New Isocoumarin Derivatives and Meroterpenoids from the Marine Sponge-Associated Fungus Aspergillus similanensis sp. nov. KUFA 0013

Two new isocoumarin derivatives, including a new 5-hydroxy-8-methyl-2H, 6H-pyrano[3,4-g]chromen-2,6-dione (1) and 6,8-dihydroxy-3,7-dimethylisocoumarin (2b), a new chevalone derivative, named chevalone E (3), and a new natural product pyripyropene S (6) were isolated together with 6, 8-dihydroxy-3-methylisocoumarin (2a), reticulol (2c), p-hydroxybenzaldehyde, chevalone B, chevalone C, S14-95 (4), and pyripyropene E (5) from the ethyl acetate extract of the undescribed marine sponge-associated fungus Aspergillus similanensis KUFA 0013. The structures of the new compounds were established based on 1D and 2D NMR spectral analysis, and in the case of compound 3, X-ray analysis was used to confirm its structure and the absolute configuration of its stereogenic carbons. Compounds 1, 2a–c and 3–6 were evaluated for their antimicrobial activity against Gram-positive and Gram-negative bacteria, Candida albicans ATCC 10231, and multidrug-resistant isolates from the environment. Chevalone E (3) was found to show synergism with the antibiotic oxacillin against methicillin-resistant Staphylococcus aureus (MRSA).

