Coumarin Derivatives Exert Anti-Lung Cancer Activity by Inhibition of Epithelial–Mesenchymal Transition and Migration in A549 Cells

A series of coumarin derivatives and isosteres were synthesized from the reaction of triflic intermediates with phenylboronic acids, terminal alkynes, and organozinc compounds through palladium-catalyzed cross-coupling reactions. The in vitro cytotoxic effect of the compounds was evaluated against two non-small cell lung carcinoma (NSCLC) cell lines (A-549 and H2170) and a normal cell line (NIH-3T3) using cisplatin as a reference drug. Additionally, the effects of the most promising coumarin derivative (9f) in reversing the epithelial-to-mesenchymal transition (EMT) in IL-1β-stimulated A549 cells and in inhibiting the EMT-associated migratory ability in A549 cells were also evaluated. 9f had the greatest cytotoxic effect (CC50 = 7.1 ± 0.8 and 3.3 ± 0.5 μM, respectively against A549 and H2170 cells) and CC50 value of 25.8 µM for NIH-3T3 cells. 9f inhibited the IL-1β-induced EMT in epithelial cells by inhibiting the F-actin reorganization, attenuating changes in the actin cytoskeleton reorganization, and downregulating vimentin in A549 cells stimulated by IL-1β. Treatment of A549 cells with 9f at 7 µM for 24 h significantly reduced the migration of IL-1β-stimulated cells, which is a phenomenon confirmed by qualitative assessment of the wound closure. Taken together, our findings suggest that coumarin derivatives, especially compound 9f, may become a promising candidate for lung cancer therapy, especially in lung cancer promoted by NSCLC cell lines.


Introduction
Cancer is characterized by cells with uncontrolled division, genome heterogeneity, and invasiveness to other tissues via blood or lymph nodes. According to the World Health

Chemistry
The synthesis of the coumarin core can be performed by different synthetic methodologies, among which the most common are Pechmann, Wittig, Knoevenagel, and Perkin reactions [11]. Cross-coupling reactions catalyzed by transition metals, such as the Suzuki-Miyaura, Negishi [40], and Sonogashira [41] reactions, have become powerful alternatives to the formation of carbon-carbon bonds [42,43] and allowed the introduction of various substituents in all positions of the basic nucleus, leading to analogous, homologous, or libraries of compounds [44].
The preparation of target compounds (coumarins, quinolones, and chromen-4-ones) involved the formation of triflic methanesulfonate derivatives as key intermediates 4, 5a-b, and 6, thanks to the cross-coupling reactions. 6-and 7-hydroxycoumarin 2a-b and 6-hydroxyquinolone 1a are commercially available, and 3-hydroxy-chromen-4-one 3 was synthesized following a reported preparation [45]. Attempts to prepare 6-OTf quinolone from 1a and triflic anhydride resulted exclusively in the formation of the 2,6 di-triflic adduct 1b. Therefore, our efforts focused on the preparation of N-Me quinolone 1c. However, N-alkylation of quinolone 1a needed a first transient protection of the phenol by an acetyl group (compounds 1d-e) according to Scheme 1. Reaction of the respective hydroxyl cores (cpds 1c, 2a-b, and 3) with triflic anhydride in the presence of pyridine afforded the corresponding triflic intermediates 4, 5a-b, and 6 in high yields (≥75%), as illustrated in Scheme 2.
