Xylariphilone: a new azaphilone derivative from the seagrass-derived fungus Xylariales sp. PSU-ES163

One new azaphilone derivative, named xylariphilone (1), along with 10 known compounds was isolated from the seagrass-derived fungus Xylariales sp. PSU-ES163. Their structures were elucidated on the basis of extensive spectroscopic analysis. The absolute and relative configurations of 1 were determined by circular dichroism spectroscopy and NOEDIFF data. The antimicrobial and cytotoxic activities of the isolated compounds were evaluated.


Results and discussion
The broth and mycelial extracts of the fungus PSU-ES163 were purified using chromatographic techniques leading to the isolation of 1 new (1) and 10 known (2 -11) compounds. All metabolites ( Figure 1) were elucidated by analysis of spectroscopic data, including UV, IR, NMR and MS. The absolute and relative configurations of 1 were determined using the circular dichroism (CD) spectroscopy and selective NOEDIFF data.
Xylariphilone (1) was obtained as a colourless gum with ½a 24 D 2 25.1 (c 0.50, CHCl 3 ). The molecular formula was deduced to be C 11 H 16 O 4 on the basis of HREIMS with four degrees of unsaturation. The UV spectrum showed a maximum absorption band at 241 nm while the IR spectrum displayed absorption bands at 3394 and 1671 cm 21 for hydroxy and conjugated carbonyl groups, respectively. The 1 H NMR spectroscopic data contained signals for two sets of nonequivalent methylene protons [d H 4.63 (d, J ¼ 16.0 Hz, 1H) and 4.14 (br d, J ¼ 16.0 Hz, 1H); 2.61 (ddd, J ¼ 18.5, 6.0 and 0.5 Hz, 1H) and 2.36 (brdd, J ¼ 18.5 and 10.5 Hz, 1H)], one oxymethine proton (d H 4.00, dd, J ¼ 10.5 and 6.0 Hz), a 1-substituted 2-oxypropyl group [d H 3.64 (dd, J ¼ 12.5 and 6.5 Hz, 1H), 2.19 (m, 1H), 2.17 (m, 1H) and 1.29 (d, J ¼ 6.5 Hz, 3H)], two hydroxy protons (d H 3.66, br s and 2.47, s) and one methyl group (d H 1.26, s). The 13 C NMR and DEPT spectra of 1 displayed 11 signals which were assigned to one ketone carbonyl (d C 199.1), three quaternary (d C 153.2, 128.5 and 77.1), two oxymethine (d C 72.6 and 69.4), three methylene (d C 63.6, 38.2 and 36.1) and two methyl (d C 21.0 and 17.8) carbons. In the 1 H-1 H COSY spectrum, the nonequivalent methylene protons, H ab -5 (d H 2.61 and 2.36), were coupled with H-6 (d H 4.00), whereas the HMBC spectrum displayed the correlations of H a -5 (d H 2.61)/C-4a (d C 153.2), C-6 (d C 72.6), C-7 (d C 77.1) and C-8a (d C 128.5) and those of H 3 -10 (d H 1.26)/C- 6, C-7 and C-8 (d C 199.1). These data established a cyclohexenone with a methyl group at C-7 and a double bond at C-4a and C-8a. The chemical shifts of C-6 and C-7 as well as the HMBC correlations from 6-OH (d H 2.47) to C-5 (d C 36.1) and 7-OH (d H 3.66) to C-6, C-7 and C-8 indicated the attachment of the hydroxy groups at these two carbons. The oxymethine proton, H-3 (d H 3.64), showed a HMBC cross peak with C-1 (d C 63.63), which correlated in the HMQC spectrum with the nonequivalent oxymethylene protons (H ab -1, d H 4.63 and 4.14). Accordingly, an ether unit (ZCH 2 CH(CH 3 )OCH 2 Z) was established. The linkage between C-1 and C-8a and that between C-4 (d C 38.2) and C-4a were indicated from the HMBC correlations of H ab -1 with C-4a and C-8a and those of H ab -4 (d H 2.19 and 2.17) with C-4a, C-5 and C-8a, thus indicating a tetrahydroisochromenone. The relative configuration of 1 was assigned from the coupling constants and NOEDIFF results (see Supplementary material). The coupling constant of 10.5 Hz between H b -5 (d H 2.36) and H-6 indicated their location at pseudoaxial positions. Irradiation of H-6 enhanced the signal intensity of H a -5, but did not affect that of H 3 -10, thus indicating that H-6 was cis to H a -5 but trans to H 3 -10. According to the coupling constants of 12.5 and 6.5 Hz of H-3, H-3 was located at a pseudoaxial position. Irradiation of H b -1 (d H 4.14) affected signal intensity of H-3 while irradiation of H-3 and H a -5 enhanced the signal intensity of H a -4 (d H 2.19). Consequently, H-3 had a cis relationship to H-6. The CD spectrum of 1 showed a positive first cotton effect (217 nm, D1 ¼ þ7.88) and a negative second cotton effect (234 nm, D1 ¼ 2 2.61), the same sign as those of (þ )-(R)-2-acetyl-3,6-dihydroxycyclohex-2-enone, CD (c ¼ 1.00 £ 10 23 mol/L, MeOH): l max : 216 nm (D1 ¼ þ1.1) and 232 nm (D1 ¼ 2 0.3) (Zaitsev & Mikhal'chuk 2001), indicating the absolute configuration at C-7 to be R. Therefore, the absolute configurations at C-3 and C-6 were assigned as R and S, respectively. It is worth to note that the absolute configuration at C-3 of 1 and the cometabolites 6, 9 and 10 have identical R configuration. Consequently, xylariphilone (1) was identified as a new azaphilone derivative.
The isolated compounds 3-7 and 10 were evaluated for antimicrobial activity against Staphylococcus aureus ATCC25923, methicillin-resistant S. aureus SK1 (a clinical isolate), Candida albicans NCPF3153, Cryptococcus neoformans ATCC90113 and Microsporum gypseum. The remaining compounds were not tested due to their small quantities. None of them showed antimicrobial activity against the tested pathogenic bacteria and fungi at the concentration of 200 mg/mL. In addition, compounds 7 and 10 were tested for cytotoxic activity against KB, MCF-7 and noncancerous Vero (African green monkey kidney fibroblast) cells. Both were inactive against these cell lines.

