Highly oxygenated chromones from mangrove-derived endophytic fungus Rhytidhysteron rufulum

Phytochemistry xxx (2015) xxx–xxx

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Highly oxygenated chromones from mangrove-derived endophytic fungus Rhytidhysteron rufulum Supichar Chokpaiboon a, Siwattra Choodej b, Nattawut Boonyuen c, Thapong Teerawatananond d, Khanitha Pudhom b,⇑ a

Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Pathumthani 12120, Thailand d Faculty of Science and Technology, Valaya Alongkorn Rajabhat University under Royal Patronage, Pathumtani 13138, Thailand b c

a r t i c l e

i n f o

Article history: Received 15 July 2015 Received in revised form 4 December 2015 Accepted 14 December 2015 Available online xxxx Keywords: Rhytidhysteron rufulum Endophytic fungi Chromone Cytotoxicity

a b s t r a c t Five highly oxygenated chromones, rhytidchromones A–E, were isolated from the culture broth of a mangrove-derived endophytic fungus, Rhytidhysteron rufulum, isolated from Thai Bruguiera gymnorrhiza. Their structures were determined by analysis of 1D and 2D NMR spectroscopic data. The structure of rhytidchromone A was further confirmed by single-crystal X-ray diffraction analysis. These compounds were evaluated for cytotoxicity against four cancer cell lines (MCF-7, Hep-G2, Kato-3 and CaSki). All compounds, except for rhytidchromone D, displayed cytotoxicity against Kato-3 cell lines with IC50 values ranging from 16.0 to 23.3 lM, while rhytidchromones A and C were active against MCF-7 cells with IC50 values of 19.3 and 17.7 lM, respectively. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Endophytic fungi, microorganisms which reside in the healthy tissue of their host harmoniously without causing any apparent negative effect (Strobel and Daisy, 2003; Faeth, 2002), are known as an exceptionally valuable resource for the discovery of structurally interesting and biologically active secondary metabolites, some of which are promising candidates for drug development or agrochemical applications (Zhang et al., 2006; Tan and Zou, 2001; Kusari and Spiteller, 2011; Strobel et al., 2004). Currently, an increasing number of fungal endophytes have been isolated and their metabolites are receiving considerable attention, with a number of structurally unique and biologically active compounds having been thus obtained from their cultures (Kharwar et al., 2011; Macías-Rubalcava et al., 2014; Liu et al., 2015; Amrani et al., 2014; Li et al., 2011). Among plant-derived fungi, those associated with a marine habitat, including mangrove plants, have received much interest from natural product researchers due to this ecosystem (Debbab et al., 2013; Xu, 2015; Zhou et al., 2014; Xiao et al., 2013). Moreover, Thailand has some of the largest mangrove formations in the world, with only those found in Bangla-

⇑ Corresponding author. E-mail address: [email protected] (K. Pudhom).

desh, Brazil, Indonesia and India being larger. This prompted us to embark on the study of bioactive metabolites from Thai mangrove-derived fungi (Pudhom and Teerawatananond, 2014; Pudhom et al., 2014; Chokpaiboon et al., 2011, 2010). Recently, a chemical investigation of the endophytic fungal strain BG2-Y was performed. This fungus was isolated from the leaves of the Thai mangrove plant Bruguiera gymnorrhiza and was identified as Rhytidhysteron rufulum. Five new chromones, namely rhytidchromones A–E (1–5), were obtained from the ethyl acetate (EtOAc) extract of the culture broth grown in a Sabouraud dextrose broth (SDB). These compounds contained a highly oxygenated side-chain in their structures. Herein, the isolation, structure elucidation, and cytotoxic activity against human cancer cell lines of these metabolites are described. 2. Results and discussion The EtOAc extract of the mangrove-derived endophytic R. rufulum cultured in SDB was subjected to chromatographic fractionation over Sephadex LH20 and silica gel to yield compounds 1–5 (Fig. 1). Rhytidchromone A (1), obtained as colorless crystals, was assigned a molecular formula of C16H16O7 by the HRESIMS ion at m/z 343.0784 [M+Na]+, implying nine degrees of unsaturation. The 1H NMR spectrum (Table 1) contained signals for one phenolic

http://dx.doi.org/10.1016/j.phytochem.2015.12.010 0031-9422/Ó 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Chokpaiboon, S., et al. Highly oxygenated chromones from mangrove-derived endophytic fungus Rhytidhysteron rufulum. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.12.010