With triflic intermediates 4, 5a,b, and 6 in hand, our attention next turns to the Suzuki-Miyaura cross-coupling reaction. Reaction with various boronic acids enables the preparation of a small library of 6-and 7-substituted coumarins (cpds 8a-f, 9a-g). The use of a catalytic amount of tetrakis(triphenylphosphine) palladium(0) (5.0 mol %) in the presence of NaHCO 3 as a base led efficiently to the target compounds (see Table 1). However, for the introduction of a pyridine moiety in the coumarin structure, K 3 PO 4 was preferred over NaHCO 3 (Table 1, cpds 8d and 9g). For these 2 cpds, the reaction was performed in Toluene/EtOH/H 2 O and yielded the expected compounds 8d and 9g in 74% and 82% yields, respectively. Starting from the Otf-flavone derivative 6, the use of Pd(Oac) 2 (5.0 mol %) in the presence of KF furnished 10 in moderate yield (50%) (Scheme 3). Reaction of the respective hydroxyl cores (cpds 1c, 2a-b, and 3) with triflic anhydride in the presence of pyridine afforded the corresponding triflic intermediates 4, 5a-b, and 6 in high yields (≥75%), as illustrated in Scheme 2. With triflic intermediates 4, 5a,b, and 6 in hand, our attention next turns to the Suzuki-Miyaura cross-coupling reaction. Reaction with various boronic acids enables the preparation of a small library of 6-and 7-substituted coumarins (cpds 8a-f, 9a-g). The use of a catalytic amount of tetrakis(triphenylphosphine) palladium(0) (5.0 mol %) in the presence of NaHCO3 as a base led efficiently to the target compounds (see Table 1). However, for the introduction of a pyridine moiety in the coumarin structure, K3PO4 was preferred over NaHCO3 (Table 1, cpds 8d and 9g). For these 2 cpds, the reaction was performed in Toluene/EtOH/H2O and yielded the expected compounds 8d and 9g in 74% and 82% yields, respectively. Starting from the Otf-flavone derivative 6, the use of Pd(Oac)2 (5.0 mol %) in the presence of KF furnished 10 in moderate yield (50%) (Scheme 3).  Reaction of the respective hydroxyl cores (cpds 1c, 2a-b, and 3) with triflic anhydride in the presence of pyridine afforded the corresponding triflic intermediates 4, 5a-b, and 6 in high yields (≥75%), as illustrated in Scheme 2. With triflic intermediates 4, 5a,b, and 6 in hand, our attention next turns to the Suzuki-Miyaura cross-coupling reaction. Reaction with various boronic acids enables the preparation of a small library of 6-and 7-substituted coumarins (cpds 8a-f, 9a-g). The use of a catalytic amount of tetrakis(triphenylphosphine) palladium(0) (5.0 mol %) in the presence of NaHCO3 as a base led efficiently to the target compounds (see Table 1). However, for the introduction of a pyridine moiety in the coumarin structure, K3PO4 was preferred over NaHCO3 (Table 1, cpds 8d and 9g). For these 2 cpds, the reaction was performed in Toluene/EtOH/H2O and yielded the expected compounds 8d and 9g in 74% and 82% yields, respectively. Starting from the Otf-flavone derivative 6, the use of Pd(Oac)2 (5.0 mol %) in the presence of KF furnished 10 in moderate yield (50%) (Scheme 3).  Sonogashira reaction with different terminal alkynes resulted in seven compounds ( Table 2). Palladium-catalyzed Sonogashira cross-coupling is a widely used method to synthesize functional molecules containing an alkyne unit. Traditional Sonogashira coupling with Pd(PPh 3 ) 2 Cl 2 (3.0 mol %) and Et 3 N typically requires the use of a Cu(I) halide salt as a cocatalyst to have high reaction productivity. So, starting from coumarin 5b and under these conditions, the phenyl acetylene moiety was introduced under microwave irradiation in 75% yield (cpd 12a). However, with Obn propargylalcohol, the same conditions yielded 12b in only 38% yield. Recently, Chorley et al. highlighted the efficacy of Pd(Oac) 2 and 2-dicyclohexylphosphino-2 ,6 -dimethoxybiphenyl (Sphos) as an effective catalytic system for the Sonogashira cross-coupling reaction [46]. In addition, the presence of tetrabutylammonium iodide (TBAI) as an additive increased the yield of the reaction [47]. Under these conditions and without the protection of propargylic alcohol, we isolated the target alkyne derivative 12c in 75% yield. These last conditions applied to Otf intermediates 4 and 5b in the presence of various terminal alkynes, yielding target compounds 11 and 13a-c in satisfactory yields (>70%, see Table 2) (Scheme 3).
