Structurally Diverse Polycyclic Salicylaldehyde Derivative Enantiomers from a Marine-Derived Fungus Eurotium sp. SCSIO F452

To enlarge the chemical diversity of Eurotium sp. SCSIO F452, a talented marine-derived fungus, we further investigated its chemical constituents from a large-scale fermentation with modified culture. Four pairs of new salicylaldehyde derivative enantiomers, euroticins F-I (1–4), as well as a known one eurotirumin (5) were isolated and characterized. Compound 1 features an unprecedented constructed 6/6/6/5 tetracyclic structures, while 2 and 3 represent two new types of 6/6/5 scaffolds. Their structures were established by comprehensive spectroscopic analyses, X-ray diffraction, 13C NMR, and electronic circular dichroism calculations. Selected compounds showed significant inhibitory activity against α-glucosidase and moderate cytotoxic activities against SF-268, MCF-7, HepG2, and A549 cell lines.


Introduction
Natural product discovery from marine-derived fungi has attracted more and more attention in recent decades. An increasing number of secondary metabolites with intriguing scaffolds and promising bioactivities have been isolated and characterized from marine fungi [1][2][3]. However, an inevitable problem of duplicated isolation of some common compounds has also emerged, resulting in wasted of time and labor. To improve the discovery efficiency of novel skeletal compounds, various strategies like genome mining and heterologous expression have been developed [4]. These strategies focus on manipulating genes responsible for the synthesis of secondary metabolites and employing heterologous expression or in vitro enzymatic studies to mine novel scaffolds. However, gene manipulation and bioinformatic analysis may deter natural-product chemists. Therefore, more traditional approaches, including One Strain Many Compounds (OSMAC) and large-scale culture are employed to enlarge the chemical space of microorganism [5]. The OSMAC can be better accumulated and isolable [5,7]. Therefore, it can serve as a complementary method to OSMAC, enabling natural product chemists to access more chemical entities.
Compound 5 was isolated as a yellow solid. It was identified as eurotirumin [19] by comparison of HRMS and NMR data (Table S3). It was not demonstrated as a racemate or not. In our separation, we proposed it to be a racemate because of its low optical rotation. Then we successfully separated it by chiral HPLC (Figures S4 and S5) and fortunately obtained suitable crystals of (−)-5 from MeOH for single-crystal X-ray diffraction experiment, which confirmed its planar and absolute configuration as 1"R,2"R,5"R,6"S with the Flack parameters of −0.06(15) (CCDC 2087463) (Figure 7). Thus, (−)-5 can be assigned as 1"S,2"S,5"S,6"R.

Proposed Biosynthesis Pathway
The putative biosynthetic pathway of 1-5 is proposed in Scheme 1. For 1, intermediate Ia was formed by polyketide pathway from the starting precursor (one acetyl-CoA, seven malonyl-CoA) and cyclization. Then Ia underwent prenylation to produce IIa. A

Proposed Biosynthesis Pathway
The putative biosynthetic pathway of 1-5 is proposed in Scheme 1. For 1, intermediate Ia was formed by polyketide pathway from the starting precursor (one acetyl-CoA, seven malonyl-CoA) and cyclization. Then Ia underwent prenylation to produce IIa. A series of selective reduction, dehydration, hydrogenation, and oxidation of IIa could produce IIIa, which went through nucleophilic addition and oxidation to become Va. Another nucleophilic addition of Va would produce compound 1 [17] For 2-5, a similar PKS pathway with one acetyl-CoA and six malonyl-CoA and prenylation gave IIb, which could generate three different precursors (IIIb1, IIIb2, IIIb3) for three different routes. In route 1, an aldol condensation of IIIb1 furnished IVb1, which can be oxidized to Vb1. Subsequent nucleophilic addition occurred between 2"-OH and C-7 or 5-OH and C-6" would produce compounds 2 and 5, respectively. In route 2, IIIb2 can transform to Vb2 by oxidation and aldol concentration. Dehydration and oxidation of Vb2 could yield compound 3. In route 3, decarbonylation of IIIb3 gave IVb3 [25]. It can transform to Vb3 by tautomerism with loss of one H 2 . Another decarbonylation of Vb3 yielded VIb3, which went through oxidation and reduction to furnish compound 4.

Purification
The EtOAc extract (356 g) was subjected to a silica gel column using step gradient elution with CH 2 Cl 2 /Acetone (1:0 to 0:1) and yielded five fractions Frs.

