Cytochalasans from the Endophytic Fungus Phomopsis sp. shj2 and Their Antimigratory Activities

Cytochalasans from the endophytic fungi featured structure diversity. Our previous study has disclosed that cytochalasans from the endophytic fungus Phomopsis sp. shj2 exhibited an antimigratory effect. Further chemical investigation on Phomopsis sp. shj2 has led to the discovery of seven new cytochalasans (1–7), together with four known ones. Their structures were elucidated through extensive spectroscopic data interpretation and single-crystal X-ray diffraction analysis. Compounds 1–3 and 8–11 exhibited antimigratory effects against MDA-MB-231 in vitro with IC50 values in the range of 1.01−10.42 μM.

Tumour spread is a major concern in cancer therapeutics as cancer metastasis is responsible for 90% of deaths from solid tumours [15]. Natural products with antimigratory activity represent a highly interesting field to explore for cancer chemoprevention and therapy. Fungi are emerging as a natural source, such as Diaporthe [16], Isaria [17], and Phenicillium [18,19] genera. Chemical investigations on endophytes of Isodon species have disclosed structurally diverse and bioactive natural products [19][20][21][22]. Phomopchalasins B and C were isolated from the endophytic fungus Phomopsis sp. shj2 from the stems of Isodon eriocalyx var. laxiflora and exhibited in vitro antimigratory effects against MDA-MB231 [19]. In our continuous efforts for more bioactive structures, the strain was further investigated by one strain-many compounds strategy (OSMAC), which led to the isolation of seven new cytochalasans (1-7), along with four known ones ( Figure 1). Herein, we report the isolation, structure elucidation, and antimigratory activities of these cytochalasans.

Fungal Material
The culture of Phomopsis sp. shj2 was isolated from the stems of Isodon eriocalyx var. laxiflora collected from Kunming Botanical Garden, Kunming, People's Republic of China, in December 2012. The isolate was identified based on sequence (GenBank Accession No. KU533636) analysis of the ITS region of the rDNA. The fungal strain was cultured on slants of potato dextrose agar at 25 °C for 7 days. Agar plugs were cut into small pieces (about 0.5 × 0.5 × 0.5 cm 3 ) under aseptic conditions, and 15 pieces were used to inoculate three Erlenmeyer flasks (250 mL), each containing 50 mL of media (0.4% glucose, 1% malt extract, and 0.4% yeast extract); the final pH of the media was adjusted to 6.5, and the flasks were sterilized by autoclave. Three flasks of the inoculated media were incubated at 28 °C on a rotary shaker at 180 rpm for 5 days to prepare the seed culture. Fermentation was carried out in 125 Fernbach flasks (500 mL), each containing 80 g of rice. Spore inoculum was prepared in sterile, distilled H2O to give a final spore/cell suspension of 1 × 10 6 /mL. Distilled H2O (120 mL) was added to each flask, and the contents were soaked overnight before autoclaving at 15 psi for 30 min. After cooling to room temperature, each flask was inoculated with 5.0 mL of the spore inoculum and incubated at 28 °C for 42 days.

Extraction and Isolation
The fermented material was extracted with EtOAc (4 × 10.0 L) and the organic solvent was evaporated to dryness under vacuum to afford a crude extract (170 g). The crude extract was purified by CC (column chromatography on SiO2 with CHCl3/acetone gradient system 1:0, 9:1, 8:2, 7:3, 6:4 and 1:1) to yield six main fractions, Fr.s A-F. Fr. B was subjected to chromatography over silica gel CC (petroleum ether-EtOAc) to give