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S. Chokpaiboon et al. / Phytochemistry xxx (2015) xxx–xxx

Fig. 1. Structures of compounds 1–5.

Table 1 H (400 MHz) and

1

Position

13

C (100 MHz) NMR spectroscopic data for 1–3 (CDCl3). 1 dC, type

2 3 4 4a 5 6 7 8 8a 10 20

167.1, qC 108.7, CH 182.5, qC 104.9, qC 163.4, qC 95.2, CH 162.9, qC 103.4, qC 155.7, qC 69.2, CH 33.6, CH2

30 40 2-Me 5-OH 7-OMe 10 -OMe 30 -OMe 40 -OMe

76.1, CH 174.9, qC 20.4, CH3

2 dH, mult (J in Hz) 6.04, s

6.36, s

6.00, 2.55, 2.86, 4.29,

dd (7.2, 9.6) ddd (7.2, 10.0, 12.8) ddd (7.2, 9.6, 12.8) t (10.0)

56.3, CH3

2.35, s 13.04, br s 3.89, s

58.6, CH3

3.64, s

3

dC, type 166.9, qC 108.4, CH 182.9, qC 105.3, qC 162.5, qC 95.3, CH 164.1, qC 104.8, qC 156.1, qC 70.6, CH 36.9, CH2 78.2, CH 172.8, qC 20.5, CH3 56.2, 56.7, 58.1, 51.8,

CH3 CH3 CH3 CH3

proton bonded to a carbonyl group (dH 13.04 s), one aromatic proton (dH 6.36 s) attributed to a pentasubstituted aromatic ring, one olefinic proton (dH 6.04 s) assigned to a trisubstituted olefin, two methoxy singlets (dH 3.64 and 3.89), and one methyl singlet (dH 2.35). Combined analysis of 13C NMR and HSQC data further established the presence of one conjugated ketone carbonyl (dC 182.5), one ester carbonyl (dC 174.9), six quaternary carbons (dC 167.1, 163.4, 162.9, 155.7, 104.9 and 103.4), four methines (dC 108.7, 95.2, 76.1 and 69.2), two methoxys (dC 58.6 and 56.3), one methylene (dC 33.6), and one methyl (dC 20.4). In addition, the UV absorption maxima at 237, 255, 290, and 319 nm indicated that compound 1 should be a chromone derivative. According to its double bond equivalent (DBE), the seven units of unsaturation for a chromone skeleton and one unit for an ester carbonyl, suggested that compound 1 contains an additional ring in the structure. The 1H–1H COSY spectrum indicated the existence of one isolated spin system, CH-10 –CH2-20 –CH-30 , as well as the HMBC cross-peaks from H-10 (dH 6.00, dd, J = 7.2, 9.6 Hz) and H-30 (dH 4.29, t, J = 10.0 Hz) to an ester carbonyl at dC 174.9 (C-40 ); this led to the corroboration of the c-lactone ring (Fig. 2). Further HMBC correlations from H-10 to C-7, C-8 and C-8a indicated that the C-10 oxymethine of the c-lactone ring was attached on C-8 of the chromone nucleus. One methoxy group (dH 3.64, dC 58.6) was located on C-30 due to its HMBC correlation with C-30 , whereas another methoxy group (dH 3.89, dC 56.3) was placed on C-7 from its HMBC cross-peak to C-7. The singlet methyl proton (dH 2.35) showed HMBC correlations with C-2 and C-3, indicating the location of the methyl group at C-2. Additionally, observed HMBC

dH, mult (J in Hz) 6.05, s

6.40, s

5.10, 2.30, 2.60, 3.63,

t (7.2) m dt (7.2, 14.0) dd (4.8, 12.8)