Compound 3 was isolated as white crystals (mp, 262-263 °C), and its molecular formula C26H38O4 was established on the basis of the (+)-HRESIMS m/z 415.2851 [M + H] + , indicating eight degrees of unsaturation. The IR spectrum showed absorption bands for hydroxyl (3300 cm −1 ), conjugated carbonyl (1664 cm −1 ) and olefin (1607, 1570 cm −1 ) groups. The 13 C NMR (Supplementary Information, Figure S9), DEPTs and HSQC spectra revealed the presence of one conjugated ketone carbonyl (δC 180.6), three quaternary sp 2 (δC 162.6, 160.5, 98.5), one methine sp 2 (δC 111.9), one oxygen bearing quaternary sp 3 Figure S8), and 13 C NMR spectra of 3 resembled those of chevalone C [9], except for the chemical shift values of the oxygen bearing methine carbon (C-3) which appeared at lower frequencies (δC 78.7; δH 3.21, dd, J = 11.1, 5.0 Hz) than those of chevalone C [9]. Furthermore, the 1 H and 13 C NMR spectra of compound 3 did not exhibit the signals of the acetyl group. Taking together the IR, HRMS and NMR data, it was possible to conclude that compound 3 is a deacetyl analog of chevalone C. Since this is the first report of isolation of this chevalone analog, we have named it chevalone E. Final proof of the structure and the stereochemistry assigned to chevalone E (3) was provided by an X-ray analysis (Figure 2), and since the diffraction data were collected with a Gemini PX Ultra equipped with CuKα radiation, it was possible to establish the absolute configuration of C-3, C-5, C-8, C-9, C-10, C-13 and C-14, respectively as 3S, 5R, 8R, 9R, 10R, 13S and 14S.  The (+)-HRESIMS of compound 6 indicated the [M + H] + peak at m/z 566.2416, corresponding to C31H36NO9. Thus, the molecular formula of compound 6 was C31H35NO9, indicating fifteen degrees of unsaturation. The IR spectrum showed absorption bands for ester carbonyl (1742 cm −1 ), conjugated carbonyl (1671 cm −1 ), aromatic (1586, 1508, 1465 cm −1 ) and olefin (1625 cm −1 ) groups. The 13 C NMR ( Supplementary Information, Figure S11), DEPTs and HSQC spectra (Table 3) Figure S10), 13 C, HSQC and HMBC spectra ( , a hexasubstituted decahydronaphthalene ring system. That two of the acetoxyl groups were on C-1 and C-7, and the three methyl groups were on C-4, C-6 and C-10 of the decahydronaphthalene moiety was substantiated by the HMBC cross peaks of the Me-15 singlet (δH 0.88, s) to the carbon signals at δC 40.6 (C-10), 72.2 (C-1), 64.7 (C-11), of the Me-12 singlet (δH 1.26, s) to the carbon signals at δC 35.5 (C-3), 38.8 (C-4), 41.1 (C-9), and of the Me-14 singlet (δH 1.59, s) to the carbon signals at δC 77.7 (C-7), 83.9 (C-6), and 144.5 (C-5). That another substituent on C-10 was the acetoxymethylene group was evidenced by the HMBC cross peaks of H-11 signals (δH 3.75, d, J = 11.9 Hz; 3.79, d, J = 11.9 Hz) to C-1, C-9, and the signal of the carbonyl at δC 171.0. On the other hand, since Me-14 singlet gave cross peaks to the signals of the oxyquaternary carbon at δC 83.9 (C-6) and the quaternary sp 2 carbon at δC 144.5 (C-5), the double bond was on C-5, and C-6 was oxygen bearing. This was corroborated by the HMBC cross peaks of the signal of the olefinic proton at δH 6.36, s (H-13) to C-4 and C-6. Moreover, the HMBC spectrum also exhibited a cross peak of H-13 signal to the signals of a conjugated carbonyl carbon at δC 161.3 (C-2′) and the quaternary sp 2 carbon at δC 161.2 (C-4′). On the other hand, there were also HMBC cross peaks of another olefinic proton at δH 6.54, s (H-5′) to C-4′ and the signals of another two quaternary sp 2 carbon at δC 101.1 (C-3′) and 157.2 (C-6′). Taken together the HMBC correlations, it was clear that the decahydronaphthalene ring system was fused, on C-5 and C-6, with 2H, 5H-pyrano [4, 3-b]pyran-5-one ring system. The COSY and HMBC spectra also indicated the presence of the 3-substituted pyridine ring. That this pyridine ring was connected to the pyranone ring through C-3 of the former and C-6′ of the later was evidenced by the HMBC correlations of the H-5′ singlet to C-3″ (δC 127.4), as well as of the signal of H-2″ (δH 8.14, dt, J = 7.8, 2.4, 2.4 Hz) to C-6′. Literature search revealed that compound 6 was previously obtained by treatment of pyripyropene A with HCl under anhydrous condition [15]; however, there were neither reports of the 1 H and 13 C data nor other description of this compound. Since 6 is a new natural product, we have named it pyripyropene S. It is interesting to point out that 6 is the first natural pyripyropene that lacks a hydroxyl group on C-13. OAc-11 171.0, CO -20.8, CH 3 2.10, s CO (OAc-11) Compounds 1, 2a-c and 3-6 were tested for their antimicrobial activity against Gram positive (Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 6633) and Gram negative (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853) bacteria, Candida albicans ATCC 10231, and multidrug-resistant isolates from the environment. All the compounds tested exhibited neither antibacterial nor antifungal activities, i.e., their MIC values were found to be higher than 256 µg/mL. Like chevalone C, chevalone E (3) does not possess the structural requirements for the antibacterial activity of this group of meroditerpenes, i.e., the presence of the β-acetoxyl group on C-3 and the presence of a free 4-hydroxy-6-methyl-2H-pyran-2-one ring on C-15 [9]. Therefore, it is not surprising that chevalone E (3) did not exhibit significant antibacterial activity. The fact that chevalone C did not show significant antibacterial activity but demonstrated synergistic effect with the antibiotics against three multidrug-resistant isolates [9] led us explore if some of these compounds could possibly have synergistic effects with antibiotics, i.e., by using a disc diffusion method to assess if, in combination with antibiotics, they could cause an increase in the growth inhibition of multidrug-resistant strains. The results (Table 4) showed that no synergistic effects were observed between the tested compounds and antibiotics for multidrug-resistant E. coli and E. faecalis; however chevalone E (3) was found to exhibit potential synergy with oxacillin and ampicillin against the MRSA strain. Table 4. Antibacterial efficacy of combined effect of antibiotics with the compounds (15 µg/disc) against three multidrug-resistant isolates, using the disc diffusion method.

