Coumarin Sulfonamides and Amides Derivatives: Design, Synthesis, and Antitumor Activity In Vitro

Coumarins possesses immeasurable antitumor potential with minimum side effects depending on the substitutions on the basic nucleus, which exhibits great prospects for antitumor drug development. In an attempt to develop novel antitumor candidates, a series of coumarin sulfonamides and amides derivatives were designed and synthetized. The majority of these derivatives showed good cytotoxic activity against MDA-MB-231 and KB cell lines, among which compound 9c was the most potent against MDA-MB-231 cells, with IC50 value of 9.33 μM, comparable to 5-fluorouracil. Further investigation revealed that compound 9c had versatile properties against tumors, including inhibition of cell migration and invasion as well as inducing apoptosis. Reactive oxygen species (ROS) assay and western blotting analysis suggested that compound 9c promoted cancer cell apoptosis by increasing ROS levels and upregulating the expression of caspase-3 in MDA-MB-231 cells. These results indicated that compound 9c could be promising lead compound for further antitumor drug research.


Introduction
Cancer is a widespread and lethal disease characterized by uncontrolled growth of abnormal cells. It has been expected to be the leading cause of death in the future [1,2]. Although cancer research resulted in a range of innovative and promising therapeutic approaches, the existing drugs for cancer treatment are frequently associated with several drawbacks such as severe side effects, multidrug resistance, poor bioavailability. Therefore, there is an urgent need for the development of more effective and safer anticancer agents.
Coumarins are a wide family of secondary metabolites found in various species of plants but also fungi and microorganisms [3,4], which exhibit diverse pharmacological effects, especially anticancer activity [5][6][7]. Many coumarins and their derivatives have attracted considerable attention as cancer chemopreventive agents and also as cancer therapeutics [8][9][10][11][12][13][14]. Additionally, they have several attractive features, such as low molecular weight, simple structure, high bioavailability, high solubility in most of the organic solvents, and low toxicity, which ensure them a prominent role as lead compounds in drug research and development [15].
The promising biological profile of coumarins as antitumor agents and their easy synthetic modifications paved the way for design and synthesis of various coumarin derivatives [16,17]. In the last decade, coumarin derivatives have been increasingly reported to possess antitumor activity through different pharmacological mechanisms, among which coumarin sulfonamides and amides derivatives have attracted great interest because of their potency. Sabt et al. reported a series of novel coumarin-6-sulfonamides as antiproliferative agents [18] and found that compound I (Figure 1) was able to induce apoptosis In order to discover more potent and selective antitumor agents for further development and as a continuation of our previous studies [21], we designed and synthesized coumarin derivatives bearing sulfonamide and amide moieties. Their cytotoxic activity was screened, and the antitumor effects of the most potent compound on MDA-MB-231 cell line were further investigated.

Chemical Synthesis
The synthesis of coumarin sulfonamides derivatives 7a-7b and 9a-9c were outlined in Scheme 1. Compound 3 were obtained by the sulfonylation of the commercially available aromatic amine 1 with methyl 2-chlorosulfonylacetate 2 in triethylamine at room temperature. The alkylation of 2,4-dihydroxybenzaldehyde 4 with bromide 5 in anhydrous DMF at 110 °C provided compound 6. The condensation of compound 3a with substituted salicylaldehyde 6 in anhydrous ethanol in the presence of piperidine gave coumarin sulfonamides 7a-7b. The reaction of compound 3 and 4-(diethylamino) salicylaldehyde 8 in the same condition afforded the desired compound 9. The coumarin amides derivatives 12a-12c were synthesized as described in Scheme 2. The ethyl coumarin-3-carboxylate 11 was prepared by the condensation of 4-(diethylamino) salicylaldehyde 8 with ethyl malonate 10 in anhydrous ethanol in the presence of piperidine. The ammonolysis of compound 11 with aromatic amines 1 in anhydrous ethanol under reflux led to desired compound 12. In order to discover more potent and selective antitumor agents for further development and as a continuation of our previous studies [21], we designed and synthesized coumarin derivatives bearing sulfonamide and amide moieties. Their cytotoxic activity was screened, and the antitumor effects of the most potent compound on MDA-MB-231 cell line were further investigated.