Negishi cross-coupling reactions represent an extremely versatile tool for the introduction of alkyl substituents. As reported by Knochel et al. [48] and in the presence of Sphos (10.0 mol %) and Pd(Oac) 2 (5.0 mol %), it was possible to perform at room temperature an efficient cross-coupling reaction between Otf coumarin 5a-b and benzyl zinc reagent (see Scheme 3, cpds 14 and 15).
Lastly, the subsequent reduction of alkynes 12a and 13a was performed by catalytic hydrogenation, leading respectively to cpds 16 and 17, as depicted in Scheme 3.
All synthesized compounds had their chemical structures confirmed by 1 H and 13 C NMR and mass spectrometry, and all spectra data are available in the Supplementary Material.
Among these two compounds, 9f was the most promising, reducing the A549 cells viability to less than 20%. Thus, this compound was selected and had its concentration that reduces the viable cell number by 50% (CC50) determined against two cancer cell lines (human lung adenocarcinoma A549 and H2170 cell lines) and one non-cancer cell line (NIH-3T3), and showed CC50 values (mean ± S.D.) of 7.1 ± 0.8 μM, 3.3 ± 0.5 μM, and 25.8 ± 1.7 μM against A549, H2170, and NHI-3T3 cells, respectively (Supplementary Materials II). Distinct explanations might be used to sustain this fact, including biochemical and metabolic changes between cell lines. While normal cells follow a set of organized metabolic programs, cancer cells show intrinsic or acquired resistance to apoptosis and also a metabolic reprogramming in order to meet the increased energy demands [49]. Facing these results, we can notate that 9f showed to be the most potent against cancer cells (A549 and H2170 cell lines) than against healthy cell (NIH-3T3 cell line). The results showed that compounds 8b, 9c, 9g, 12c, 13b, 13c, 14, 15, and 16 showed little or no cytotoxicity. Compounds 7, 8a, 8c, 8d, 8e, 9a, 9b, 9d, 9e, 10, 11, 12a-b, 13a, and 17 showed a moderate cytotoxicity by inducing a reduction in cell viability within 80-60%. Compounds 8f and 9f, which have in common the presence of a 3,4-dichloro-phenyl group ((3,4-Cl)-Ph), induced a strong cytotoxicity by decreasing the A549 cells viability to values below 50%.
Among these two compounds, 9f was the most promising, reducing the A549 cells viability to less than 20%. Thus, this compound was selected and had its concentration that reduces the viable cell number by 50% (CC 50 ) determined against two cancer cell lines (human lung adenocarcinoma A549 and H2170 cell lines) and one non-cancer cell line (NIH-3T3), and showed CC 50 values (mean ± S.D.) of 7.1 ± 0.8 µM, 3.3 ± 0.5 µM, and 25.8 ± 1.7 µM against A549, H2170, and NHI-3T3 cells, respectively (Supplementary Materials II). Distinct explanations might be used to sustain this fact, including biochemical and metabolic changes between cell lines. While normal cells follow a set of organized metabolic programs, cancer cells show intrinsic or acquired resistance to apoptosis and also a metabolic reprogramming in order to meet the increased energy demands [49]. Facing these results, we can notate that 9f showed to be the most potent against cancer cells (A549 and H2170 cell lines) than against healthy cell (NIH-3T3 cell line).
On the basis of cytotoxic effect, the concentration of 7 µM of 9f was chosen for further to characterize the antitumor activity by investigating their effects on the process of inhibition of the EMT-associated migratory ability and epithelial-to-mesenchymal transition (EMT) in IL-1β-stimulated A549 cells.

Effect of 9f on IL-1β-Induced EMT in A549 Cells
The EMT process is characterized by the phenotypic conversion of epithelial into mesenchymal cells that occurs with great frequency in fibrotic tissues, embryonic cells, and cancer. This transition increases the invasion capacity and the migratory potential of cells, which are characteristic of metastatic cancer, contributing additionally to the development of drug resistance in cancer [50][51][52][53][54][55].