Fungal Material
The culture of Phomopsis sp. shj2 was isolated from the stems of Isodon eriocalyx var. laxiflora collected from Kunming Botanical Garden, Kunming, People's Republic of China, in December 2012. The isolate was identified based on sequence (GenBank Accession No. KU533636) analysis of the ITS region of the rDNA. The fungal strain was cultured on slants of potato dextrose agar at 25 • C for 7 days. Agar plugs were cut into small pieces (about 0.5 × 0.5 × 0.5 cm 3 ) under aseptic conditions, and 15 pieces were used to inoculate three Erlenmeyer flasks (250 mL), each containing 50 mL of media (0.4% glucose, 1% malt extract, and 0.4% yeast extract); the final pH of the media was adjusted to 6.5, and the flasks were sterilized by autoclave. Three flasks of the inoculated media were incubated at 28 • C on a rotary shaker at 180 rpm for 5 days to prepare the seed culture. Fermentation was carried out in 125 Fernbach flasks (500 mL), each containing 80 g of rice. Spore inoculum was prepared in sterile, distilled H 2 O to give a final spore/cell suspension of 1 × 10 6 /mL. Distilled H 2 O (120 mL) was added to each flask, and the contents were soaked overnight before autoclaving at 15 psi for 30 min. After cooling to room temperature, each flask was inoculated with 5.0 mL of the spore inoculum and incubated at 28 • C for 42 days.

X-ray Crystal Structure Analysis
The intensity data for 1 and 3 were collected on a Bruker APEX DUO diffractometer using graphite-monochromated Cu Kα radiation. The structures of these compounds were solved by direct methods (SHELXS97), expanded using difference Fourier techniques, and refined by the program and full-matrix least-squares calculations. The non-hydrogen atoms were refined anisotropically, and hydrogen atoms were fixed at calculated positions.

Antimigration Assay
Cell migration was determined using the Oris™ Pro Cell Migration Assay (Platypus Technologies, Madison, WI, USA), according to the manufacturer's protocol. Briefly, MDA-MB-231 cells were seeded and incubated (37 • C, 5% CO 2 ) for 1 h, and then indicated concentrations of compounds were added and incubated with cells for an additional 18 h. At the end of incubation, the cell viability was evaluated with MTS assays and the migration area of each group was calculated and analysed, and the results of each subgroup were presented as a percentage of DMSO-treated cells.