2.38, s 13.01, br s 3.90, s 3.17, s 3.29, s 3.73, s

dC, type 166.9, qC 108.4, CH 182.9, qC 105.8, qC 162.3, qC 95.3, CH 164.0, qC 104.7, qC 155.9, qC 70.2, CH 37.4, CH2 77.9, CH 173.3, qC 20.5, CH3 56.2, 56.6, 58.4, 51.8,

CH3 CH3 CH3 CH3

dH, mult (J in Hz) 6.04, s

6.38, s

5.09, 1.97, 2.70, 4.06,

dd (3.9, 9.9) ddd (3.9, 9.7, 14.1) ddd (3.7, 9.9, 14.1) dd (3.7, 9.7)

2.38, s 13.03, s 3.89, s 3.19, s 3.43, s 3.71, s

correlations of 5-OH with C-4a, C-5, and C-6 verified the location of the hydroxy group on C-5. This assignment was also supported by the appearance of the hydroxy proton downfield due to a chelation effect. Thus, the structure of 1 was established as shown. The proposed structure of 1 was further confirmed, along with the establishment of its relative configuration by single-crystal X-ray diffraction analysis using Mo Ka radiation, and a perspective ORTEP plot is depicted in Fig. 3. To the best of our knowledge, chromones possessing a c-lactone ring as a side chain seem to be rare. The only precedents include lachnones C–D from a filamentous fungus Lachnum sp. (Rukachaisirikul et al., 2006). Rhytidchromone B (2) was obtained as a pale yellow gum. The HRESIMS afforded an [M+Na]+ ion peak at m/z 389.1207, consistent with a molecular formula of C18H22O8, indicating eight degrees of

Fig. 2. 1H–1H COSY and key HMBC correlations of 1 and 2.

Please cite this article in press as: Chokpaiboon, S., et al. Highly oxygenated chromones from mangrove-derived endophytic fungus Rhytidhysteron rufulum. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.12.010

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S. Chokpaiboon et al. / Phytochemistry xxx (2015) xxx–xxx Table 2 H (400 MHz) and

1

Position

13

C (100 MHz) NMR spectroscopic data for 4–5 (CDCl3).

4 dC, type

Fig. 3. ORTEP drawing of compound 1.

unsaturation. The 1H and 13C NMR spectroscopic data (Table 1) as well as the HMBC and 1H–1H COSY correlations (Fig. 2) were similar to those of compound 1. However, compound 2 had no c-lactone ring in the structure due to the lack of the HMBC correlation from H-10 to an ester carbonyl at dC 172.8. This was correlated to its DBE, implying that the structure of 2 contained the oxygenated alkyl side-chain in place of the c-lactone ring in 1. In addition, proton signals of two methoxy groups were observed. The HMBC correlation of the methoxy protons at dH 3.17 with C-10 signified the attachment of this methoxy on the C-10 carbon, while the methoxy protons at dH 3.73 displayed a HMBC cross-peak with the ester carbonyl carbon (dC 172.8), thus indicating the presence of the methyl ester at C-40 . The relative configurations of compound 1 were determined on the basis of the NOESY data. In the NOESY spectrum (Fig. S12, see Supporting information), the NOE correlation between 2-Me and 40 -OMe, in combination with the lack of correlation between 10 -OMe and 30 -OMe, indicated the different orientations of the methoxy groups at C-10 and C-30 . Thus, rhytidchromone B (2) was established as shown. Rhytidchromone C (3), a pale yellow gum, had the molecular formula of C18H22O8 from the HRESIMS ion peak at m/z 389.1211, being the same as that of compound 2. The 1H and 13C NMR spectroscopic data (Table 1) of compound 3 closely resembled those of compound 2. Moreover, detailed 2D NMR analysis revealed that they shared the same overall structure. The obvious differences were the chemical shift of the proton signals for the side-chain. This indicated compounds 2 and 3 differed in the configurations at C-10 and/or C-30 . The observed NOE correlation (Fig. S19, See Supporting information) between 10 -OMe and 30 -OMe, as well as between 2-Me and 40 -OMe, supported the same orientation of the methoxy groups at C-10 and C-30 . This assignment was supported by the opposite signs of their specific rotations ([a]20 D 24.8 for 2 vs +7.2 for 3). Rhytidchromone D (4), a pale yellow gum, had the molecular formula of C17H28O8 as established by HRESIMS at m/z 375.1050. The NMR spectroscopic data (Table 2) of compound 4 were closely related to those of compound 2, except for the disappearance of signals for a methoxy group. The location of three methoxy groups at C-7, C-10 , and C-30 was confirmed by the HMBC experiment. Thus, compound 4 was identified as a carboxylic acid derivative of rhytidchromones B or C from its molecular formula, although the intensity of the carboxylic carbonyl in the 13C NMR spectrum was quite low and no HMBC cross-peak from any proton to the carbonyl carbon was observed (Fig. S21, see Supporting information). Finally, the configurations at C-10 and C-30 in compound 4 were

