Dichloroisocoumarins with Potential Anti-Inflammatory Activity from the Mangrove Endophytic Fungus Ascomycota sp. CYSK-4

Three new isocoumarins—dichlorodiaportintone (1), desmethyldichlorodiaportintone (2) and desmethyldichlorodiaportinol (3)—as well as six known analogues (4–9) were isolated from the culture of the mangrove endophytic fungus Ascomycota sp. CYSK-4 from Pluchea indica. Their structures were elucidated by analysis of spectroscopic data. The absolute configuration of compounds 1 and 2 were determined by the modified Mosher’s method. Compound 2 showed significant anti-inflammatory activity by inhibiting the production of NO in LPS-induced RAW 264.7 cells with IC50 value of 15.8 μM, while compounds 1, 5, and 6 exhibited weak activities with IC50 values of 41.5, 33.6, and 67.2 μM, respectively. In addition, compounds 1, 5, and 6 showed antibacterial effects against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, and Acinetobacter calcoaceticus with the MIC values in the range of 25–50 μg·mL−1.


Introduction
Fungi in the genus Ascomycota sp. produce various structurally novel metabolites, including ascomindones A-C and ascomfurans A-B [1], ascomycotin A [2], wortmannilactone E [3], orsellinic acid [4], isosclerone [5], and chaetocyclinone [6]. Furthermore, most of these compounds possess a wide range of biological activities. For example, polyketone ascomindones A exhibited more potent capacity in scavenging DPPH radical [1]. Diphenyl ether barceloneic acid A showed modest inhibition of FPTase enzyme [7]. As part of our ongoing investigation on bioactive natural products from mangrove-derived fungi, an endophytic fungus Ascomycota sp. CYSK-4, which was isolated from the healthy branch of the marine semimangrove Pluchea indica, attracted our attention because an EtOAc extract of the fungal culture exhibited significant anti-inflammatory activity. Bioassay-guided fractionation of the EtOAc extract led to the isolation of three new isocoumarin derivatives, compounds 1-3, together with six known isocoumarin analogues 4-9 ( Figure 1). In the in vitro assays, compounds 1-2 and 5-6 showed inhibitory activities against the lipopolysaccharides (LPS) induced nitric oxide (NO) production in murine macrophage RAW 264.7. Herein, the details of the isolation, structural elucidation and anti-inflammatory evaluation of these compounds were reported. nitric oxide (NO) production in murine macrophage RAW 264.7. Herein, the details of the isolation, structural elucidation and anti-inflammatory evaluation of these compounds were reported.

