Diisoprenyl Cyclohexene-Type Meroterpenoids with Cytotoxic Activity from a Mangrove Endophytic Fungus Aspergillus sp. GXNU-Y85

Five new diisoprenyl cyclohexene-type meroterpenoids, aspergienynes J–N (1–5), along with three known analogues (6–8), were obtained from the mangrove endophytic fungal strain Aspergillus sp. GXNU-Y85. The chemical structures, including their absolute configurations, were established via spectroscopic data and comparison of experimental and calculated ECD spectra. Cytotoxicity assay results indicated that compound 8 had strong cytotoxicity against HeLa cancer cells, and its IC50 value was 11.8 μM. In addition, flow cytometry analysis revealed that the cytotoxicity of 8 was due to the induction of G1 cell cycle arrest and apoptosis in HeLa cells.

As part of our continuing research to find mangrove endophytic fungi-derived bioactive constituents, the chemical constituents of Aspergillus sp.Y85 were investigated.The chemical investigation of the secondary metabolites of Aspergillus sp.Y85 from the fruit of Kandelia candel, a mangrove plant, resulted in the isolation of five undescribed diisoprenyl cyclohexene-type meroterpenoids, aspergienynes J-N (1-5), together with three previously reported analogues (6-8) (Figure 1).Compounds 1-8 were evaluated for their cytotoxic activity against five human cancer cell lines.Herein, the isolation, structure elucidation, and cytotoxicity of (1-8) are described.