a

2 3 4 4a 5 6 7 8 8a 10 20

167.1, qC 108.4, CH 182.8, qC 105.1, qC 162.5, qC 95.3, CH 164.0, qC 104.8, qC 156.1, qC 70.6, CH 36.0, CH2

30 40 a 2-Me 5-OH 7-OMe 10 -OMe 30 -OMe

78.2, CH 175.0, qC 20.5, CH3 56.2, CH3 56.6, CH3 58.1, CH3

5 dH, mult (J in Hz) 6.05, s

6.39, s

5.16, 2.30, 2.64, 3.68,

t (6.8) m m br br s

2.38, s 13.05, br s 3.89, s 3.18, s 3.34, s

dC, type 167.0, qC 108.4, CH 182.8, qC 105.2, qC 162.5, qC 95.3, CH 163.9, qC 104.8, qC 156.0, qC 70.6, CH 37.0, CH2 77.8, CH 175.0, qC 20.5, CH3 56.2, CH3 56.5, CH3 58.7, CH3

dH, mult (J in Hz)

6.05, s

6.37, s

5.10, 2.00, 2.78, 4.05,

dd (4.0, 10.0) ddd (4.0, 8.4, 14.0) ddd (4.0, 10.0, 14.0) dd (4.0, 8.4)

2.38, s 13.04, br s 3.88, s 3.20, s 3.48, s

Ambiguous signal was assigned.

