Two New Secondary Metabolites from the Endophytic Fungus Endomelanconiopsis endophytica

Two new secondary metabolites, endomeketals A–B (1–2), a new natural product (3), and a known compound (4) were isolated from the ethyl acetate extract of the endophytic fungus Endomelanconiopsis endophytica A326 derived from Ficus hirta. Their structures were determined on the basis of extensive spectroscopic analysis. All compounds were evaluated for their cytotoxic activities against SF-268, MCF-7, NCI-H460, and HepG-2 tumor cell lines. However, no compound showed cytotoxic activity against these human tumor cell lines.


Introduction
Endophytes are microorganisms that reside in the tissues of living plants asymptomatically, and the mutualistic interaction that occurs between the two may have benefits to each other. These organisms represent a huge and largely untapped resource of natural products with chemical structures that have been optimized by evolution for biological and ecological relevance [1]. Over the past 20 years, a surprisingly high number of metabolites have been described from endophytes [2][3][4][5][6][7][8]. Several secondary metabolites, including alkaloids [9], sesquiterpenoids [10], and azaphilones [11], exhibit a variety of biological activities. Ficus hirta Vahl. (Moraceae), endemic to Guangdong province of China, is known as "Wu Zhi Mao Tao" in traditional Chinese medicine for the treatment of cough, asthma, and inflammatory diseases [12]. As part of an ongoing program aimed at exploring bioactive secondary metabolites from endophytic fungi [13][14][15], we undertook a detailed chemical analysis on the ethyl acetate (EtOAc) extract of Endomelanconiopsis endophytica A326 derived from the medicinal plant Ficus hirta, which led to the isolation of two new secondary metabolites (1-2) and two known compounds (3)(4). All compounds were evaluated for their cytotoxic activities against SF-268, MCF-7, NCI-H460, and HepG-2 tumor cell lines. Herein, details of the isolation and structural elucidation of these compounds are described.

Results and Discussion
The liquid culture of the fungus E. endophytica was centrifuged to separate broth and mycelia. The broth was exhaustively extracted with ethyl acetate (EtOAc), and the concentrated EtOAc extract was further purified by various chromatographic methods to yield four compounds (1-4) ( Figure 1). Two new compounds, endomeketals A-B (1-2), were identified by spectroscopic analyses physicochemical properties, while a new natural product and a known compound were identified as 2,3-dimethylcyclopent-2-en-1-one (3) [16,17] and 2-hydroxymethyl-3-methylcyclopent-2-enone (4) [18] by comparison of their spectroscopic data with those in the literature.
Detailed 2D-NMR studies (HSQC, 1 H-1 H-COSY, and HMBC experiments) afforded the gross structures of two sub-units (A and B) as depicted in Figure 2. 1 H-1 H-COSY correlation between H-4 and H-5 and HMBC correlations from H3-7 to C-2/C-3/C-4, from H-5 to C-1, and from H-6 to C-1/C-2/C-3, revealed unit A was highly similar to 2-hydroxymethyl-3-methylcyclopent-2-enone (4) (Figure 2), which was further confirmed by comparison of their 1D-NMR data. As for unit B, the structural fragments (C-1′→C-7′) were established by the 1 H-1 H-COSY correlations of C-1′/C-2′/C-3′/C-4′/C-5′/C-1′, and C-2′/ C-6′ and C-3′/C-7′. The connectivities of two sub-units were mainly achieved by HMBC interactions from H-6 to C-1′ and C-6′. The relative configuration of 1 was determined by a NOESY experiment. NOESY correlations of H-6/H-1′/H-2′/H-3′ and Me-7′/H-6′ indicated that H-6, H-1′, H-2′, and H-3′ were cofacial and designated as α-oriented ( Figure S6). Thus, compound 1 was determined as depicted and given the trivial name endomeketal A. HRESIMS analysis of 2 revealed an adduct ion [M + Na] + consistent with a molecular formula (C11H16O3) requiring four double bond equivalents ( Figure S14). The 1D-NMR data of 2 were similar to those of 1 except for the absence of signals for the saturated pentacyclic unit and the presence of the signals for two methyls and two sp 3 methines bearing heteroatoms (Table 1, Figures S8 and S9). The gross structure of 2 was established by analyses of its 2D-NMR data (Figures S10-S13). COSY correlations revealed a 1 H spin system from C-1′ to C-4′, so that the sub-structure B in 1 was substituted in 2 by a butane-2,3-diyl residue. The carbon chemical shifts assigned to unit A in 2 were nearly identical to the corresponding carbon chemical shifts assigned to this fragment in 1. The HMBC interactions from H-6 to C-1, C-2, and C-3, and from H-2′ to C-6 connected unit B to unit A. As three of the four degrees of unsaturation were accounted for by the unsaturated pentacyclic unit, the remaining double bond equivalent required that unit B and unit A in 2 were connected via a 1,3-dioxolane. The relative configuration of 2 was determined by a NOESY experiment. NOESY correlations of H-6/H-3′ and H-3′/H3-1′ indicated that H3-1′, H-3′, and H-6 were cofacial and designated as α-oriented, while correlation of H-2′/H3-4′ assigned H-2′ and H3-4′ as β. Thus, compound 2 was assigned as depicted and named endomeketal B.