Chemical Synthesis
The synthesis of coumarin sulfonamides derivatives 7a-7b and 9a-9c were outlined in Scheme 1. Compound 3 were obtained by the sulfonylation of the commercially available aromatic amine 1 with methyl 2-chlorosulfonylacetate 2 in triethylamine at room temperature. The alkylation of 2,4-dihydroxybenzaldehyde 4 with bromide 5 in anhydrous DMF at 110 • C provided compound 6. The condensation of compound 3a with substituted salicylaldehyde 6 in anhydrous ethanol in the presence of piperidine gave coumarin sulfonamides 7a-7b. The reaction of compound 3 and 4-(diethylamino) salicylaldehyde 8 in the same condition afforded the desired compound 9. The coumarin amides derivatives 12a-12c were synthesized as described in Scheme 2. The ethyl coumarin-3-carboxylate 11 was prepared by the condensation of 4-(diethylamino) salicylaldehyde 8 with ethyl malonate 10 in anhydrous ethanol in the presence of piperidine. The ammonolysis of compound 11 with aromatic amines 1 in anhydrous ethanol under reflux led to desired compound 12.

Cytotoxicity
The in vitro cytotoxicity of the synthesized compounds (Table 1) was evaluated using MTT assay against three human cancer cell lines MDA-MB-231 (human breast cancer cell line), KB (human oral epidermoid carcinoma cell line), and HCT-116 (human colon cancer cell line). 5-Fluorouracil was included as positive control. As shown in Table 2, the majority of the compounds exhibited good inhibitory activities against MDA-MB-231 and KB cell lines, while weak cytotoxicity was observed against HCT-116 cell line except for compounds 9a and 9b. Almost all the compounds were more active than 5-fluorouracil against KB cells. In particular, compounds 9b, 9c and 12b showed stronger inhibitory effects than the rest of the compounds. The most potent compound 9c with the IC 50 values of 9.33 and 13.66 µM against MDA-MB-231 and KB cells, respectively, was chosen for further investigation. majority of the compounds exhibited good inhibitory activities against MDA-MB-231 and KB cell lines, while weak cytotoxicity was observed against HCT-116 cell line except for compounds 9a and 9b. Almost all the compounds were more active than 5-fluorouracil against KB cells. In particular, compounds 9b, 9c, and 12b showed stronger inhibitory effects than the rest of the compounds. The most potent compound 9c with the IC50 values of 9.33 and 13.66 μM against MDA-MB-231 and KB cells, respectively, was chosen for further investigation.  In addition, it was observed that among coumarin sulfonamides 7a-7b and 9a-9c, 7-diethylaminocoumarin sulfonamides 9a-9c exhibited higher activity than 7-alkoxycoumarin sulfonamides 7a-7b, which implied the diethylamino group at C-7 position improved potency. Coumarin sulfonamides 9a-9c had better cytotoxicity compared to amides derivatives 12a-12c, suggesting the sulfonamide moiety at C-3 position of the coumarin core is favored to the activity. Furthermore, the IC50 values of compounds 9a-9c and 12a-12c indicated that substituents on the benzene ring in the sulfonamides or amides had little impact on the potency. 9c majority of the compounds exhibited good inhibitory activities against MDA-MB-231 and KB cell lines, while weak cytotoxicity was observed against HCT-116 cell line except for compounds 9a and 9b. Almost all the compounds were more active than 5-fluorouracil against KB cells. In particular, compounds 9b, 9c, and 12b showed stronger inhibitory effects than the rest of the compounds. The most potent compound 9c with the IC50 values of 9.33 and 13.66 μM against MDA-MB-231 and KB cells, respectively, was chosen for further investigation.  In addition, it was observed that among coumarin sulfonamides 7a-7b and 9a-9c, 7-diethylaminocoumarin sulfonamides 9a-9c exhibited higher activity than 7-alkoxycoumarin sulfonamides 7a-7b, which implied the diethylamino group at C-7 position improved potency. Coumarin sulfonamides 9a-9c had better cytotoxicity compared to amides derivatives 12a-12c, suggesting the sulfonamide moiety at C-3 position of the coumarin core is favored to the activity. Furthermore, the IC50 values of compounds 9a-9c and 12a-12c indicated that substituents on the benzene ring in the sulfonamides or amides had little impact on the potency. 7b and KB cell lines, while weak cytotoxicity was observed against HCT-116 cell line except for compounds 9a and 9b. Almost all the compounds were more active than 5-fluorouracil against KB cells. In particular, compounds 9b, 9c, and 12b showed stronger inhibitory effects than the rest of the compounds. The most potent compound 9c with the IC50 values of 9.33 and 13.66 μM against MDA-MB-231 and KB cells, respectively, was chosen for further investigation.