identified as the same as those in rhytidchromone B (2) from the almost identical chemical shift, splitting, and coupling patterns of H-10 , H2-20 , and H-30 , as well as the same sign of their specific rotations ([a]20 D 18.2 for 4 vs 24.8 for 2). Rhytidchromone E (5) was obtained as a pale yellow gum. Its molecular formula of C17H28O8 was deduced by the HRESIMS ion peak at m/z 375.1061, being the same that of compound 4. The NMR spectroscopic data (Table 2) of compound 5 were very similar to those of compound 3. The difference was the loss of signals for a methoxy group. Interpretation of 2D NMR spectroscopic data established the same overall structure as compound 4. Similarly, the identical chemical shift, splitting, and coupling patterns of H10 , H2-20 , and H-30 to those in compound 3 and the same sign of their specific rotations led to the designation of the same configurations at C-10 and C-30 . The existence of the carboxylic acid functionality as C-40 in both rhytidchromones D (4) and E (5) could be verified by an additional piece of evidence. Both compounds 4 and 5 were gradually converted into a nearly 1:1 mixture of 1 and 1a, a pair of C-10 epimers, in CDCl3 over a few days (Figs. S30 and S31, see Supporting information). The relative configurations of 1a at C-10 and C-30 were determined by the NOE correlations. The NOESY spectrum (Fig. S32, see Supporting information) of the 1 and 1a mixture showed correlations of H-10 (dH 6.00)/H-2b0 (dH 2.86) and H-20 b/ H-30 (dH 4.29) for 1, consistent with its ORTEP diagram. In the case of correlations for 1a, the correlations of H-10 (dH 6.25)/H-20 b (dH 2.70) and H-20 b (dH 2.52)/H-30 (dH 4.29), with the lack of correlation between H-20 a/H-30 , suggested the a-orientation at C-10 of the c-lactone ring. Considering the 1/1a ratio of the obtained mixture indicated, upon exposure to the acidity of CDCl3, the benzylic carbocation might first be formed owing to its stability, and the lactone ring is subsequently formed by the attack of the hydroxyl lone pair of 40 -COOH on carbocation, which can occur from either side of the planar, sp2-hybridized carbocation to afford compounds 1 and 1a as shown in Scheme 1. Therefore, it seems reasonable to postulate that the biosynthetic origin of 1 would probably be compounds 4 and 5. This also supports the relative configurations of compounds 2 and 3, the methyl ester derivatives of 4 and 5, respectively. The isolated compounds 1–5 were evaluated for their cytotoxicity against human breast (MCF-7), liver (Hep-G2), cervical (CaSki)

Please cite this article in press as: Chokpaiboon, S., et al. Highly oxygenated chromones from mangrove-derived endophytic fungus Rhytidhysteron rufulum. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.12.010

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Scheme 1. Proposed mechanism for the formation of a mixture of 1 and 1a from 4 or 5.

Table 3 Cytotoxicity of compounds 1–5. Compounds

1 2 3 4 5 Doxorubicin

IC50 (lM) MCF-7

Hep-G2

Kato-3

CaSki

19.3 ± 2.5 >25 >25 >25 17.7 ± 3.7 1.0 ± 0.1

>25 >25 >25 >25 >25 1.0 ± 0.1

23.3 ± 1.1 21.4 ± 2.3 >25 16.8 ± 3.6 16.0 ± 1.9 2.7 ± 0.5

>25 >25 >25 >25 >25 1.1 ± 0.5

and gastric (KATO-3) cancer cell lines by the MTT method (Table 3). Doxorubicin was used as a positive control, since it is an anticancer drug widely used in the clinic for the treatment of a broad spectrum of cancers (Buzdar et al., 1985). Compound 1 showed cytotoxicity against MCF-7 and Kato-3 with IC50 values of 19.3 ± 2.5 and 23.3 ± 1.1 lM, respectively, while compound 5 gave IC50 of 17.7 ± 3.7 and 16.0 ± 1.9 lM, respectively. Compounds 2 and 4 were active only against Kato-3 cells with IC50 values of 21.4 ± 2.3 and 16.8 ± 3.6 lM, respectively. Only compound 3 was inactive against all tested cell lines, and none of them was active on Hep-G2 and CaSki cells (>25 lM).

reference. HRESIMS were performed on a Bruker micrOTOF. Single-crystal X-ray diffraction analysis was performed on a Bruker APEX II diffractometer. Silica gel (Merck, 230–400 mesh), Sephadex LH20 (GE Healthcare), and ODS (WAKOÒ100C18) were used for open-column chromatography (CC). Analytical TLC was performed using precoated silica gel 60 GF254 plates (Merck). 4.2. Fungal material The fungal strain BG2-Y was isolated from the healthy leaves of B. gymnorrhiza collected from Pak Nam Pran, Prachuab Kiri Khan Province, Thailand, in July 2012. The fungus was identified based on the internal transcribed spacer (ITS1-5.8S-ITS2) rDNA and on partial 28S rRNA analyses using universal fungal primers. Its partial 28S rRNA sequence (GenBank accession number KP994148) showed affinity within the class of Dothideomycetes; sub-class Pleosporomycetidae; order Hysteriales; family Hysteriaceae. The ITS sequence (GenBank accession number KP994147) of this isolate demonstrated a high 98–99% matching similarity with R. rufulum from both GenBank (http://www.ncbi.nlm.nih.gov/) and CBSKNAW Fungal Biodiversity Centre (http://www.cbs.knaw.nl/). Thus, the fungus was identified as R. rufulum. 4.3. Fermentation, extraction, and isolation