Cytotoxicity Assay
The in vitro cytotoxicities of compounds 1-4 were evaluated against four tumor cell lines,  Detailed 2D-NMR studies (HSQC, 1 H-1 H-COSY, and HMBC experiments) afforded the gross structures of two sub-units (A and B) as depicted in Figure 2. 1 H-1 H-COSY correlation between H-4 and H-5 and HMBC correlations from H3-7 to C-2/C-3/C-4, from H-5 to C-1, and from H-6 to C-1/C-2/C-3, revealed unit A was highly similar to 2-hydroxymethyl-3-methylcyclopent-2-enone (4) (Figure 2), which was further confirmed by comparison of their 1D-NMR data. As for unit B, the structural fragments (C-1′→C-7′) were established by the 1 H-1 H-COSY correlations of C-1′/C-2′/C-3′/C-4′/C-5′/C-1′, and C-2′/ C-6′ and C-3′/C-7′. The connectivities of two sub-units were mainly achieved by HMBC interactions from H-6 to C-1′ and C-6′. The relative configuration of 1 was determined by a NOESY experiment. NOESY correlations of H-6/H-1′/H-2′/H-3′ and Me-7′/H-6′ indicated that H-6, H-1′, H-2′, and H-3′ were cofacial and designated as α-oriented ( Figure S6). Thus, compound 1 was determined as depicted and given the trivial name endomeketal A. HRESIMS analysis of 2 revealed an adduct ion [M + Na] + consistent with a molecular formula (C11H16O3) requiring four double bond equivalents ( Figure S14). The 1D-NMR data of 2 were similar to those of 1 except for the absence of signals for the saturated pentacyclic unit and the presence of the signals for two methyls and two sp 3 methines bearing heteroatoms (Table 1, Figures S8 and S9). The gross structure of 2 was established by analyses of its 2D-NMR data ( Figures S10-S13). COSY correlations revealed a 1 H spin system from C-1′ to C-4′, so that the sub-structure B in 1 was substituted in 2 by a butane-2,3-diyl residue. The carbon chemical shifts assigned to unit A in 2 were nearly identical to the corresponding carbon chemical shifts assigned to this fragment in 1. The HMBC interactions from H-6 to C-1, C-2, and C-3, and from H-2′ to C-6 connected unit B to unit A. As three of the four degrees of unsaturation were accounted for by the unsaturated pentacyclic unit, the remaining double bond equivalent required that unit B and unit A in 2 were connected via a 1,3-dioxolane. The relative configuration of 2 was determined by a NOESY experiment. NOESY correlations of H-6/H-3′ and H-3′/H3-1′ indicated that H3-1′, H-3′, and H-6 were cofacial and designated as α-oriented, while correlation of H-2′/H3-4′ assigned H-2′ and H3-4′ as β. Thus, compound 2 was assigned as depicted and named endomeketal B.