Experimental 3.1. General experimental procedures
The IR spectra were recorded on a Perkin-Elmer 783 FTS 165 FT-IR spectrometer. Optical rotations were measured on a JASCO P-1020 polarimeter. Ultraviolet (UV) spectra were recorded on a Shimadzu UV-160A spectrophotometer. CD spectrum was recorded on a JASCO model J-810 polarimeter. EIMS mass spectrum of 1 was acquired using a MAT 95 XL mass spectrometer (Thermofinnigan). 1 H and 13 C NMR spectra were recorded on a 300 or 500 MHz Bruker FTNMR Ultra Shield spectrometer. Thin layer chromatography (TLC) and precoated TLC (PTLC) were performed on silica gel GF 256 (Merck). Column chromatography (CC) was carried out on silica gel (Merck) type 60 (230 -400 mesh ASTM), Sephadex LH-20 or on reverse phase silica gel C-18.

Fungal material
The endophytic fungus PSU-ES163 (BCC47786) was isolated from the leaves of the seagrass Halophila ovalis collected from Trang Province, Thailand. This isolate was identified based on the nuclear ribosomal internal transcribed spacer (ITS) regions, the large (28S) subunit ribosomal RNA (LSU) and the small (18S) subunit ribosomal RNA (SSU). The sequences of this isolate were found to belong to Order Xylariales. The ITS sequence (accession number JN116682) showed the highest similarity with EF448415 Annulohypoxylon atroroseum (86.2%). The LSU sequence (accession number JQ419763) was closely related to Annulohypoxylon atroroseum (DQ840060) with 96.6% nucleotide identity. However, the SSU sequence (accession number JQ419764) gave the highest similarity (98.5%) with Xylaria acuta. Thus, this isolate could be identified to an order level as Xylariales sp.