Compd
Structure Compd Structure In addition, it was observed that among coumarin sulfonamides 7a-7b and 9a-9c, 7-diethylaminocoumarin sulfonamides 9a-9c exhibited higher activity than 7-alkoxycoumarin sulfonamides 7a-7b, which implied the diethylamino group at C-7 position improved potency. Coumarin sulfonamides 9a-9c had better cytotoxicity compared to amides derivatives 12a-12c, suggesting the sulfonamide moiety at C-3 position of the coumarin core is favored to the activity. Furthermore, the IC50 values of compounds 9a-9c and 12a-12c indicated that substituents on the benzene ring in the sulfonamides or amides had little impact on the potency. 12a and KB cell lines, while weak cytotoxicity was observed against HCT-116 cell line except for compounds 9a and 9b. Almost all the compounds were more active than 5-fluorouracil against KB cells. In particular, compounds 9b, 9c, and 12b showed stronger inhibitory effects than the rest of the compounds. The most potent compound 9c with the IC50 values of 9.33 and 13.66 μM against MDA-MB-231 and KB cells, respectively, was chosen for further investigation.

Compd
Structure Compd Structure In addition, it was observed that among coumarin sulfonamides 7a-7b and 9a-9c, 7-diethylaminocoumarin sulfonamides 9a-9c exhibited higher activity than 7-alkoxycoumarin sulfonamides 7a-7b, which implied the diethylamino group at C-7 position improved potency. Coumarin sulfonamides 9a-9c had better cytotoxicity compared to amides derivatives 12a-12c, suggesting the sulfonamide moiety at C-3 position of the coumarin core is favored to the activity. Furthermore, the IC50 values of compounds 9a-9c and 12a-12c indicated that substituents on the benzene ring in the sulfonamides or amides had little impact on the potency. 9a and KB cell lines, while weak cytotoxicity was observed against HCT-116 cell line except for compounds 9a and 9b. Almost all the compounds were more active than 5-fluorouracil against KB cells. In particular, compounds 9b, 9c, and 12b showed stronger inhibitory effects than the rest of the compounds. The most potent compound 9c with the IC50 values of 9.33 and 13.66 μM against MDA-MB-231 and KB cells, respectively, was chosen for further investigation.  In addition, it was observed that among coumarin sulfonamides 7a-7b and 9a-9c, 7-diethylaminocoumarin sulfonamides 9a-9c exhibited higher activity than 7-alkoxycoumarin sulfonamides 7a-7b, which implied the diethylamino group at C-7 position improved potency. Coumarin sulfonamides 9a-9c had better cytotoxicity compared to amides derivatives 12a-12c, suggesting the sulfonamide moiety at C-3 position of the coumarin core is favored to the activity. Furthermore, the IC50 values of compounds 9a-9c and 12a-12c indicated that substituents on the benzene ring in the sulfonamides or amides had little impact on the potency. 12b and KB cell lines, while weak cytotoxicity was observed against HCT-116 cell line except for compounds 9a and 9b. Almost all the compounds were more active than 5-fluorouracil against KB cells. In particular, compounds 9b, 9c, and 12b showed stronger inhibitory effects than the rest of the compounds. The most potent compound 9c with the IC50 values of 9.33 and 13.66 μM against MDA-MB-231 and KB cells, respectively, was chosen for further investigation.  In addition, it was observed that among coumarin sulfonamides 7a-7b and 9a-9c, 7-diethylaminocoumarin sulfonamides 9a-9c exhibited higher activity than 7-alkoxycoumarin sulfonamides 7a-7b, which implied the diethylamino group at C-7 position improved potency. Coumarin sulfonamides 9a-9c had better cytotoxicity compared to amides derivatives 12a-12c, suggesting the sulfonamide moiety at C-3 position of the coumarin core is favored to the activity. Furthermore, the IC50 values of compounds 9a-9c and 12a-12c indicated that substituents on the benzene ring in the sulfonamides or amides had little impact on the potency. 