To determine whether compound 9f acts as an inhibitory compound of EMT in epithelial cells, the morphological changes induced by IL1-β on A549 cells was observed. As shown in Figure 2A,B, the A549 cells maintained to culture medium (DMEM) or treated with compound 9f exhibited, in a confluent monolayer, a cobblestone-like cell morphology, which is characteristic of epithelial cells. Cells treated with 1 ng/mL IL-1β exhibited an evident morphological change and acquired a spindle-like morphology with the loss of cell-cell interactions that is a characteristic feature of mesenchymal cells ( Figure 2C). A549 cells treated with 9f exhibited an impairment in changes in its mesenchymal characteristics induced by IL-1β ( Figure 2D), suggesting that 9f possesses inhibitory effects on IL-1β-induced F-actin reorganization.
To evaluate the effect of compound 9f on actin cytoskeleton organization, A549 cells were IL-1β-stimulated and evaluated by staining with FITC-labeled phalloidin. As presented in Figure 2E,F, the A549 cells maintained to culture medium (DMEM) or treated with 9f exhibited an abundant deposition of actin filament in the cortical region, which determines a cellular cobblestone-like morphology typical of epithelial cells. Stimulation with IL-1β induced a cytoskeleton reorganization, leading to the activation of actin polymerization and the morphologic cell reorganization, which indicate a differentiation from the epithelial to mesenchymal phenotype ( Figure 2G). Treatment with 9f attenuated the changes in the actin cytoskeleton reorganization in A549 cells stimulated by IL-1β ( Figure 2H).
To corroborate whether this morphological transformation represents EMT, immunofluorescent staining was used to quantify the vimentin, which is a mesenchymal marker most commonly associated with EMT and involved in cancer progression [56].
As shown in Figure 3A,B, 24 h incubation with 1 ng/mL IL-1β increased significantly the expression of vimentin in A549 cells compared with those maintained in DMEM medium (control). We found that the treatment of cells with 9f (7 µM) significantly diminished the expression of mesenchymal marker vimentin in IL-1β-stimulated A549 cells ( Figure 3A,B), which is a phenomenon confirmed by the quantitative assessment by flow cytometry ( Figure 3B-C). The treatment of cells with 9f did not change the levels of vimentin expression in unstimulated cells with IL-1β ( Figure 3A-C). This result showed that 9f treatment suppresses IL-1β-induced EMT in A549 cells through downregulating vimentin.
with compound 9f exhibited, in a confluent monolayer, a cobblestone-like cell morphol ogy, which is characteristic of epithelial cells. Cells treated with 1 ng/mL IL-1β exhibited an evident morphological change and acquired a spindle-like morphology with the los of cell-cell interactions that is a characteristic feature of mesenchymal cells ( Figure 2C) A549 cells treated with 9f exhibited an impairment in changes in its mesenchymal charac teristics induced by IL-1β ( Figure 2D), suggesting that 9f possesses inhibitory effects on IL-1β-induced F-actin reorganization.  Given the good results of 9f in inhibiting the IL-1β-induced EMT in epithelial cells, we investigated whether 9f could affect the EMT-associated migratory ability in A549 cells. For this, in vitro wound-healing assay was performed to evaluate whether 9f acts as an anti-metastatic agent in A549 cells.   As shown in Figure 4A,B, IL-1β-treated cells exhibited an increase in wound closure within 24 h compared with those not treated with IL-1β (control). Treatment of cells with 9f at 7 µM for 24 h significantly reduced the migration of IL-1β-stimulated cells, which is a phenomenon confirmed by qualitative assessment of the wound closure ( Figure 4A,B). Given the good results of 9f in inhibiting the IL-1β-induced EMT in epithelial cells, we investigated whether 9f could affect the EMT-associated migratory ability in A549 cells. For this, in vitro wound-healing assay was performed to evaluate whether 9f acts as an anti-metastatic agent in A549 cells.