Cytotoxicity Assay
The in vitro cytotoxicities of compounds 1-4 were evaluated against four tumor cell lines, Detailed 2D-NMR studies (HSQC, 1 Figure 2. 1 H-1 H-COSY correlation between H-4 and H-5 and HMBC correlations from H3-7 to C-2/C-3/C-4, from H-5 to C-1, and from H-6 to C-1/C-2/C-3, revealed unit A was highly similar to 2-hydroxymethyl-3-methylcyclopent-2-enone (4) (Figure 2), which was further confirmed by comparison of their 1D-NMR data. As for unit B, the structural fragments (C-1′→C-7′) were established by the 1 H-1 H-COSY correlations of C-1′/C-2′/C-3′/C-4′/C-5′/C-1′, and C-2′/ C-6′ and C-3′/C-7′. The connectivities of two sub-units were mainly achieved by HMBC interactions from H-6 to C-1′ and C-6′. The relative configuration of 1 was determined by a NOESY experiment. NOESY correlations of H-6/H-1′/H-2′/H-3′ and Me-7′/H-6′ indicated that H-6, H-1′, H-2′, and H-3′ were cofacial and designated as α-oriented ( Figure S6). Thus, compound 1 was determined as depicted and given the trivial name endomeketal A. HRESIMS analysis of 2 revealed an adduct ion [M + Na] + consistent with a molecular formula (C11H16O3) requiring four double bond equivalents ( Figure S14). The 1D-NMR data of 2 were similar to those of 1 except for the absence of signals for the saturated pentacyclic unit and the presence of the signals for two methyls and two sp 3 methines bearing heteroatoms (Table 1, Figures S8 and S9). The gross structure of 2 was established by analyses of its 2D-NMR data (Figures S10-S13). COSY correlations revealed a 1 H spin system from C-1′ to C-4′, so that the sub-structure B in 1 was substituted in 2 by a butane-2,3-diyl residue. The carbon chemical shifts assigned to unit A in 2 were nearly identical to the corresponding carbon chemical shifts assigned to this fragment in 1. The HMBC interactions from H-6 to C-1, C-2, and C-3, and from H-2′ to C-6 connected unit B to unit A. As three of the four degrees of unsaturation were accounted for by the unsaturated pentacyclic unit, the remaining double bond equivalent required that unit B and unit A in 2 were connected via a 1,3-dioxolane. The relative configuration of 2 was determined by a NOESY experiment. NOESY correlations of H-6/H-3′ and H-3′/H3-1′ indicated that H3-1′, H-3′, and H-6 were cofacial and designated as α-oriented, while correlation of H-2′/H3-4′ assigned H-2′ and H3-4′ as β. Thus, compound 2 was assigned as depicted and named endomeketal B.

Cytotoxicity Assay
The in vitro cytotoxicities of compounds 1-4 were evaluated against four tumor cell lines,  Detailed 2D-NMR studies (HSQC, 1 H-1 H-COSY, and HMBC experiments) afforded the gross structures of two sub-units (A and B) as depicted in Figure 2. 1 H-1 H-COSY correlation between H-4 and H-5 and HMBC correlations from H3-7 to C-2/C-3/C-4, from H-5 to C-1, and from H-6 to C-1/C-2/C-3, revealed unit A was highly similar to 2-hydroxymethyl-3-methylcyclopent-2-enone (4) (Figure 2), which was further confirmed by comparison of their 1D-NMR data. As for unit B, the structural fragments (C-1′→C-7′) were established by the 1 H-1 H-COSY correlations of C-1′/C-2′/C-3′/C-4′/C-5′/C-1′, and C-2′/ C-6′ and C-3′/C-7′. The connectivities of two sub-units were mainly achieved by HMBC interactions from H-6 to C-1′ and C-6′. The relative configuration of 1 was determined by a NOESY experiment. NOESY correlations of H-6/H-1′/H-2′/H-3′ and Me-7′/H-6′ indicated that H-6, H-1′, H-2′, and H-3′ were cofacial and designated as α-oriented ( Figure S6). Thus, compound 1 was determined as depicted and given the trivial name endomeketal A. HRESIMS analysis of 2 revealed an adduct ion [M + Na] + consistent with a molecular formula (C11H16O3) requiring four double bond equivalents ( Figure S14). The 1D-NMR data of 2 were similar to those of 1 except for the absence of signals for the saturated pentacyclic unit and the presence of the signals for two methyls and two sp 3 methines bearing heteroatoms (Table 1, Figures S8 and S9). The gross structure of 2 was established by analyses of its 2D-NMR data ( Figures S10-S13). COSY correlations revealed a 1 H spin system from C-1′ to C-4′, so that the sub-structure B in 1 was substituted in 2 by a butane-2,3-diyl residue. The carbon chemical shifts assigned to unit A in 2 were nearly identical to the corresponding carbon chemical shifts assigned to this fragment in 1. The HMBC interactions from H-6 to C-1, C-2, and C-3, and from H-2′ to C-6 connected unit B to unit A. As three of the four degrees of unsaturation were accounted for by the unsaturated pentacyclic unit, the remaining double bond equivalent required that unit B and unit A in 2 were connected via a 1,3-dioxolane. The relative configuration of 2 was determined by a NOESY experiment. NOESY correlations of H-6/H-3′ and H-3′/H3-1′ indicated that H3-1′, H-3′, and H-6 were cofacial and designated as α-oriented, while correlation of H-2′/H3-4′ assigned H-2′ and H3-4′ as β. Thus, compound 2 was assigned as depicted and named endomeketal B.