E. coli G1
S. aureus B1 E. faecalis W1 In order to verify if the synergism occurred with both antibiotics or with either of them, the checkerboard method was carried out. The results, represented by the fractional inhibitory concentration (FIC) index, shown in Table 5, confirmed the synergy between chevalone E (3) and oxacillin, and not between chevalone E (3) and ampicillin. It is interesting to note that while chevalone E (3) shows synergism with oxacillin against the MRSA isolate, the structurally related meroditerpene aszonapyrone exhibited synergism only with vancomycin against the VRE isolate, and not with oxacillin against the MRSA strain [9].

General Procedures
Melting points were determined on a Bock monoscope and are uncorrected. Optical rotations were determined on an ADP410 Polarimeter (Bellingham + Syanley Ltd., Tunbridge Wells, Kent, UK). Infrared spectra were recorded on an ATT Mattson Genesis Series FTIR™ using WinFIRST Software. 1 H and 13 C NMR spectra were recorded at ambient temperature on a Bruker AMC instrument (Bruker Biosciences Corporation, Billerica, MA, USA) operating at 500.13 and 125.8 MHz, respectively. High resolution mass spectra were measured with a Waters Xevo QToF mass spectrometer (Waters Corporations, Milford, MA, USA) coupled to a Waters Aquity UPLC system. A Merck silica gel GF254 was used for preparative TLC, and a Merck Si gel 60 (0.2-0.5 mm) was used for analytical chromatography.

Extraction and Isolation
The strain KUFA0013 was isolated from the marine sponge Rhabdermia sp., which was collected from the coral reef of the Similan Islands, Phang Nga Province, Thailand, by scuba diving at 10 m depth, in April 2010, and the sponge was identified by J. Buaruang (Division of Environmental Science, Faculty of Science, Ramkhamhaeng University, Bangkok 10240, Thailand). Briefly, after rinsing with sterile sea water, the sponge was dried on sterile filter paper and cut into small pieces (5 × 5 mm) and placed on the plates containing malt extract agar [MEA, 30 g of malt extract powder (Himedia, Mumbai, India), 15 g of bacto agar, distilled water 300 mL, sea water 700 mL and adjusted to the final pH at 5.5] with 70% sea water and incubated at 28 °C under 12 h light/12 h dark cycle for seven days. The fungus was identified by one of us (T. Dethoup), by morphological features, including characteristic of ascospores, conidiogenesis and colonies, as well as by DNA sequence analysis of the calmodulin gene described by the previous report [16] (GenBank Accession No. KC 920702). Since the sequence was not identical to that deposited at GenBank, the strain was not identified at species level. The pure cultures were deposited as KUFA0013 at the Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand. A. similanensis (KUFA 0013) was cultured for one week in five 90 mm Petri dishes (i.d. 90 mm) containing 25 mL of MEA with 70% sea water per dish. Thirty 1000 mL Erlenmeyer flasks, each containing white rice (200 g), water (30 mL) and sea water (70 mL), were autoclaved at 121 °C for 15 min, inoculated with ten mycelia plugs of the fungus and incubated at 28 °C for 30 days. The moldy rice was macerated in ethyl acetate (7 L total) for seven days and then filtered by filter paper. The two layers were separated using a separatory funnel, and the ethyl acetate solution was evaporated under reduced pressure to yield 97 g of crude ethyl acetate extract that was dissolved in 500 mL of a 4:1 mixture of EtOAc and CHCl3, and then washed with 5% NaHCO3 aqueous solution (2 × 300 mL) and H2O (3 × 300 mL). The organic layer was dried with anhydrous Na2SO4, filtered and evaporated under reduced pressure to give 75 g of crude extract, which was applied on a column chromatography of silica gel (640 g) and eluted with mixtures of CHCl3-petrol and CHCl3-Me2CO, 250 mL fractions were collected as follows: , 50-64 (petrol-CHCl3, 1:9), 65-77 (CHCl3-Me2CO, 9:1). Subfrs 4-5 were combined and recrystallized in a mixture of petrol and CHCl3 to give 1 (2.4 mg). Sfrs 6-10 were combined (160 mg) and recrystallized in a mixture of petrol and CHCl3 to give 2c (7.6 mg). Sfrs 11-16 were combined (108 mg) and recrystallized in a mixture of petrol to give 2b (5 mg). Sfrs 27-33 were combined (206 mg) and purified by TLC (Si gel, CHCl3: Me2CO. 93:7) to give p-hydroxybenzaldehyde (36 mg). Frs 231-247 were combined (6.7 g) and recrystallized in a mixture of petrol and Me2CO to give 1.39 g of chevalone C. Frs 272-294 were combined (1.54 g) and crystallized in a mixture of petrol and Me2CO to yield 5 (265 mg). Frs 328-335 were combined (296 mg) and applied on a column of Sephadex LH-20 (22 g) and eluted with a mixture of CHCl3-MeOH (9:1) to give 3 (11.2 mg). Frs 354-398 were combined (1.14 g) and purified by TLC (Si gel, CHCl3:MeOH:HCO2H, 95:5:0.01) to give 6 (27.3 mg). Pseudomonas aeruginosa ATCC 27853), Candida albicans ATCC 10231 and multidrug-resistant bacteria isolated from the environment, S. aureus B1 (isolated from public bus), Enterococcus faecalis W1 (isolated from river water) and E. coli G1 (isolated from seagull feces). Bacteria were grown in Mueller-Hinton agar (MH-BioKar diagnostics, Allonne, France) from stock cultures, while C. albicans was grown in Sabouraud dextrose agar (SAB-BioKar diagnostics, Allonne, France). MH and SAB plates were incubated at 37 °C prior to obtain fresh cultures for each in vitro bioassay.