9b for compounds 9a and 9b. Almost all the compounds were more active than 5-fluorouracil against KB cells. In particular, compounds 9b, 9c, and 12b showed stronger inhibitory effects than the rest of the compounds. The most potent compound 9c with the IC50 values of 9.33 and 13.66 μM against MDA-MB-231 and KB cells, respectively, was chosen for further investigation.  In addition, it was observed that among coumarin sulfonamides 7a-7b and 9a-9c, 7-diethylaminocoumarin sulfonamides 9a-9c exhibited higher activity than 7-alkoxycoumarin sulfonamides 7a-7b, which implied the diethylamino group at C-7 position improved potency. Coumarin sulfonamides 9a-9c had better cytotoxicity compared to amides derivatives 12a-12c, suggesting the sulfonamide moiety at C-3 position of the coumarin core is favored to the activity. Furthermore, the IC50 values of compounds 9a-9c and 12a-12c indicated that substituents on the benzene ring in the sulfonamides or amides had little impact on the potency. 12c for compounds 9a and 9b. Almost all the compounds were more active than 5-fluorouracil against KB cells. In particular, compounds 9b, 9c, and 12b showed stronger inhibitory effects than the rest of the compounds. The most potent compound 9c with the IC50 values of 9.33 and 13.66 μM against MDA-MB-231 and KB cells, respectively, was chosen for further investigation.  In addition, it was observed that among coumarin sulfonamides 7a-7b and 9a-9c, 7-diethylaminocoumarin sulfonamides 9a-9c exhibited higher activity than 7-alkoxycoumarin sulfonamides 7a-7b, which implied the diethylamino group at C-7 position improved potency. Coumarin sulfonamides 9a-9c had better cytotoxicity compared to amides derivatives 12a-12c, suggesting the sulfonamide moiety at C-3 position of the coumarin core is favored to the activity. Furthermore, the IC50 values of compounds 9a-9c and 12a-12c indicated that substituents on the benzene ring in the sulfonamides or amides had little impact on the potency. In addition, it was observed that among coumarin sulfonamides 7a-7b and 9a-9c, 7diethylaminocoumarin sulfonamides 9a-9c exhibited higher activity than 7-alkoxycoumarin sulfonamides 7a-7b, which implied the diethylamino group at C-7 position improved potency. Coumarin sulfonamides 9a-9c had better cytotoxicity compared to amides derivatives 12a-12c, suggesting the sulfonamide moiety at C-3 position of the coumarin core is favored to the activity. Furthermore, the IC 50 values of compounds 9a-9c and 12a-12c indicated that substituents on the benzene ring in the sulfonamides or amides had little impact on the potency.

Inhibition of Wound Healing in MDA-MB-231 Cells by Compound 9c
The wound healing assay is a classic and common method used to investigate the effect of antitumor drugs on tumor migration. We next assessed the effect of compound 9c on the migration of MDA-MB-231 cells with a wound-healing assay. The results showed that the scratch gaps in compound 9c (10 and 20 µM) treated MDA-MB-231 cells were wider than the control with increased treating time (Figure 2). The cell migration was inhibited, and the wound area was increased compared with the control in a concentrationdependent manner. effect of antitumor drugs on tumor migration. We next assessed the effect of compound 9c on the migration of MDA-MB-231 cells with a wound-healing assay. The results showed that the scratch gaps in compound 9c (10 and 20 μM) treated MDA-MB-231 cells were wider than the control with increased treating time (Figure 2). The cell migration was inhibited, and the wound area was increased compared with the control in a concentration-dependent manner.