Results and Discussion
The molecular formula of aspergienyne J ( 1 [5] revealed the same planar structure; however, some proton chemical shifts and coupling constants were different.Furthermore, the 1 H-1 H COSY of H-3/H-4 /H-5 and H-12/H-13, as well as the key HMBC signals from H-1 to C-2/C-5/C-6/C-7/C-14, from H-16 to C-1/C-14, were used to further prove the planar structure of 1 (Figure 2).The relative configuration of 1 was established via NOESY experiments.NOESY correlations H-1/H-12a, H-1/H-15, H-12a/H-15, H-13/H-16, and H-3/H-12b (Figure 3), as well as the small J H-3/H-4 value (1.8 Hz), determined the relative configurations as 1R*, 2S*, 3R*, 4R*, and 13R*.Finally, the comparison of the calculated and experimental ECD spectra allowed us to establish the absolute configurations as 1R, 2S, 3R, 4R, and 13R (Figure 4).Aspergienyne K (2) has the same molecular formula as 1, C16H20O4, as determined via (+)-HR-ESI-MS and 13 C NMR data (Table 2). 1 H and 13 C NMR spectra (Tables 1 and 2) indicated that compound 2 differs to 1 except for a perturbation in the region around carbon at position 13 suggesting a different configuration of the hydroxyl group.The comparison of calculated and experimental ECD spectra confirmed the 13S absolute configuration (Figure 4).Aspergienyne L (3) was obtained as a yellowish oil, and the molecular formula was assigned as C16H22O5 via (+)-HR-ESI-MS at m/z 317.1355 [M + Na] + (calcd for C16H22O5Na + , 317.1359) and 13 C NMR data, corresponding to six degrees of unsaturation.The 13 C NMR and HSQC data of 3 revealed sixteen carbon resonances (Table 2), including two sp carbons, four olefinic carbons, three methyls, one methylene, four methines, and two quaternary carbons.The NMR spectroscopic data of 3 were similar to that of 1, except that the C-14 was connected to the C-2 through an oxygen atom in 3. The deduction was corroborated by the key HMBC correlations from H-12/H-13 to C-2/C-14, which demonstrated the existence of a C-2-O-C-14 ring (tetrahydrofuran).NOESY correlations (Figure 3) between H-1 and H-3, between H-1 and H-12b, and between H-12b and H-16 indicated that H-1, H-3, 12b, and H-16 were on the same face.The large JH-3/H-4 value (8.1 Hz) and NOESY correlations from H-4 to H-12a/H-13/H-15 suggested the same axial orientation of these protons.Therefore, the relative configuration of 3 was determined as 1R*, 3S*, 4R*, and Aspergienyne K (2) has the same molecular formula as 1, C 16 H 20 O 4 , as determined via (+)-HR-ESI-MS and 13 C NMR data (Table 2). 1 H and 13 C NMR spectra (Tables 1 and 2) indicated that compound 2 differs to 1 except for a perturbation in the region around carbon at position 13 suggesting a different configuration of the hydroxyl group.The comparison of calculated and experimental ECD spectra confirmed the 13S absolute configuration (Figure 4).
Aspergienyne L (3) was obtained as a yellowish oil, and the molecular formula was assigned as The 13 C NMR and HSQC data of 3 revealed sixteen carbon resonances (Table 2), including two sp carbons, four olefinic carbons, three methyls, one methylene, four methines, and two quaternary carbons.The NMR spectroscopic data of 3 were similar to that of 1, except that the C-14 was connected to the C-2 through an oxygen atom in 3. The deduction was corroborated by the key HMBC correlations from H-12/H-13 to C-2/C-14, which demonstrated the existence of a C-2-O-C-14 ring (tetrahydrofuran).NOESY correlations (Figure 3) between H-1 and H-3, between H-1 and H-12b, and between H-12b and H-16 indicated that H-1, H-3, 12b, and H-16 were on the same face.The large J H-3/H-4 value (8.1 Hz) and NOESY correlations from H-4 to H-12a/H-13/H-15 suggested the same axial orientation of these protons.Therefore, the relative configuration of 3 was determined as 1R*, 3S*, 4R*, and 13R*.To assign the full relative configuration of 3, the 13 C NMR data of two potential isomers (3a and 3d) (Figure S46) were calculated based on the GIAO method, and the (1R, 2R, 3S, 4R, 13R)-configuration of 3 was suggested due to the high DP4+ probability analysis (100%) (Figure S47) and the better correlation coefficient (R 2 = 0.9982) between the experimental and calculated 13 C NMR chemical shifts (Figure S48).The absolute configurations of C-1, C-2, C-3, C-4, and C-13 in compound 3 were determined as 1R, 2R, 3S, 4R, and 13R via comparison of its calculated and experimental ECD spectra (Figure 4).