As shown in Figure 4A,B, IL-1β-treated cells exhibited an increase in wound closure within 24 h compared with those not treated with IL-1β (control). Treatment of cells with 9f at 7 μM for 24 h significantly reduced the migration of IL-1β-stimulated cells, which is a phenomenon confirmed by qualitative assessment of the wound closure ( Figure 4A,B). Figure 4. The effect of 9f on the migration of A549 cells assayed by the wound-healing assay. Cells were treated with 9f at 7 μM, and images were captured to calculate the scratch closure. In (A), representative photomicrography images showing the cell migration toward the cell-free area after treatment with DMEM (control) or 9f and after 24 h. In (B), the graph shows the percentage of scratch covered, which was measured by quantifying the total distance the cells moved from the edge of the scratch toward the center of the scratch, using ImageJ software, followed by conversion to a percentage of the wound covered. Values represent mean ± S.D. from three independent experiments. (+++) p < 0.001 compared with respective DMEM-treated cells and (***) p < 0.001 compared with IL-1β-stimulated cell vehicle-treated cells.
All other coumarin derivatives: triflic intermediates (4, 5a, 5b, 6); Suzuki-Miyaura adducts (7, 8a-g, 9a-f); Sonogashira adducts 11, 12a-c, 13 a-c); Negishi adducts (14 and 15); and alkyl coumarin derivatives obtained by catalytic hydrogenation (16 and 17) were prepared according to the synthetic procedures described in the Supplementary Material. . The effect of 9f on the migration of A549 cells assayed by the wound-healing assay. Cells were treated with 9f at 7 µM, and images were captured to calculate the scratch closure. In (A), representative photomicrography images showing the cell migration toward the cell-free area after treatment with DMEM (control) or 9f and after 24 h. In (B), the graph shows the percentage of scratch covered, which was measured by quantifying the total distance the cells moved from the edge of the scratch toward the center of the scratch, using ImageJ software, followed by conversion to a percentage of the wound covered. Values represent mean ± S.D. from three independent experiments. (+++) p < 0.001 compared with respective DMEM-treated cells and (***) p < 0.001 compared with IL-1β-stimulated cell vehicle-treated cells.

Cell Viability Assay and Treatment
The effect of coumarin derivatives on cell viability was evaluated by the MTT assay at a single dose according to the NCI testing protocol or at different concentrations for IC 50 determination [66]. Coumarin derivatives were dissolved in dimethyl sulfoxide (DMSO) and then diluted with DMEM. Briefly, cells were plated in 96-well plates (2 × 10 4 /well) and each coumarin derivative at 12 µM was added to the culture medium, and the cell cultures were continued for 24 h. Cisplatin (2.6 µM) was used as a reference drug. Thereafter, the medium was replaced with fresh DMEM containing 5 mg/mL MTT. Following an incubation period (4 h) in a humidified CO 2 incubator at 37 • C and 5% CO 2 , the supernatant was removed, and dimethyl sulfoxide solution (DMSO, 150 mL/well) was added to each cultured plate. After incubation at room temperature for 15 min, the absorbance of the solubilized MTT formazan product was spectrophotometrically measured at 540 nm. Three individual wells were assayed for each treatment, and the percentage viability relative to the control sample was determined as (absorbance of treated cells/absorbance of untreated cells) × 100%. Only the compound that reduced the viability by more than half the value of the control cells were screened in a range of concentration (10 −8 to 10 −3 M) with the A549, H2170, and NHI-3T3 cell lines. The concentration of 9f compound that reduced the viable cell number by 50% (CC 50 ) was determined using a non-linear regression approach, and the mean value of CC 50 for each cell type was calculated from triplicate.