ECD and 13 C NMR Calculation Methods
The theoretical calculations of 13 C NMR of 1 and ECD of 1, 2, and 4 were carried out using the Gaussian 09 [27] and ORCA 4.2.1 [28,29] software packages. Conformational analysis was initially performed using Spartan'14 (Wavefunction, Irvine, CA, USA). More details about the experimental procedures and the optimized conformation geometries, thermodynamic parameters, and populations of all conformations are provided in Supporting Information.
3.6. Cytotoxicity, Antioxidative, and α-Glucosidase Inhibitory Activity, and Antimicrobial Activity Assays 3.6.1. Cytotoxicity Assay The cells of SF-268 (human glioblastoma carcinoma), MCF-7 (breast cancer), HepG-2 (liver cancer), and A549 (lung cancer) were purchased from Stem Cell Bank, Chinese Academy of Sciences. The cells were cultured in DMEM medium (Gibco) containing 10% fetal bovine serum (Gibco) at 37 • C in a humidified atmosphere with 5% (v/v) CO 2 . The cells were incubated in cultural flasks until sub-confluent (~80%). Then, cells (180 µL) with a density of 3 × 10 4 cells/mL of media were seeded onto 96-well plates and incubated for 24 h at 37 • C, 5% CO 2 . Subsequently, 20 µL of different concentrations of compounds ranging from 1 to 128 µM in DMSO were added to each plate well. Equal volume of DMSO was used as a negative control. The plates were further incubated for 72 h. After incubation, cell monolayers were fixed with 50% (wt/v) trichloroacetic acid (50 µL) and stained for 30 min with 0.4% (wt/v) SRB dissolved in 1% acetic acid. Unbound dye was removed by washing repeatedly with 1% acetic acid. The protein-bound dye was dissolved in 10 mM Tris base solution (200 µL) for OD determination at 570 nm using a microplate reader. Adriamycin was used as positive control possessing potent cytotoxic activity. All data were obtained in triplicate and presented as means ± SD. IC 50 values were calculated with the Sigma Plot 10.0 software (Systat Software Inc., CA, USA) using a non-linear curve-fitting method [17].

α-Glucosidase Inhibitory Activity Assay
Inhibitory α-glucosidase activities were determined spectrophotometrically in a 96well microtiter plate based on p-nitrophenyl-α-D-glucopyranoside (PNPG) as a substrate. In brief, 20 µL 0.2 U/mL α-glucosidase enzyme solution, 50 µL 0.1 mol/L PBS (pH 6.8), and 10 µL of the test compounds in DMSO were mixed and preincubated at 37 • C prior to initiation of the reaction by adding the substrate. After 10 min of preincubation, 20 µL 5 mmol/L PNPG solution was added and then incubated together at 37 • C. After 15 min of incubation, 20 µL 0.2 mol/L Na 2 CO 3 was added to the test tubes to stop the reaction. The absorbance values were measured at 405 nm and converted into percentage inhibitory activity using the following formula: AA(%) = [1 − A sample /(A negative control − A blank )] × 100%. Acarbose was used as positive control. All data were obtained in triplicate and presented as means ± SD. IC 50 values were calculated with the Sigma Plot 10.0 software using a non-linear curve-fitting method [30].

Antimicrobial Activity Assay
All the compounds were tested for antibacterial activity against Staphylococcus aureus and Bacillus subtilis by the Mueller-Hinton broth microdilution method. Tested strains were cultured for 16 h on a rotary shaker at 37 • C. Cultures were diluted with sterilized medium to achieve an optical absorbance of 0.04-0.06 at 600 nm, then further diluted 10-fold before adding into 96-well microtiter plates. Compounds were dissolved in acetone, serially diluted to 7 concentrations (1.56-100 µg/mL), and tested in the 96-well plate in triplicate. The minimum inhibitory concentration (MIC) that completely inhibited visible growth of the tested strains were recorded after 18 h cultivation from three independent experiments, with vancomycin as the positive control and acetone as a blank control [26].

Conclusions
In conclusion, euroticins F-I (1-4), four pairs of new salicylaldehyde derivative enantiomers, as well as a known one, eurotirumin (5), were isolated from a South China Sea fungus Eurotium sp. SCSIO F452. Compound 1 features an unprecedented constructed 6/6/6/5 tetracyclic structures, while 2 and 3 represent two new types of 6/6/5 scaffolds. Compound 4 is the first dearomatized prenylated salicylaldehyde derivative. Compounds 1-5 are all occurred as racemates. Their optical pure enantiomers, except for 3 were well separated with absolute configuration unambiguously resolved by single crystal X-ray diffraction and ECD calculation for the first time. Selected compounds showed significant inhibitory activity against α-glucosidase and moderate cytotoxic activities. Our work would further enlarge the chemical diversity and pharmacological prosperity of salicylaldehyde derivatives.