Fermentation, extraction and isolation
The broth and mycelial ethyl acetate extracts of the fungus PSU-ES163 were prepared using the same procedure as described previously ). For broth BuOH extract, the aqueous residue obtained after extraction of the filtrate from broth culture with ethyl acetate was divided into five portions. Each portion was extracted twice with an equal volume of BuOH (2 £ 500 mL). The BuOH layer was then dried over anhydrous Na 2 SO 4 and evaporated to dryness to obtain a dark brown gum (1.25 g). The broth EtOAc extract (829.5 mg) was subjected to CC over Sephadex LH-20 with 100% MeOH to afford six fractions (A -F). Fraction B (145.0 mg) was purified by CC over silica gel using a gradient of MeOH/CH 2 Cl 2 to yield four subfractions (B1 -B4). Subfraction B2 (7.7 mg) was purified by PTLC using EtOAc/CH 2 Cl 2 (2:3) (3 runs) to obtain 2 (1.0 mg) while subfraction B3 (18.4 mg) was subjected to CC over silica gel using a gradient of EtOAc/CH 2 Cl 2 and PTLC using EtOAc/petroleum ether (2:3) (5 runs) to give 1 (1.4 mg). Fraction D (51.0 mg) was purified by CC over silica gel using a gradient of acetone/CH 2 Cl 2 to yield five subfractions. The second subfraction (3.6 mg) was separated by PTLC with acetone/CH 2 Cl 2 (1:99) (5 runs) to afford 3 (2.8 mg) while the fourth subfraction contained 4 (2.5 mg). Fraction E (55.7 mg) was subjected to CC over reverse phase silica gel using a gradient of MeOH/H 2 O followed by PTLC with acetone/CH 2 Cl 2 (3:97) (5 runs) to afford 6 (2.4 mg) and 7 (4.0 mg). Fraction F (25.1 mg) was chromatographed using the same procedure as fraction E to yield 5 (2.5 mg). The broth BuOH extract was initially purified by CC over Sephadex LH-20 with 100% MeOH, followed by CC over silica gel using a gradient of MeOH/ CH 2 Cl 2 and CC over Sephadex LH-20 with MeOH/CH 2 Cl 2 (1:1) to give 8 (1.2 mg). The mycelial EtOAc extract (1.70 g) was separated by CC over sephadex LH-20 with 100% MeOH to afford four fractions. The second fraction (990.5 mg) was fractionated by CC over silica gel using a gradient of MeOH/CH 2 Cl 2 to obtain 9 (1.4 mg) and 10 (9.0 mg). Compound 11 (1.1 mg) was obtained from the third fraction (97.2 mg) after purification by CC over silica gel using a gradient of MeOH/CH 2 Cl 2 and PTLC using CH 2 Cl 2 /hexane (1:1) (2 runs).

Antimicrobial assay
Antimicrobial activity was determined as described by the Clinical and Laboratory Standards Institute (Drummond & Waigh 2000;Clinical and Laboratory Standards Institute (CLSI) 2002a, 2002b, 2002c. Vancomycin, amphotericin B and miconazole were used as positive controls for bacteria, yeasts and fungus with the MIC values of 1, 0.25 and 1 mg/mL, respectively.

Cytotoxicity assay
The activity assay against African green monkey kidney fibroblast (Vero) cells was performed in triplicate employing the method described by Hunt and co-workers (Hunt et al. 1999). The activities against KB and MCF-7 cell lines were evaluated using the resazurin microplate assay (O'Brien et al. 2000).

Conclusion
One new azaphilone derivative (1), along with seven known compounds, four naphthalenone (2-5), two isocoumarin (6 -7) and one g-lactone (8) derivatives, was obtained from the broth extract of Xylariales sp. PSU-ES163. Whereas three known compounds, two d-lactone (9 -10) and one naphthalene (11) derivatives, were isolated from the mycelial extract. Compound 1 was structurally related to pestafolide A (Ding et al. 2008), peneciraistin C (Ma et al. 2012), monascusone A and FK17-P2b2 (Jongrungruangchok et al. 2004) which have a tetrahydro-1Hisochromen-8(5H)-one core structure with the methyl group at C-7 and hydroxy groups at C-6 and C-7, identical to those in 1. The absolute and relative configurations of 1 were assigned by circular dichroism spectroscopy and NOEDIFF data which indicated that C-6 and C-7 in the tetrahydroisochromenone unit have the S and R configurations, respectively, identical to those of the above structurally related compounds (Jongrungruangchok et al. 2004;Ding et al. 2008;Ma et al. 2012).

Supplementary material
The 1 H and 13 C NMR spectra as well as the selected NOEDIFF correlations of 1 are available online.