Epithelial-to-Mesenchymal Transition (EMT) Induction and Coumarin Derivatives Treatment
For induction of the EMT process, A549 cells (1 × 10 5 per well) were seeded in 24-well culture plates and treated with 1 ng/mL IL-1β (Peprotech, Rocky Hill, NJ, USA) for 24 h. In the unstimulated cells, DMEM medium was added. Then, the morphological alteration of cells was observed under a microscope. This protocol for EMT induction is as reported in the previous literature [67]. To evaluate the effects of coumarin derivative with respect to EMT induced by IL-1β, cells were pretreated with compound 9f at 7 µM, being this treatment also maintained during stimulation with IL-1β for 24 h.

Immunofluorescence Staining
After 24 h, cells were fixed for 15 min at 4 • C with 4% paraformaldehyde in PBS. Cells were permeabilized with 0.1% Triton X-100 and washed with PBS. Next, cells were incubated with FITC-conjugated phalloidin (1:100) for 2 h at room temperature and then rinsed several times with PBS. Following an additional washing step with PBS, cells were stained with 10 µg/mL DAPI at room temperature for 10 min for the visualization of cell nuclei. Cell morphology was determined using an inverted epifluorescence microscope (Nikon Eclipse 50i). Fluorescence quantification was done using ImageJ 1.47 software (NIH, Bethesda, MD, USA). Images were analyzed through the "Measure" menu, which allowed analyzing the fluorescence intensity signal per cell from original photomicrographs.
In another set of experiments, the analysis for vimentin, a well-recognized marker for its selective expression and specific role in the mesenchymal state, was performed. After treatment, cells were fixed, permeabilized, and washed as described above. Next, the slides were incubated with an anti-vimentin antibody (1:100) at 4 • C overnight. The next day, the slides were incubated with secondary antibody goat anti-rabbit-FITC (1:100) dilutions at room temperature for 1 h. Lastly, cells were stained with DAPI (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and washed with PBS. Stained cells were analyzed by a flow cytometer (FACSCanto II, Becton Dickinson, San Jose, CA, USA) accompanied with the BD FACSDIVA™ software for data analysis. The cell-associated fluorescence of 5000 cells per sample was measured as mean fluorescence intensity (MFI) in the FL1 channel. The MFI values were corrected for unspecific staining by subtracting the fluorescence of cells unstained (negative control).

In Vitro Scratch Wound Healing Assay
To evaluate the effect of 9f on epithelial motility, we performed the scratch assay as described by Cardoso et al. [68]. Cells were maintained in 24-well plates until they reached 90% confluency. Thereafter, a vertical stripe on the cell monolayer was made using a sterile pipette (200 µL) tip. The wells were washed with PBS to remove dead cells and debris, and then, 9f was added at a concentration of 7 µM. As a control, the cells were treated with cell culture medium. Photographs were captured by a digital camera connected to an inverted microscope (Olympus IX70) at 0 and 24 h after scratch. The migration gap area of the cells was measured by ImageJ software (https://imagej.nih.gov/ij/; accessed on 24 November 2020, Center for Information Technology, National Institute of Health, Bethesda, MA, USA). Each measurement was repeated three times.

Statistical Analysis
Data were expressed as mean ± standard deviation (S.D.). The statistical analysis involving two groups was done using Student's t-test. Analysis of variance followed by the Tukey's test was used to compare three or more groups. Values of p < 0.05 were considered as indicative of significance.

Conclusions
In conclusion, twenty-six coumarin derivatives were synthesized through PCCCR and were evaluated for their anti-lung cancer properties against two non-small cell lung carcinoma (NSCLC) cell lines. Coumarins 8f and 9f, presenting a 3,4-dichloro-phenyl radical, inhibited in vitro the growth of both human lung adenocarcinoma cells in low micromolar concentration. Derivative 9f regulates the epithelial-to-mesenchymal transition (EMT) suppressing the mesenchymal marker vimentin and cancer cell migration in IL-1β-stimulated A549 cells. Taken together, our findings suggest that coumarin derivatives, especially compound 9f, may become a promising hit in the process of lung cancer drug discovery, especially in lung cancer promoted by non-small cell lung carcinoma (NSCLC) cell lines.