcal/mol, respectively), indicating that the binding of proteins and small molecular ligands was a spontaneous process.
Pestalotiopsis, we proposed that they arose from the same biosynthetic pathway shared the chiral center.
Biosynthetically, a precursor polyketide substance is commonly produced microorganisms in the synthesis of aromatics and macrolides.Compounds (1-6, 11 originated from malonyl-CoA by reduction, dehydration, cyclization, and oxidatio form the different polyketide precursors and subsequently formed compounds (1-6 12) after diverse transformations (Scheme 1).In the present study, polyketide derivatives 1-12 were evaluated for their cyto activities via MTT assay (Table 3).The cytotoxicity results demonstrated that compo 7 was mildly cytotoxic to HepG2, with IC50 values of 16.2 μM; the IC50 values of 8, w was weakly cytotoxic to human cancer cells, ranged from 34.8 to 63.1 μM; unfortuna compounds 1-6, at concentrations of 100 μM, did not show cytotoxicity against t tumor cell lines.Bacteriostatic effects 1-12 were performed (Table 3).Compounds 7 a showed antibacterial effects on Staphylococcus aureus and Bacillus subtilis, with MIC va varying from 3 to 50 mg/mL.No obvious bioactivities were found in the other compou at 100 μM or 100 mg/mL.The results showed that there was considerable potenti develop compound 7 as a lead drug with antimicrobial activity.
In order to further verify the activity of compounds 7 and 8 against HepG2, the apoptotic protein Bcl-2 (PDB ID: 4LVT) was used as a target for molecular dock Compounds 7 and 8 bound to ASN9, TRP192, ILE186, TRP141, PHE195, TRP141, LEU TYR199, and GLN187 residues and formed hydrogen bonds and non-polar interact respectively.Noticeably, the docking results showed that compounds 7 and 8 were

General Experimental Procedures
An MCP500 polarimeter (Anton) was used to record optical rotations in CH 3 OH.IR and HRESIMS were performed on a Shimadzu IR and a Bruker maXis TOF-Q mass spectrometer, respectively.Two different Bruker spectrometers (AVANCE III-400 and AV-600) were used for the NMR data collection, and the NMR data were recorded using TMS as an internal standard, d in ppm rel at 25 • C. Column chromatography involved normal-phase silica gel (100-300 mesh), Sephadex LH-20 (MeOH), and reversed-phase YMC ODS-A (50 µm) being used, while precoated silica gel GF254 plates (0.20-0.25 mm in thickness) were used for thin-layer chromatography (TLC) analyses, and the spots were visualized by UV light (254 nm) and colored by spraying heated silica gel plates with 10% H 2 SO 4 in ethanol.Preparative HPLC was conducted on a SAIPURUISHE system equipped with a UV detector, an ODS column (YMC-5µm, ODS-A, 250 mm × 10 mm), a flow rate of 2.0 mL/min, and a column temperature of 25 • C. Circular dichroism (CD) spectra were recorded on a Chirascan circular dichroism spectrometer.

Fungal Material
The strain (SWMU-WZ04-1) was obtained from the sponge collected on Weizhou Island, China, and identified as Pestalotiopsis sp.SWMU-WZ04-1 by sequence alignment of the 18S rRNA gene.During its initial growth stage on a PDA plate, the mycelium of Pestalotiopsis sp.SWMU-WZ04-1 showed a french grey, which gradually transitioned to a brown color.As growth progressed, the mycelium produced conidia along with small oil droplets on the surface.
The crude extract was fractionated on a normal-phase column using a stepped gradient elution with petroleum ether/EtOAc (30: 1 to 0: 1, v/v) to obtain 8 fractions (Fr.1-Fr.8).Fr.3 was applied to a normal-phase column (petroleum ether/EtOAc, 15:1-3:1) to obtain three subfractions (Frs.The Mo 2 (AcO) 4 solution was prepared with DMSO; subsequently, compounds 1 and 2 were added to the stock solution, respectively.The CD spectra of compounds 1 and 2 were recorded immediately after every 10 min for 30 min to form stationary Mo 2 (AcO) 4 -induced CD spectra [32].

Cytotoxicity Assay
The MTT method was applied to analyze the cytotoxicity of compounds 1-12 against four cancer cell lines, involving (7860), (HepG2), (H1975), and (Hela).The MTT assay was depicted as a previously used method [37].The cells were seeded in complete medium per well within a 96-well plate.The cells were incubated at 37 • C for 12-24 h to facilitate adherent cell growth.Following this, compounds 1-12 solutions were added, and cells were cultured for 48 h.Subsequently, MTT solution was introduced into the wells and incubated for 4 h at 37 • C. DMSO was added to each well after the cell medium was discarded.The absorbance was measured at a wavelength of 570 nm.

Antimicrobial Assay
The antimicrobial assay against three bacteria (Bacillus subtilis, Staphylococcus aureus, and Escherichia coli) and one fungus (Candida albicans) was assessed for adopting the microbroth dilution reported previously [38].The MIC was defined as the lowest concentration of the antimicrobial agent that completely inhibited the visual growth of an organism.Ciprofloxacin and amphotericin B were used as positive controls against bacteria and fungi, respectively.

Molecular Docking
The molecular docking of compounds 7 and 8 was performed (Figure 4).The initial models for Bcl-2 (PDB ID:4LVT) was gained from the Protein Data Bank (http://www.rcsb.org,accessed on 16 November 2023).The 3D structures of compounds 7 and 8 were obtained from ChemBio 3D Ultra 14.0.AutoDock Vina (Center for Computational Structural Biology, La Jolla, the US, accessed on 16 November 2023), and AutoDockTools-1.5.6 were used to generate docking input files [39].The docking results were then analyzed for interaction patterns using PyMOL 2.3.0.
C , Type δ H (J in Hz) δ C , Type δ H (J in Hz) δ C , Type δ H (J in Hz)

Table 3 .
Antibacterial activities and cytotoxicity of 1