Structure Elucidation
The molecular formula of 18-acetoxycytochalasin H (1) was determined to be C 32 H 41 NO 6 (Table 2) displayed resonances for 32 carbons, ascribed to 5 methyls, 4 methylenes (including 1 olefinic), 11 methines (4 olefinic and 2 oxygenated), 61 quaternary carbons (1 olefinic, 1 amide and 2 ester carbonyls), and 6 other signals assignable to the single-substituted phenyl group. Thus, the above-mentioned results indicated that 1 should be a new tetracyclic cytochalasin including a benzene ring, with structural similarity with cytochalasin H [23]. The manifest difference of the structure of 1 from that of cytochalasin H was an additional acetoxy group linked at C-18 (δ C 84.4) in 1, which was further supported by the HMBC correlation from OAc (δ H 2.24, s) to C-18. And the planar structure of 1 was established by extensive analysis of its 2D NMR spectra ( Figure 2); its relative configuration was determined by the ROESY correlations ( Figure 3) and comparative analysis of those of cytochalasin H. Fortunately, suitable crystals of 1 were obtained and subjected to X-ray diffraction analysis using Cu Kα radiation (Figure 4), which confirmed the above deductions and unambiguously determined the absolute configuration of 1 as 3S,4R,5S,7S,8R,9R,16S,18R,21R with the Hooft parameter 0.15 (11) for 1883 Bijvoet pairs (CCDC 2169670). quaternary carbons (1 olefinic, 1 amide and 2 ester carbonyls), and 6 other signals assignable to the single-substituted phenyl group. Thus, the above-mentioned results indicated that 1 should be a new tetracyclic cytochalasin including a benzene ring, with structural similarity with cytochalasin H [23]. The manifest difference of the structure of 1 from that of cytochalasin H was an additional acetoxy group linked at C-18 (δC 84.4) in 1, which was further supported by the HMBC correlation from OAc (δH 2.24, s) to C-18. And the planar structure of 1 was established by extensive analysis of its 2D NMR spectra ( Figure 2); its relative configuration was determined by the ROESY correlations ( Figure 3) and comparative analysis of those of cytochalasin H. Fortunately, suitable crystals of 1 were obtained and subjected to X-ray diffraction analysis using Cu Kα radiation (Figure 4), which confirmed the above deductions and unambiguously determined the absolute configuration of 1 as 3S,4R,5S,7S,8R,9R,16S,18R,21R with the Hooft parameter 0.15 (11) for 1883 Bijvoet pairs (CCDC 2169670). 18-Ethoxycytochalasin H (2) was obtained as a white powder; its molecular formula was established as C32H43NO5 on the basis of the HRESIMS ion peak at m/z 544.3030 [M + Na] + (calcd for C32H43NO5Na, 544.3039), indicating 12 degrees of unsaturation. Analyses of the NMR data of 2 with those of 1 indicated their structural similarities, except for an ethoxy group located at C-18 in 2 rather than the 18-OAc group in 1, which was confirmed by the 1 H-1 H COSY correlation of CH2 (δH 3.38, m; 2.65, m)/CH3 (1.14, t, J = 6.9 Hz) in the ethoxy group and the HMBC correlations from CH2-18-OEt (δH 3.38, m; 2.65, m) to C-18 (δC 78.5) (Figure 2). The relative configurations of C-3, C-4, C-5, C-7, and C-8 in 2 were determined to be the same as those of 1 by analysis of the ROESY spectrum ( Figure 3). Considering the almost complete consistent CD spectra of 1 and 2 (see Supplementary Materials), the absolute configuration of 2 was determined as shown.       (Tables 1 and 2) of 3 were similar to those of cytochalasin J [24], except for an additional acetoxy group located at C-18 in 3. The above deduction was further confirmed by the changed chemical shift of C-18, compared with the 13 C NMR data of cytochalasin J [24], and the HMBC correlation from CH3-18-OAc (δH 1.96, s) to 18-OAc carbonyl (δC 170.4) ( Figure 2); its structure including the relative configuration was finally established as shown by X-ray diffraction analysis (Figure 4). Considering the similar CD spectra of 1 and 3 (SI), the absolute configuration of 3 was determined to be 3S,4R,5S,7S,8R,9R,16S,18R,21R.  (Tables 1  and 2) suggested their similar structures, except for an ethoxy group located at C-18 in 4 rather than an acetoxy group in 3. The above deduction was supported by the 1 H-1 H COSY 1 3 18-Ethoxycytochalasin H (2) was obtained as a white powder; its molecular formula was established as C 32 H 43 NO 5 on the basis of the HRESIMS ion peak at m/z 544.3030 [M + Na] + (calcd for C 32 H 43 NO 5 Na, 544.3039), indicating 12 degrees of unsaturation. Analyses of the NMR data of 2 with those of 1 indicated their structural similarities, except for an ethoxy group located at C-18 in 2 rather than the 18-OAc group in 1, which was confirmed by the 1 H-1 H COSY correlation of CH 2 (δ H 3.38, m; 2.65, m)/CH 3 (1.14, t, J = 6.9 Hz) in the ethoxy group and the HMBC correlations from CH 2 -18-OEt (δ H 3.38, m; 2.65, m) to C-18 (δ C 78.5) (Figure 2). The relative configurations of C-3, C-4, C-5, C-7, and C-8 in 2 were determined to be the same as those of 1 by analysis of the ROESY spectrum ( Figure 3). Considering the almost complete consistent CD spectra of 1 and 2 (see Supplementary Materials), the absolute configuration of 2 was determined as shown.
18-Acetoxycytochalasin J (3) had the molecular formula of C 30 H 39 NO 5 based on the positive HRESIMS at m/z 516.2726 [M + Na] + (calcd 516.2720), corresponding to 12 degrees of unsaturation. The 1D NMR data (Tables 1 and 2) of 3 were similar to those of cytochalasin J [24], except for an additional acetoxy group located at C-18 in 3. The above deduction was further confirmed by the changed chemical shift of C-18, compared with the 13 C NMR data of cytochalasin J [24], and the HMBC correlation from CH 3 -18-OAc (δ H 1.96, s) to 18-OAc carbonyl (δ C 170.4) ( Figure 2); its structure including the relative configuration was finally established as shown by X-ray diffraction analysis ( Figure 4). Considering the similar CD spectra of 1 and 3 (SI), the absolute configuration of 3 was determined to be 3S,4R,5S,7S,8R,9R,16S,18R,21R.  (Tables 1 and 2) suggested their similar structures, except for an ethoxy group located at C-18 in 4 rather than an acetoxy group in 3. The above deduction was supported by the 1 H-1 H COSY correlations of CH 2 /CH 3 (18-OEt) and the HMBC correlation from CH 2 -18-OEt (δ H 3.41, m; 3.37, m) to C-18 (δ C 78.5) (Figure 2). The absolute configuration of 4 was determined to be the same as that of 1 by analysis of their CD spectra; thus, the structure of 4 was established as shown (Figure 1 (Figure 3). Considering the same biogenetic pathway of 1 and 5, the structure of 5 was determined as shown (Figure 1).
Cytochalasin H 3 (6) had the molecular formula of C 30 H 39 NO 5 with 12 degrees of unsaturation, which was determined by the positive HRESIMS (m/z 516.2719 [M + Na] + , calcd 516.2720). Detailed analysis of 1 H and 13 C NMR data of 6 (Tables 1 and 2) indicated that 6 possessed a similar structure to that of 5, except for the presence of a saturated C-C bond between C-6 (δ C 45.8) and C-12 (δ C 16.0) in 6 rather than a terminal double bond in 5, which was further confirmed by the 1 H-1 H COSY correlation of H-6/H 3 -12 and the HMBC correlations from H 3 -12 (δ H 1.12, d, J = 7.0 Hz) to C-5 (δ C 35.7) and C-7 (δ C 214.0) (Figure 2 Figure 3). By analysis of the similar CD spectra of 6 and 1 and biogenetic consideration, the structure of 6 was determined as shown.
The molecular formula of cytochalasin H 4 (7) was deduced to be C 32 H 41 NO 6 with 13 degrees of unsaturation based on the positive HRESIMS (m/z 536.3014 [M + H] + , calcd 536.3007). The 13 C NMR data (Tables 1 and 2 of 7 displayed resonances for 32 carbons, ascribed to five methyls, four methylenes (including one oxygenated), 11 methines (5 olefinic and one oxygenated), six quaternary carbons (one olefinic, one amide and two ester carbonyls), and 6 other signals assignable to the single-substituted phenyl group. The above-mentioned results indicated the presence of an additional acetoxy group and an oxymethine group compared to those of the known RKS-1778 (10) [25]. The ROESY correlations of H-3/H 3 -11, H 2 -10/H-4, and H-4/H-8 implied the α-orientation of H-3 and β-orientations of H-4, H-5, and H-8 ( Figure 3). The absolute configuration of 7 was determined as shown by analysis of their similar CD spectra of 7 and 10 and biogenetic consideration.

