Dibenzofuran, 4-Chromanone, Acetophenone, and Dithiecine Derivatives: Cytotoxic Constituents from Eupatorium fortunei

Five new compounds, eupatodibenzofuran A (1), eupatodibenzofuran B (2), 6-acetyl-8-methoxy-2,2-dimethylchroman-4-one (3), eupatofortunone (4), and eupatodithiecine (5), have been isolated from the aerial part of Eupatorium fortunei, together with 11 known compounds (6‒16). Compounds 1 and 2 featured a new carbon skeleton with an unprecedented 1-(9-(4-methylphenyl)-6-methyldibe nzo[b,d]furan-2-yl)ethenone. Among the isolates, compound 1 exhibited potent inhibitory activity with IC50 values of 5.95 ± 0.89 and 5.55 ± 0.23 μM, respectively, against A549 and MCF-7 cells. The colony-formation assay demonstrated that compound 1 (5 μM) obviously decreased A549 and MCF-7 cell proliferation, and Western blot test confirmed that compound 1 markedly induced apoptosis of A549 and MCF-7 cells through mitochondrial- and caspase-3-dependent pathways.

To further confirm whether apoptosis was triggered, annexin V/propidium iodide (PI) assay was performed and the expression levels of apoptosis-associated proteins, Bcl-2, Bax, and caspase-3 were analyzed by Western blot analysis after A549 and MCF-7 cells were treated with compound 1. As shown in Figure 8A,B, compound 1 significantly induced the cell apoptosis in A549 and MCF-7 cells, respectively. Furthermore, compound 1 increased the expression of Bax and cleaved-caspase-3, and decreased Bcl-2 and pro-caspase-3 levels in a dose-dependent manner in both A549 and MCF-7 cells (Figure 9A,B). The above results confirm that compound 1 markedly induces apoptosis of A549 and MCF-7 cells through mitochondrial-and caspase-3-dependent pathways (Scheme 1).

Figure 7.
Compound 1 inhibited the growth of human non-small-cell lung cancer cell (A549) and human breast cancer cell (MCF-7) in a dose-dependent manner (1.25-10 μM) by the colony-formation assay. The data were expressed as mean ± SEM (n = 3). Asterisks indicate significant differences (* p < 0.05 and *** p < 0.001) compared with the control group.
To further confirm whether apoptosis was triggered, annexin V/propidium iodide (PI) assay was performed and the expression levels of apoptosis-associated proteins, Bcl-2, Bax, and caspase-3 were analyzed by Western blot analysis after A549 and MCF-7 cells were treated with compound 1. As shown in Figure 8A,B, compound 1 significantly induced the cell apoptosis in A549 and MCF-7 cells, respectively. Furthermore, compound 1 increased the expression of Bax and cleaved-caspase-3, and decreased Bcl-2 and pro-caspase-3 levels in a dose-dependent manner in both A549 and MCF-7 cells ( Figure  9A,B). The above results confirm that compound 1 markedly induces apoptosis of A549 and MCF-7 cells through mitochondrial-and caspase-3-dependent pathways (Scheme 1).
To further understand the mechanism of compound 1 in this study, we predicted potential targets using the similarity ensemble approach server [20]. This approach predicts possible target proteins of a compound by comparing chemical similarities. Compound 1 was predicted to target four proteins (Supplementary Materials Table S2), including PON1, CELA1, CBR1, and NQO1. The Tanimoto coefficients (Tc) of chemical similarity were generated for the predicted targets. The Tc is a pairwise score between the compound and the predicted target. The Tc score ranges from 0.0 (no similarity) to 1.0 (total similarity). The P-value indicates the prediction reliability, and a value approaching zero means a reliable prediction. The possible targets may account for the inhibition mechanisms of compound 1. Figure 7. Compound 1 inhibited the growth of human non-small-cell lung cancer cell (A549) and human breast cancer cell (MCF-7) in a dose-dependent manner (1.25-10 µM) by the colony-formation assay. The data were expressed as mean ± SEM (n = 3). Asterisks indicate significant differences (* p < 0.05 and *** p < 0.001) compared with the control group.
To further understand the mechanism of compound 1 in this study, we predicted potential targets using the similarity ensemble approach server [20]. This approach predicts possible target proteins of a compound by comparing chemical similarities. Compound 1 was predicted to target four proteins (Supplementary Materials Table S2), including PON1, CELA1, CBR1, and NQO1. The Tanimoto coefficients (Tc) of chemical similarity were generated for the predicted targets. The Tc is a pairwise score between the compound and the predicted target. The Tc score ranges from 0.0 (no similarity) to 1.0 (total similarity). The P-value indicates the prediction reliability, and a value approaching zero means a reliable prediction. The possible targets may account for the inhibition mechanisms of compound 1.