3. Conclusions Rhytidchromones A–E (1–5), five chromones with a highly oxygenated side-chain, were isolated from the mycelial and culture broth EtOAc extracts of a mangrove-derived endophytic fungus, R. rufulum, obtained from Thai B. gymnorrhiza. Their structures were determined on the basis of extensive spectroscopic analyses. In addition, the structure of compound 1 was further confirmed by single-crystal X-ray diffraction analysis, along with the establishment of its relative configuration. Compound 1 might be derived from compounds 4 and 5 via a C-10 benzylic carbocation. However, only compound 1 was obtained from the EtOAc crude extract isolation, while a 1:1 mixture of 1 and 1a was observed when compounds 4 and 5 were exposed to the acidity of CDCl3. Based on this fact, it could be concluded that compound 1 is not an artifact produced during the separation, but genuine natural product. 4. Experimental section 4.1. General experimental procedures Melting points were determined on a M565 melting point apparatus and are uncorrected. Optical rotations were measured on a JASCO P-1010 polarimeter. UV spectra were recorded on a Spekol 1200 Analytic Jena spectrophotometer. IR spectra were recorded on a Bruker ALPHA FT-IR spectrometer. NMR spectra were acquired on a Varian Mercury-400 Plus NMR spectrometer with TMS as

The fungal strain was cultured on potato dextrose agar at room temperature for 7 days. Then, six pieces (6  6 mm2) of agar plugs were inoculated in an Erlenmeyer flask (1000 mL) containing 200 mL of Sabouraud dextrose broth (SDB) (25). Ultimately, the fungus was statically cultured at room temperature for 21 days. After incubation, the fungal culture (5 L) was filtered to remove mycelia, and the culture broth was extracted twice with an equal volume of EtOAc. The combined EtOAc extract was concentrated under reduced pressure to afford a dark brown gum (4.62 g). The separated mycelia were successively extracted with hexane and EtOAc (3 for each). The EtOAc soluble part was evaporated under reduced pressure and dried under vacuum to yield a dark brown gum (0.87 g). The EtOAc crude extract of culture broth (4.62 g) was subjected to Sephadex LH20 CC and eluted with MeOH to yield five fractions (A1–A5). Fraction A3 (3.58 g) was applied to a Sephadex LH20 column eluting with MeOH again to yield six subfractions (B1–B6). Subfraction B4 (0.36 g) was further isolated by silica gel CC, eluting with a 1:1 mixture of EtOAc-hexane to yield nine fractions (C1– C9). Fraction C2 (72.0 mg) was subsequently purified by Sephadex LH20 CC, eluting with MeOH to afford compound 1 (28.7 mg). Fraction A2 (0.36 g) was subjected to reversed phase ODS CC with MeOH:H2O (1:1) to yield seven subfractions (D1–D7). Subfraction D6 (95.0 mg) was applied to a silica gel column and eluted with MeOH–CH2Cl2 (1:19), then the major fraction was further purified by a reversed phase ODS column eluting with MeOH:H2O (3:7) to

Please cite this article in press as: Chokpaiboon, S., et al. Highly oxygenated chromones from mangrove-derived endophytic fungus Rhytidhysteron rufulum. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.12.010