Determination of Minimum Inhibitory and Bactericidal/Fungal Concentrations
The minimum inhibitory concentrations (MIC) of the compounds were determined using a broth microdilution technique, following the recommendations of the Clinical and Laboratory Standards Institute [17]. Stock solutions of 10 mg/mL, prepared in dimethylsulfoxide (DMSO-Applichem GmbH, Darmstadt, Germany), were serially diluted in Mueller-Hinton broth (MHB-BioKar diagnostics, Allonne, France) to achieve in-test concentrations ranging from 2 to 256 µg/mL. Each bacterial inoculum was prepared in MHB, while C. albicans inoculum was prepared in RPMI-1640 with L-glutamine, with MOPS and without NaHCO3 (Lonza, Walkersville, MD, USA). All inocula were standardized in order obtain a concentration of 5 × 10 5 CFU/mL in each inoculated well of the microtiter plate. The concentration of DMSO in the highest in-test concentration did not affect the microbial growth. The MIC was defined as the lowest concentration of compound that has inhibited the visible growth.

Screening of Combined Effect between the Compounds and Antibiotics
A screening susceptibility test to assess the combined effect between the compounds and antibiotics was conducted using the disc diffusion method on MH, according to the procedure already described by Gomes et al. [9].

Synergy Test: Checkerboard Method
Based on the results of the previous assay, potential synergy between 3 and oxacillin or ampicillin (Sigma-Aldrich, St. Louis, MO, USA) was checked using a broth microdilution checkerboard method and tested in MRSA isolate (S. aureus B1), as has been already described [9]. Two independent experiments in duplicate were performed. The fractional inhibitory concentration (FIC) was calculated as follows: FIC of drug A (FIC A) = MIC of drug A in combination/MIC of drug A alone, and FIC of drug B (FIC B) = MIC of drug B in combination/MIC of drug B alone. The FIC index (ΣFIC), calculated as the sum of each FIC, was interpreted as follows: ΣFIC ≤ 0.5, synergy; 0.5 < ΣFIC ≤ 4, no interaction; 4 < ΣFIC, antagonism [18].

Conclusions
Although several analogs of chevalone have been reported from several members of the genus Aspergillus, this is the first report of isolation of isocoumarin derivatives from a member of this genus.
The synergism of chevalone E with the antibiotic oxacillin against MRSA can be considered relevant for anti-infective marine natural products research.