Inhibition of Compond 9c on the Invasion of MDA-MB-231 Cells
Transwell invasion assay was used to investigate the effect of compound 9c on the invasion of MDA-MB-231 cells. Treatment of compound 9c significantly reduced (p < 0.01) the number of invaded cells at the concentration of 20 μM (Figure 3).

Inhibition of Compond 9c on the Invasion of MDA-MB-231 Cells
Transwell invasion assay was used to investigate the effect of compound 9c on the invasion of MDA-MB-231 cells. Treatment of compound 9c significantly reduced (p < 0.01) the number of invaded cells at the concentration of 20 µM (Figure 3).

Compound 9c Induces Apoptosis in MDA-MB-231 Cells
To further evaluate whether compound 9c can induce apoptosis, the Annexin V-FITC/PI double-staining assay was applied with flow cytometer. After treatment with compound 9c for 48 h, MDA-MB-231 cells were dyed by Annexin V-FITC and showed green fluorescence, which indicates the early phase of apoptosis. The cells dyed by Annexin V-FITC and PI showed red fluorescence inside with green fluorescence outside, which corresponded to the late phase of apoptosis (Figure 4a). The cells treated with compound 9c (10 µM) had stronger fluorescence intensity compared with the control group, which indicated that compound 9c promoted apoptosis of MDA-MB-231 cells. Meanwhile, as shown in Figure 4b

Compound 9c Induces Apoptosis in MDA-MB-231 Cells
To further evaluate whether compound 9c can induce apoptosis, the Annexin V-FITC/PI double-staining assay was applied with flow cytometer. After treatment with compound 9c for 48 h, MDA-MB-231 cells were dyed by Annexin V-FITC and showed green fluorescence, which indicates the early phase of apoptosis. The cells dyed by Annexin V-FITC and PI showed red fluorescence inside with green fluorescence outside, which corresponded to the late phase of apoptosis (Figure 4a). The cells treated with compound 9c (10 μM) had stronger fluorescence intensity compared with the control group, which indicated that compound 9c promoted apoptosis of MDA-MB-231 cells. Meanwhile, as shown in Figure 4b,c, when treated with different concentrations of compound 9c, the percentages of apoptotic MDA-MB-231 cells increased from 3.85% in control cells to 7.72%, 20.11%, 40.71%, 53.50%, and 57.46%, respectively. The results implied that compound 9c could induce the apoptosis of MDA-MB-231 cells in a concentration-dependent manner.

Western Blot Analysis
Next, Western blot analysis was applied to understand the molecular mechanism of apoptosis induced by compound 9c. The protein expression levels of caspase-3 and Bcl-2 were detected since caspase-3 played a crucial role in apoptotic pathways by cleaving a variety of key cellular proteins, and Bcl-2 is the well-known anti-apoptotic marker. After exposure of MDA-MB-231 cells to compound 9c, the expression level of caspase-3 was dramatically increased, while no obvious effect on the expression of Bcl-2 was observed ( Figure 6).

Western Blot Analysis
Next, Western blot analysis was applied to understand the molecular mechanism of apoptosis induced by compound 9c. The protein expression levels of caspase-3 and Bcl-2 were detected since caspase-3 played a crucial role in apoptotic pathways by cleaving a variety of key cellular proteins, and Bcl-2 is the well-known anti-apoptotic marker. After exposure of MDA-MB-231 cells to compound 9c, the expression level of caspase-3 was dramatically increased, while no obvious effect on the expression of Bcl-2 was observed ( Figure 6). apoptosis induced by compound 9c. The protein expression levels of caspase-3 and Bcl-2 were detected since caspase-3 played a crucial role in apoptotic pathways by cleaving a variety of key cellular proteins, and Bcl-2 is the well-known anti-apoptotic marker. After exposure of MDA-MB-231 cells to compound 9c, the expression level of caspase-3 was dramatically increased, while no obvious effect on the expression of Bcl-2 was observed ( Figure 6).