Plant Material
The aerial part of E. fortunei collected from Dihua St., Datong Dist., Taipei City, Taiwan, in May 2019 and identified by Prof. J.-J. Chen. A voucher specimen was deposited in the Department of Pharmacy, National Yang Ming Chiao Tung University, Taipei, Taiwan.

Extraction and Isolation
The aerial part of E. fortunei (5.0 kg) was pulverized and extracted three times with MeOH (30 L each) for 3 days. The MeOH extract was concentrated under reduced pressure at 35 °C, and the residue (123.7 g) was partitioned between EtOAc and H2O (1:1) to Scheme 1. The mechanism of apoptosis for compound 1 in A549 and MCF-7 cells.

General Experimental Procedures
Ultraviolet (UV) spectra were obtained on a Jasco UV-240 spectrophotometer. Infrared (IR) spectra (neat or KBr) were recorded on a Perkin Elmer 2000 FT-IR spectrometer.

Plant Material
The aerial part of E. fortunei collected from Dihua St., Datong Dist., Taipei City, Taiwan, in May 2019 and identified by Prof. J.-J. Chen. A voucher specimen was deposited in the Department of Pharmacy, National Yang Ming Chiao Tung University, Taipei, Taiwan.

Cell Viability Assay
The cell viability was conducted by the MTT assay as previously described method [23]. Briefly, 5 × 10 3 cells in 200 µL per well were plated in 96-well culture plates and cultured in complete medium overnight. After 24 h, cells were treated with different concentrations (3.125, 6.25, 12.5, 25, 50, and 100 µM) of compounds 1-16. Fluorouracil (5-FU) (Sigma-Aldrich, St. Louis, MO, USA) was used as a positive control against A549 and MCF-7 cells with IC 50 values of 10.57 ± 1.89 and 8.59 ± 1.03 µM, respectively. The optical density at 570 nm was measured by ELISA plate reader (µ Quant) and the IC 50 value was calculated. The optical density of formazan formed in control (untreated) cells was taken as 100% viability.

Colony-Formation Assay
The colony-formation assay was determined by the reference method with a slight modification [24]. In this assay, A549 and MCF-7 cells were seeded in 6-well plates with 1 × 10 3 cells per well and incubated for 12 h. The cells were then treated with the indicated concentrations of compound 1, and cultured for 10 days. The cells were washed three times using PBS and fixed using 95% methanol for 30 min. After washing three times with distilled water, the cells were stained using 0.2% crystal violet dye for 20 min and rinsed with distilled water to wash away the excess dye. The visible colonies were compared with the control samples and photographed using a standard camera under natural light.

Flow Cytometry
Annexin V/PI assay was used to determine the apoptotic and necrotic cells. The A549 and MCF-7 cells were seeded on 6-well microplates at a density of 10 6 cells/mL respectively. After 24 h incubation, the cells were treated with following concentrations of 0, 5, and 10 µM for compound 1. After 24 h, they were washed and re-suspended in PBS solution (500 µL). Then, Annexin V-FITC (5 µL) and PI staining solution (5 µL) were introduced to the mixture, and the incubation process was followed under the dark condition for 5 min at 25 • C. Finally, flow cytometer analysis (Beckman Coulter ® , Miami, FL, USA) was performed using an AnnexinV-FITC Apoptosis Detection Kit (Strong Biotech Corporation, Taipei, Taiwan) and Flowjo version 7.6.1. Software.