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yield compounds 2 (4.5 mg) and 3 (4.0 mg). Fraction B5 (1.63 g) was subjected to silica gel CC eluting with EtOAc–hexane (2:3), and subsequently further purified on a Sephadex LH20 column eluting with MeOH to afford compound 4 (4.0 mg). The mycelial EtOAc crude extract (0.87 g) was subjected to Sephadex LH20 CC eluting with MeOH to yield six fractions (E1– E6). Fraction E3 (0.18 g) was further fractionated by ODS CC eluting with MeOH:H2O (1:1) to give seven subfractions (F1–F7). Subfraction F7 (49.1 mg) was further purified by silica gel CC eluting with MeOH–CH2Cl2 (1:19) to afford compound 5 (4.0 mg). 4.4. Rhytidchromone A (1) Colorless crystals; mp 149–150 °C; UV (MeOH) kmax (log e) 237 (3.53), 255 (3.48), 290 (3.20), 319 (3.06); IR (neat) tmax 3358, 1732, 1640, 1423, 1212 cm1; for 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS m/z 343.0784 [M+Na]+ (calcd. for C16H16O7Na, 343.0788). 4.5. Rhytidchromone B (2) Pale yellow gum; UV (MeOH) kmax (log e) 246 (3.58), 265 (3.64), 291 (3.50); IR (neat) tmax 3346, 1743, 1638, 1539, 1404, 1242 cm1; for 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS m/z 389.1207 [M+Na]+ (calcd. for C18H22O8Na, 389.1212). 4.6. Rhytidchromone C (3) Pale yellow gum; UV (MeOH) kmax (log e) 245 (3.62), 266 (3.68), 292 (3.58); IR (neat) tmax 3365, 1721, 1642, 1536, 1403, 1236 cm1; for 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS m/z 389.1211 [M+Na]+ (calcd. for C18H22O8Na, 389.1212). 4.7. Rhytidchromone D (4) Pale yellow gum; UV (MeOH) kmax (log e) 247 (3.59), 265 (3.64), 289 (3.52); IR (neat) tmax 3349, 1727, 1639, 1535, 1403, 1236 cm1; for 1H and 13C NMR spectroscopic data, see Table 2; HRESIMS m/z 375.1056 [M+Na]+ (calcd. for C17H28O8Na, 375.1061). 4.8. Rhytidchromone E (5) Pale yellow gum; UV (MeOH) kmax (log e) 244 (3.55), 260 (3.61), 289 (3.46); IR (neat) tmax 3359, 1725, 1635, 1540, 1405, 1235 cm1; for 1H and 13C NMR spectroscopic data, see Table 2; HRESIMS m/z 375.1050 [M+Na]+ (calcd. for C17H28O8Na, 375.1061). 4.9. Compound 1a 1

H NMR data dH 13.07 (brs, 5-OH), 6.38 (s, H-6), 6.25 (dd, J = 4.8, 6.8 Hz, H-10 ), 6.06 (s, H-3), 4.19 (dd, J = 2.8, 6.4 Hz, H-30 ), 3.89 (s, 7-OMe), 3.61 (s, 30 -OMe), 2.69 (ddd, J = 5.2, 6.4, 11.2 Hz, H-20 ), 2.52 (m, H-20 ). 4.10. X-ray crystallographic analysis of rhytidchromone A (1) All crystallographic data were collected at 298 K on a Bruker APEX II diffractometer with Mo Ka radiation (k = 0.71073). The structures were solved by direct methods using SHELXS-97 and refined by full-matrix least-squares on all F2 data using SHELXS97 to final R values (Sheldrick, 1997a,b). All hydrogen atoms were added at calculated positions and refined using a rigid model. Crystallographic data for 1 have been deposited with the Cambridge Crystallographic Data Centre with the deposition number CCDC 1406338. Copies of the data can be obtained, free of charge, via www.ccdc.cam.ac.uk/data_request/cif, by e-mailing data_