Cytotoxicity Assay
The in vitro cytotoxicities of compounds 1-4 were evaluated against four tumor cell lines,  Figure S14). The 1D-NMR data of 2 were similar to those of 1 except for the absence of signals for the saturated pentacyclic unit and the presence of the signals for two methyls and two sp 3 methines bearing heteroatoms (Table 1, Figures S8 and S9). The gross structure of 2 was established by analyses of its 2D-NMR data (Figures S10-S13). COSY correlations revealed a 1 H spin system from C-1 1 to C-4 1 , so that the sub-structure B in 1 was substituted in 2 by a butane-2,3-diyl residue. The carbon chemical shifts assigned to unit A in 2 were nearly identical to the corresponding carbon chemical shifts assigned to this fragment in 1. The HMBC interactions from H-6 to C-1, C-2, and C-3, and from H-2 1 to C-6 connected unit B to unit A. As three of the four degrees of unsaturation were accounted for by the unsaturated pentacyclic unit, the remaining double bond equivalent required that unit B and unit A in 2 were connected via a 1,3-dioxolane. The relative configuration of 2 was determined by a NOESY experiment. NOESY correlations of H-6/H-3 1 and H-3 1 /H 3 -1 1 indicated that H 3 -1 1 , H-3 1 , and H-6 were cofacial and designated as α-oriented, while correlation of H-2 1 /H 3 -4 1 assigned H-2 1 and H 3 -4 1 as β. Thus, compound 2 was assigned as depicted and named endomeketal B.

Fungal Material
The endophytic fungal strain A326 was isolated from the twigs of Ficus hirta, which was collected at Luofu Mountain Nature Reserve, Huizhou, Guangdong province of China, in October 2010. The isolated strain was identified as Endomelanconiopsis endophytica based on a morphological study, and sequence analysis of rDNA ITS (internal transcribed spacer), showing 100% similarity to the strain of E. endophytica (Accession No. EU687005). The strain is preserved at the Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Institute of Microbiology.

Extraction and Isolation
E. endophytica A326 was grown on potato-dextrose agar (PDA) medium at 28˝C for five days and then inoculated into five flasks (500 mL) containing potato-dextrose (PD) medium (250 mL). After five days of incubation at 28˝C on a rotary shaker at 120 r/min, a portion of the liquid culture was aseptically transferred into each of a total of 200 flasks (500 mL) containing potato-dextrose (PD) medium (250 mL). Following seven days of cultivation at 28˝C and 120 r/min on a rotary shaker, the culture (a total of 50 L) was filtered to give the filtrate and mycelia. The broth was exhaustively extracted with ethyl acetate (EtOAc) for four times, and then the EtOAc layers were combined and evaporated under reduced pressure at a temperature not exceeding 40˝C to yield a dark brown gum (11.7 g). The crude extract was subjected to silica gel column chromatography with a CH 2 Cl 2 /MeOH gradient (9:1Ñ1:9) to afford six fractions (Fr. I-VI). Fr. II (1.1 g) was subjected to column chromatography on silica gel using n-hexane as the first eluent, and then EtOAc of increasing polarity, to give nine sub-fractions (Fr. II a´F r. II i ). Fr.II d was further separated by RP-HPLC equipped with a semi-preparative column (CH 3 CN, 3 mL/min) to afford 2 (5 mg, t R 7.6 min) and 3 (19 mg, t R 9.1 min). Fr. II e was chromatographed by RP-HPLC equipped with a semi-preparative column (MeOH/H 2 O, 70:30, 3 mL/min) to afford 4 (36 mg, t R 7.2 min) and 1 (24 mg, t R 13.0 min).  Figure S7); 1 H-and 13 C-NMR data, see Table 1.  Figure S14); 1 H-and 13 C-NMR data, see Table 1.

Conclusions
Fungal endophytes live inside plant tissues for all, or part, of their life without causing apparent disease symptoms. Endophytic fungi have been found within host tissues at high density and diversity in all tropical and temperate ecosystems where endophytes have been sought [20]. Endomelanconiopsis is a new anamorph genus in the Botryosphaeriaceae, and usually lives inside plant tissues [21]. Previous investigation onto E. endophytica A326 in our research group led to the isolation of a series of xyloketals, which were the first chemical constituents of the genus Endomelanconiopsis [22]. As the fermentation products of the fungus E. endophytica displayed considerable structural diversity, continued investigation of this fungus has resulted in the isolation of two further new secondary metabolites, which were named as endomeketals A and B. The structures were determined by spectroscopic analysis. All of the isolates were evaluated for in vitro cytotoxicity against SF-268, MCF-7, NCI-H460, and HepG-2 cell lines. This study not only enriches the chemical diversity of the genus Endomelanconiopsis, but also implies that this endophytic fungus may be a potential source for producing a series of ketals.