Results
Compound 1 was obtained as an amorphous solid. Its molecular formula was established as C 16 (Table 2) exhibited 16 carbon resonances assignable to one methyl, two methylenes, two sp 3 and three sp 2 methines, six quaternary carbons and two carbonyl carbons. These spectroscopic features suggested that 1 belong to the isocoumarin class [8][9][10]. Analysis of the 1 H-1 H COSY spectrum ( Figure 2) suggested the presence of one independent spin system H 2 -11/H-12. Together with the HMBC cross-peaks ( Figure 2) of H-12/C-13; H-11/C-10, C-13; H-9/C-3, C-10; H-14/C-9, C-10, and C-11 indicated the side chain of isocaproicacid moiety location at C-3. The chemical shift of H-14 (δ H 6.52) indicated the dichloro substitution at C-14 [8]. Apart from a carbonyl group and isocoumarin group, the remaining one indices of hydrogen deficiency was proved to be α-hydroxyl-γ-lactone ring. The chemical shift of quaternary carbon δ C 86.0 (C-10) confirmed the ring bridging C-10 and C-13. Moreover, the methoxy group was placed to C-6 based on the HMBC correlation of its proton to C-6. Thus, the constitution of 1 was established ( Figure 1).  6.52, s 6.52, s 6-OCH 3 3.92, s 8-OH 11.0, br s 11.0, br s 10.9, br s The relative configuration of 1 was determined by NOESY data (Figure 3). The cross-peak of H-12 and H-14 indicated the syn relationship between H-12 and H-14. The absolute configuration of C-12 was further confirmed by the modified Mosher ester method [11]. The (S)-and (R)-MTPA esters of 1 (1a and 1b) were prepared using (R)-and (S)-MTPA chloride, respectively. The differences in the 1 H NMR chemical shifts of 1a and 1b were summarized to determine the absolute configuration of this position, which was clearly established as 12R. Taking the data discussed above into account, the absolute configuration of 1 was assigned as 10R,12R ( Figure 4). Thus, compound 1 was determined as dichlorodiaportintone ( Figure 1).    The relative configuration of 1 was determined by NOESY data (Figure 3). The cross-peak of H-12 and H-14 indicated the syn relationship between H-12 and H-14. The absolute configuration of C-12 was further confirmed by the modified Mosher ester method [11]. The (S)-and (R)-MTPA esters of 1 (1a and 1b) were prepared using (R)-and (S)-MTPA chloride, respectively. The differences in the 1 H NMR chemical shifts of 1a and 1b were summarized to determine the absolute configuration of this position, which was clearly established as 12R. Taking the data discussed above into account, the absolute configuration of 1 was assigned as 10R,12R ( Figure 4). Thus, compound 1 was determined as dichlorodiaportintone ( Figure 1).   Figure 3) from H-12 to H-14 proved that compounds 2 and 1 shared the same relative configuration. The absolute configuration at C-12 was also determined as R by the modified Mosher's method. Thus, the absolute configuration of 2 was determined as 10R,12R ( Figure 4). Therefore, the compound 2 was assigned as desmethyldichlorodiaportintone ( Figure 1).  Figure 3) from H-12 to H-14 proved that compounds 2 and 1 shared the same relative configuration.
The absolute configuration at C-12 was also determined as R by the modified Mosher's method. Thus, the absolute configuration of 2 was determined as 10R,12R ( Figure 4). Therefore, the compound 2 was assigned as desmethyldichlorodiaportintone ( Figure 1).  Tables 1 and 2) of 3 were similar to those of dichlorodiaportinol A (4), except for the absence of the methoxy group (δC 56.3, δH 3.90) at C-6 in 3. The structure of 3 was also confirmed using 1 H-1 H COSY and HMBC spectra ( Figure 2). The configuration of two stereocenters (C-9 and C-10) were determined by coupling constants and NOESY experiments ( Figure 3). Protons H-9 and H-10 displayed a large coupling constant ( 3 JH-9,H-10 = 8.8 Hz), indicating them to be in an anti configuration. This allowed for only two of the six possible relative configuration for C-9 and C-10 could be satisfied. As well as the NOE correlation of H-4/OH-10 in DMSO, the relative configuration of C-9 and C-10 was unambiguously determined as 9R* and 10S* ( Figure 5) and named desmethyldichlorodiaportinol ( Figure 1).   This allowed for only two of the six possible relative configuration for C-9 and C-10 could be satisfied. As well as the NOE correlation of H-4/OH-10 in DMSO, the relative configuration of C-9 and C-10 was unambiguously determined as 9R* and 10S* ( Figure 5) and named desmethyldichlorodiaportinol ( Figure 1).  Figure 3) from H-12 to H-14 proved that compounds 2 and 1 shared the same relative configuration.
The absolute configuration at C-12 was also determined as R by the modified Mosher's method. Thus, the absolute configuration of 2 was determined as 10R,12R ( Figure 4). Therefore, the compound 2 was assigned as desmethyldichlorodiaportintone ( Figure 1).  Tables 1 and 2) of 3 were similar to those of dichlorodiaportinol A (4), except for the absence of the methoxy group (δC 56.3, δH 3.90) at C-6 in 3. The structure of 3 was also confirmed using 1 H-1 H COSY and HMBC spectra (Figure 2). The configuration of two stereocenters (C-9 and C-10) were determined by coupling constants and NOESY experiments (Figure 3). Protons H-9 and H-10 displayed a large coupling constant ( 3 JH-9,H-10 = 8.8 Hz), indicating them to be in an anti configuration. This allowed for only two of the six possible relative configuration for C-9 and C-10 could be satisfied. As well as the NOE correlation of H-4/OH-10 in DMSO, the relative configuration of C-9 and C-10 was unambiguously determined as 9R* and 10S* ( Figure 5) and named desmethyldichlorodiaportinol ( Figure 1).  The other known compounds were identified as dichlorodiaportinol (4) [9], desmethyldichlorodiaportin (5) [8], dichlorodiaportin (6) [10], mucorisocoumarin B (7) [12], citroisocoumarin (8) [13], and diaportinol (9) [11] by comparison with NMR data in the literature.
The anti-inflammatory activities of all compounds were evaluated against nitric oxide (NO) production in the lipopolysaccharide (LPS)-stimulated mouse macrophage RAW 264.7. The results suggest that the compound 2 showed potent inhibitory activities with IC 50 value of 15.8 µM, and compounds 1, 5, and 6 exhibited weak inhibitory activity in comparison with the indomethacin (the positive control, IC 50 = 37.5 µM). Other compounds showed no inhibitory effect (IC 50 > 100 µM) ( Table 3). All compounds showed no cytotoxic effect at the tested concentration. Compounds 2 and 5 which have a hydroxyl group at C-6 showed batter than compounds 1 and 6 with a methoxy group at C-6. The structure-activity relationships of these dichloroisocoumarins indicated that a hydroxyl group was more significance than a methoxy group on anti-inflammatory activity. Isocoumarins were previously reported to have radical scavenging and antioxidant [14], anti-HIV [15], antimicrobial [16], anti-γ-secretase [17], antitumor [18], immunomodulatory [19], antifungal [20], toxicity to zebrafish embryos [13], and α-glucosidase inhibitory activities [21]. This is the first report of anti-inflammatory activity of dichloroisocoumarins. The antibacterial activities of the isolated compounds 1-9 against two Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) and three Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, and Acinetobacter calcoaceticus) were tested (Table 4). Compounds 5 and 6 showed antibacterial activities with the MIC values between 25 and 50 µg·mL -1 against S. aureus, B. subtilis, E. coli, K. pneumoniae, and A. calcoaceticus. Compound 1 exhibited antibacterial activities with the MIC values at 50 µg·mL −1 against S. aureus, E. coli and K. pneumoniae. Other compounds did not exhibit obvious activity at 50 µg·mL −1 .