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[email protected], or by contacting the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax:+44 1223 336033. Crystal data of rhytidchromone A (1): colorless crystal, C16H16O7, Mr = 320.30, monoclinic, a = 8.0040(4) Å, b = 8.6152(4) Å, c = 11.7898(4) Å, space group P21, Z = 2, V = 766.45(6) Å3, and l (Mo Ka) = 0.11 mm1. Crystal dimensions: 0.50  0.20  0.20 mm. Independent reflections: 2347 (Rint = 0.016). The final R1 values were 0.030, wR2 = 0.060 (I > 2r (I)). 4.11. Cytotoxic assay Cytotoxicity of isolated compounds against human breast (MCF-7), liver (Hep-G2), cervical (CaSki), and gastric (Kato-3) cancer cell lines was assessed using the MTT (3-[4,5-dimethylthiazol2-yl-2,5-diphenyltetrazolium] bromide) assay according to previously described procedures (Scudiero et al., 1988). Briefly, freshly trypsinized cell suspensions were seeded into 96-well culture plates at densities of 1  104 cells in the presence or absence of test compounds. After incubation at 37 °C for 72 h, MTT solution (10 lL, 5 mg in 1 mL PBS) was added to each well for 4 h. Cell-free supernatant was removed, and DMSO was then added to dissolve the formazan crystals. Absorbance values were measured with a microplate reader at 540 nm. Experiments were operated in triplicate, and data are described as the mean ± SEM of three independent experiments. Doxorubicin was used as a positive control. Acknowledgements This research was financially supported by the Thailand Research Fund (Grant No. RSA5580023) and the 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund). The authors wish to thank Dr. Rick Attrill, Department of Chemistry, Faculty of Science, Chulalongkorn University, for language proving. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2015. 12.010. References Amrani, M.E., Lai, D., Debbab, A., Aly, A.H., Siems, K., Seidel, C., Schnekenburger, M., Gaigneaux, A., Diederich, M., Feger, D., Lin, W., Proksch, P., 2014. Protein kinase and HDAC inhibitors from the endophytic fungus Epicoccum nigrum. J. Nat. Prod. 77, 49–56. Buzdar, A.U., Marcus, C., Smith, T.L., Blumenschein, G.R., 1985. Cancer 55, 2761– 2765. Chokpaiboon, S., Sommit, D., Bunyapaiboonsri, T., Matsubara, K., Pudhom, K., 2011. Antiangiogenic effect of chamigrane endoperoxides from a Thai mangrovederived fungus. J. Nat. Prod. 74, 2290–2294. Chokpaiboon, S., Sommit, D., Teerawatananond, T., Muangsin, N., Bunyapaiboonsri, T., Pudhom, K., 2010. Cytotoxic nor-chamigrane and chamigrane endoperoxides from a basidiomycetous fungus. J. Nat. Prod. 73, 1005–1007. Debbab, A., Aly, A.H., Proksch, P., 2013. Mangrove derived fungal endophytes: a chemical and biological perception. Fungal Divers. 61, 1–27. Faeth, S.H., 2002. Are endophytic fungi defensive plant mutualists? Okios 98, 25–36. Kharwar, R.N., Mishra, A., Gond, S.K., Stierle, A., Stierle, D., 2011. Anticancer compounds derived from fungal endophytes: their importance and future challenges. Nat. Prod. Rep. 28, 1208–1228. Kusari, S., Spiteller, M., 2011. Are we ready for industrial production of bioactive plant secondary metabolites utilizing endophytes? Nat. Prod. Rep. 28, 1203– 1207. Li, J., Li, L., Si, Y., Jiang, X., Guo, L., Che, Y., 2011. Viragatolides A–C, benzannulated spiroketals from the plant endophytic fungus Pestalotiopsis virgatula. Org. Lett. 13, 2670–2673. Liu, L., Chen, X., Li, D., Zhang, Y., Li, L., Guo, L., Cao, Y., Che, Y., 2015. Bisabolane sesquiterpenoids from the plant endophytic fungus Paraconiothyrium brasiliense. J. Nat. Prod. 78, 746–753.

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Please cite this article in press as: Chokpaiboon, S., et al. Highly oxygenated chromones from mangrove-derived endophytic fungus Rhytidhysteron rufulum. Phytochemistry (2015), http://dx.doi.org/10.1016/j.phytochem.2015.12.010