Antimigratory Activity
Our previous studies have revealed that phomopchalasins B and C displayed antimigratory effects [19]. In order to explore the potential of the cytochalasans on antimigration against tumours, eight compounds in sufficient natural amounts (Table 3) were evaluated for antimigratory activities against MDA-MB-231 in vitro. As a result, 1-3 and 8-11 exhibited in vitro antimigratory effects with IC 50 values in the range of 1.01-10.42 µM (cytochalasin D as the positive control); it suggested the activity decreased when the C-18 hydroxy group was substituted with the acetoxy, ethoxy or methoxy group (8 vs . 1, 2, and 9). When a double bond was introduced between C-17 and C-18 rather than an ethoxy or methoxy group at C-18, the activity slightly improved (11 vs . 2 and 9). Compound 3 displayed antimigratory activity with an IC 50 value of 6.38 µM. The introduction of an acetoxy group at C-21 may enhance the activity (1 vs. 3). When the unit of a terminal double bond (C6-C12) and a hydroxy group at C-7 was replaced by a trisubstituted alkene (C12-C6-C7), the activity slightly improved (8 vs. 10), but the further introduction of an acetoxy group at C-12 decreased the activity (10 vs. 7).

Conclusions
Seven new cytochalasans (1-7), together with four known ones, cytochalasin H (8), cytochalasin J 1 (9), RKS-1778 (10), and 21-acetoxycytochalasin J 2 (11), were isolated from Phomopsis sp. shj2. Their structures were elucidated through extensive spectroscopic data interpretation and single-crystal X-ray diffraction analysis. In the present study, eight cytochalasans were evaluated for their antimigratory activity. Compounds 1-3 and 8-11 exhibited antimigratory activity against MDA-MB-231 in vitro with IC 50 values in the range of 1.01−10.42 µM. The results will lay a foundation for further study of the structureactivity relationship for the discovery of antitumour lead compounds.