Fungal Material and Fermentation
The fungus CYSK-4 used in this study was isolated from healthy branch of the marine semimangrove Pluchea indica, which was collected in July 2015 from Shankou Mangrove Nature Reserve in Guangxi Province, China. It was obtained using the standard protocol for isolation [21]. Initially, the plant tissue was washed with sterile water and surface-sterilized in a 200 mL beaker with 75% ethanol for 1 min. This was followed by dipping the sample into 5% sodium hypochlorite for 1 min, then the plant parts were rinsed with sterile water, cut into 3 mm sections, and plated on PDA with penicillin (100 units per mL) and streptomycin (0.08 mg·mL −1 ). The plates were incubated at 25 ± 1 • C. The endophytic fungal strains were isolated by routine microbiological methods. The fungal isolates were numbered and stored at 4 • C in triplicate on PDA slants. Fungal identification was carried out using a molecular biological protocol by DNA amplification and sequencing of the ITS region [22]. The sequence data obtained from the fungal strain have been deposited at Gen Bank with accession no. MG571637. A BLAST search result showed that the sequence was the most similar (99%) to the sequence of Ascomycota sp. (compared to KT240142.1 EF060747.1). A voucher strain was deposited in the Guangdong Microbial Culture Center under patent depository number GDMCC 60100. The fungus Ascomycota sp. CYSK-4 was cultured on autoclaved rice solid-substrate medium (60 × 500 mL Erlenmeyer flasks, each containing 50 g rice and 50 mL 3‰ of saline water) for 30 days at room temperature.

Nitric Oxide Production Assay
Murine macrophage RAW 264.7 cells purchased from the Shanghai Institutes for Biological Sciences in DMEM (high glucose) medium supplemented with 10% (v/v) fetal bovine serum, 100 µg·mL -1 penicillin and streptomycin, and 10 mM HEPES at 37 • C in a 5% CO 2 atmosphere [23]. Cells were pretreated with different samples dissolved in serum-free culture medium containing 0.5% DMSO (10, 5, 2.5, 1.25, and 0.625 µM) for 4 h, followed by stimulation with 1 µg·mL -1 LPS for 24 h. Fifty µL of cell culture medium was mixed with 100 µL of Griess reagent I and II and incubated at room temperature for 10 min with horizontal shaking, after which the absorbance at 540 nm was measured in a microplate reader. Indomethacin was used as a positive control and was purchased from Sigma-Aldrich Co. (CAS number: 53-86-1, EINECS number: 200-186-5; Buchs, Switzerland).
Wells with DMSO were used as a negative control (final DMSO concentration was 0.1%). The NO production inhibition rate was calculated by the flowing formula: NO production inhibition rate (%) = LPS group − Compoud group LPS group − DMSO group × 100 (1) IC 50 was defined as the concentration of compound that inhibited 50% NO production relative to the LPS group and was calculated using SPSS 16.0 software. All assays were performed in triplicate.

Antimicrobial Activity
Antimicrobial activities were evaluated by the conventional broth dilution assay [24]. Two Gram-positive-S. aureus (ATCC 12228) and B. subtilis (ATCC 6633)-and three Gram-negative-E. coli (ATCC 25922), K. pneumoniae (ATCC 13883), and A. calcoaceticus (ATCC 23055)-were used. Overnight cultures of five bacterial strains were made up in 0.9% saline to an inoculum density of 5 × 10 5 cfu by comparison with a MacFarland standard. All compounds were dissolved in DMSO and diluted by Mueller Hinton broth to a starting concentration of 2 mg·mL -1 . Ninety-five µL of MHB and 5 µL of test compounds or the antibiotic were dispensed into wells as well as the 100 µL bacterial suspension. After incubation at 37 • C for 24 h, the inhibitory effect was evaluated by optical density measurement. The MIC was determined as the concentration which the growth was inhibited 80% of bacterial. One hundred µL bacterial suspension were added to the solutions in 96-well to achieve a final volume of 200 µL and final sample concentrations from 50 to 0.125 µg·mL -1 . The blank well was also incubated with only medium under the same conditions. OD measurement was record at 595 nm. All experiments were performed in triplicate and with ciprofloxacin and gentamicin as the positive control.