Aspergienyne M (4) was obtained as a yellowish oil.3), one sp 3 methylene carbon (δ C 7.9), and three sp 3 methyl carbons (δ C 31.7, 29.7, 23.6).The data of 4 (Tables 1 and 2) were highly similar to those of monosporasol B (7) [6], except for the absence of a peroxide bridge.This assumption was supported by the oxymethine (δ H /δ C 3.73/74.1,C-1), and the non-protonated carbons (δ C 70.1, C-14) were significantly shifted to the upfield region in 4 compared to 7. Accordingly, the planar structure of 4 was assigned and confirmed via analysis of key HMBC correlations from H-12 to C-2/C-3, from H-13 to C-1, and from H-15/H-16 to C-13/C-14, (Figure 2).In the NOESY experiment, the correlations from H-1 to H-3 and H-1 to H-13, as well as the small value of J H-3/H-4 (5.2 Hz), indicated that H-1, H-3, H-4, and H-13 were on the same face (Figure 3).Therefore, the relative configuration of 4 was established as shown in Figure 3.The absolute configuration 1S, 2R, 3S, 4R, and 12S was deduced via the comparison of calculated and experimental ECD spectra (Figure 4).
The (+)-HR-ESI-MS spectrum of Aspergienyne N (5), isolated as a yellowish oil, showed a pseudo molecular ion peak at m/z 303.1567 [M + Na] + corresponding to a molecular formula C 16 H 24 O 4 .Comprehensive analysis of the NMR spectral information of biscognienyne D (8) and 5 (Tables 1 and 2) indicated that they are structural analogues.The comparison of the NMR data of compounds 5 and 8 [4] revealed the lack of D5,6 double bond and the replacement of the oxirane ring in 8 with two hydroxyl group at C-2 and C-3 in compound 5.This speculation was further corroborated via two oxymethines at δ C 75.3 (C-2) and 76.3 (C-3), along with the 1 H-1 H COSY correlations of H-3/H-4/H-5/H-6 and the crucial HMBC signals from H-6 to C-1/C-5/C-7/C-8 (Figure 2).NOESY correlations between H-1/H-3, H-1/H-5b, H-1/H-12 b, and H-3/H-5 b indicated that each is b positioned.The NOESY correlations from H-4 to H-6 suggested that these protons were on the same side.Therefore, the absolute configurations of C-1, C-2, C-3, C-4, and C-6 in compound 5 were defined as 1R, 2R, 3S, 4R, and 6R on the basis of the calculated ECD spectrum (Figure 4).Three known compounds were identified as biscognienyne D (6) [5], monosporasol B (7) [6], and biscognienyne D (8) [4] via comparison of their spectroscopic data with those reported in the literature.
To verify whether the observed growth inhibition was owing to cell cycle arrest, HeLa cells were treated with 8 (5, 10, and 15 µM) for 24 h, and the distribution of cells in the cell cycle was established based on flow cytometric analysis.As shown in Figure 5, treatment of the HeLa cells with 8 led to an observable increase in the population of cells in the G1 phase (from 46.72% to 57.78%), with a gradual decrease in the cell number in the G2 phase (6.60-0.18%).These findings suggest that 8 may be more likely to induce cell cycle arrest in the G1 phase of HeLa cells.To further determine whether the induction of apoptosis contributed to the cytotoxicity in HeLa cells, flow cytometry was performed to detect apoptosis with Annexin V and propidium iodide (PI) double staining.Following treatment of HeLa cells with different concentrations of 8 for 24 h, the percentages of apoptotic cells increased in a concentrationdependent manner to 17.72% (5 μM), 22.24% (10 μM), and 31.4% (15 μM) compared with 6.68% in the control (Figure 6).
These results indicated that the cytotoxicity of 8 against HeLa cells resulted from the cell cycle arrest and the induction of apoptosis.To further determine whether the induction of apoptosis contributed to the cytotoxicity in HeLa cells, flow cytometry was performed to detect apoptosis with Annexin V and propidium iodide (PI) double staining.Following treatment of HeLa cells with different concentrations of 8 for 24 h, the percentages of apoptotic cells increased in a concentrationdependent manner to 17.72% (5 µM), 22.24% (10 µM), and 31.4% (15 µM) compared with 6.68% in the control (Figure 6).
These results indicated that the cytotoxicity of 8 against HeLa cells resulted from the cell cycle arrest and the induction of apoptosis.

General Experimental Procedures
The UV and IR spectra of the new compounds were obtained using a PerkinElmer 650 spectrophotometer (PerkinElmer, Waltham, MA, USA) and a PerkinElmer Spectrum Two FT-IR spectrometer (PerkinElmer).The NMR data were acquired on a Bruker 400 MHz (Bruker, Bremen, Germany).Optical rotations were measured using a JASCO P-2000 a polarimeter (Jasco, Tokyo, Japan).An LC-MS spectrometer (Agilent 6545 Q-TOF, Santa Clara, CA, USA) was used to obtain HR-ESI-MS data.The detailed ECD instrument are described in the Supplementary Materials.The other measuring instruments used for the purification of the isolates and the materials used in the separation procedure were identical to our previous reports [9,10].

Fungal Material
The fungal strain GXNU-Y85 was obtained from the fresh fruit of the mangrove plant Kandelia candel, in Beihai, and was identified as Aspergillus sp.via the sequence of its internal transcribed spacer region (ITS) and morphology.ITSrDNA of GXNU-Y85 was submitted to GenBank, and the accession number is OR999402.

General Experimental Procedures
The UV and IR spectra of the new compounds were obtained using a PerkinElmer 650 spectrophotometer (PerkinElmer, Waltham, MA, USA) and a PerkinElmer Spectrum Two FT-IR spectrometer (PerkinElmer).The NMR data were acquired on a Bruker 400 MHz (Bruker, Bremen, Germany).Optical rotations were measured using a JASCO P-2000 a polarimeter (Jasco, Tokyo, Japan).An LC-MS spectrometer (Agilent 6545 Q-TOF, Santa Clara, CA, USA) was used to obtain HR-ESI-MS data.The detailed ECD instrument are described in the Supplementary Materials.The other measuring instruments used for the purification of the isolates and the materials used in the separation procedure were identical to our previous reports [9,10].

Fungal Material
The fungal strain GXNU-Y85 was obtained from the fresh fruit of the mangrove plant Kandelia candel, in Beihai, and was identified as Aspergillus sp.via the sequence of its internal transcribed spacer region (ITS) and morphology.ITSrDNA of GXNU-Y85 was submitted to GenBank, and the accession number is OR999402.

ECD and NMR Calculations
The ECD and NMR calculations of all the new isolates were performed as previously described [10,11].The detailed procedure is described in the Supplementary Materials.

Cell Viability Assay
The cytotoxic activity of 1-8 against five cancer cell lines were tested via the MTT method as in previous reports [12].Etoposide was used as the positive control.

Cell Cycle Analysis
After treatment with 8 for 24 h, HeLa cells were collected, washed with PBS, and fixed with ice-cold 70% EtOH.Propidium iodide (PI) mixed with RNase was used to stain each set of cells, and flow cytometry analysis was carried out as previously reported [12].

Apoptosis Analysis
After 24 h of treatment with 8, HeLa cells were collected, washed twice with cold PBS, and resuspended in 1 × binding buffer.Annexin V-FITC and PI were used to stain each set of cells, which were then cultivated for 30 min at room temperature.The apoptosis assay was performed based on flow cytometry as described previously [12].

Conclusions
In summary, aspergienynes J-N (1-5), five new diisoprenyl cyclohexene-type meroterpenoids, along with three previously described analogues, were isolated from the mangrove endophytic fungus Aspergillus sp.GXNU-Y85.Their structures, including the absolute configurations of all the new isolates, were unambiguously established through a combination of HR-ESI-MS and NMR spectroscopic data, as well as ECD calculations.The results showed that the isolates 3, 5, and 8 displayed significant cytotoxicity against HeLa cancer cell lines, with IC 50 values of 11.8-34.7 µM.Furthermore, compound 8 induced apoptosis in HeLa cells.Additionally, compound 8 inhibited the growth and proliferation of HeLa cells and resulted in G1 phase arrest.

11 Figure 5 .
Figure 5. Effects of 8 on the cell cycle distribution in the HeLa cells.The cell cycle distributions of HeLa cells treated with 8 for 24 h were analyzed with flow cytometry.The graphs show the quantified results.Data are presented as the mean fold changes ± SD of three independent experiments (p < 0.001).

Figure 5 .
Figure 5. Effects of 8 on the cell cycle distribution in the HeLa cells.The cell cycle distributions of HeLa cells treated with 8 for 24 h were analyzed with flow cytometry.The graphs show the quantified results.Data are presented as the mean fold changes ± SD of three independent experiments (p < 0.001).

Figure 6 .
Figure 6.Effects of 8 on the induction of apoptosis in the HeLa cells.Cells were seeded, treated with 8 for 24 h, collected, and stained with annexin V-FITC/PI to detect the apoptotic cell population using flow cytometry.Data are presented as the mean fold changes ± SD of three independent experiments (p < 0.001).

Figure 6 .
Figure 6.Effects of 8 on the induction of apoptosis in the HeLa cells.Cells were seeded, treated with 8 for 24 h, collected, and stained with annexin V-FITC/PI to detect the apoptotic cell population using flow cytometry.Data are presented as the mean fold changes ± SD of three independent experiments (p < 0.001).