Discussion
Coumarins and their derivatives have been regarded as an important family of anticancer lead compounds, which exhibit diverse effects such as anti-proliferation, anti-invasion, anti-metastasis, anti-angiogenesis and inducing apoptosis. The various
In this study, coumarin sulfonamide 9c with the best antiproliferative activity against MDA-MB-231 and KB cell lines were obtained by modification of the basic structure of coumarin. The effects of compound 9c on the migration and invasion of MDA-MB-231 cells have been further investigated since the ability of cancer cells to migrate and invade into the surrounding tissue is the hallmark of tumor metastasis [29]. Treatment using compound 9c has shown significant reduction in the percentage of migration and invasion of MDA-MB-231 cells in a concentration-dependent manner.
Apoptosis, an immunogenic programmed cell death, eliminates unhealthy, unwanted cells and maintains surrounding tissue [30]. In cancer, evasion of cell death is one of the major mechanisms for abnormal cells to transform themselves into malignant ones [31]. Thus, drugs that can induce apoptosis, specifically in malignant and metastatic cells, would be the most promising cancer treatment candidates. Annexin V-FITC/PI double-staining assay combined with flow cytometry analysis confirmed that compound 9c induced apoptosis in MDA-MB-231 cells significantly in a concentration-dependent manner. Most antitumor reagents that can induce apoptosis, including many first-line chemotherapy drugs such as cisplatin, are known to provoke oxidative stress by generating excess ROS, which suggests that abnormal ROS generation is closely associated with apoptosis [32]. Our results of ROS assay showed that ROS levels in MDA-MB-231 cells were significantly increased after treatment with compound 9c.
The activation of the key apoptotic protein caspase-3 [33] can cleave regulatory proteins essential for cell survival and maintenance as well as poly (ADP-ribose) polymerase (PARP), which is involved in DNA repair and programmed cell death, thereby promoting apoptosis. Bcl-2 as an anti-apoptotic protein has been shown to enhance cell survival by inhibiting apoptosis and enhance the sensitivity of tumor cells to chemotherapy drugs [34][35][36]. Western blotting analysis demonstrated that the expression of caspase-3 was markedly upregulated, whereas little changes in the expression level of Bcl-2 were observed after treating with compound 9c. These results suggested that compound 9c induced apoptosis by increasing ROS levels and upregulating caspase-3 expression in MDA-MB-231 cancer cells.
However, the anticancer mechanism of coumarin sulfonamides needs to be further elucidated. Their anticancer potential, security and pharmacokinetic properties in vivo were also left to be investigated. Anyway, this study offers a start point for further elaboration to develop anticancer reagents with coumarin sulfonamide scaffold. More researches which link the anticancer effect, safety, and bioavailability with structure modification are expected to accelerate the development of coumarin derivatives for cancer treatment.

Syntheses
All melting points were taken on an X-6 microscope melting point instrument (Bjfuka, Beijing, China) and are uncorrected. Infrared spectra were determined with a TENSORII spectrometer (Bruker, Karlsruhe, Germany). NMR spectra were measured using 300 MHz Bruker AV III spectrometers (Bruker, Karlsruhe, Germany) and 400 MHz JNM-ECZ400S/L1 spectrometers (Varian INOVA, Palo Alto, CA, USA) with tetramethylsilane (TMS) as an internal standard. The following abbreviations were used to designate the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, brs = broad singlet. Coupling constant (J) is reported in Hertz (Hz). HR-MS spectra were obtained on Finnigan LCQ and Micromass Auto Spec Ultima-Tof spectrometer (Thermo, Boston, Massachusetts, USA). Analytic thin-layer chromatography (TLC) was performed on precoated silica gel 60 F254 plates (Qingdao Haiyang, Qingdao, Shandong Province, China) using petroleum ether-ethyl acetate as solvent system. All the chemicals were of analytic grade. The detailed preparation procedures of intermediates, byproducts, and target compounds are described below.

General Procedure for the Synthesis of Compounds 3a-3c
Methyl 2-chlorosulfonylacetate 2 (500 mg, 2.90 mmol) was added dropwise to a solution of aromatic amines 1 (4.35 mmol) in Et 3 N (10 mL). The reaction mixture was stirred at room temperature for 3 h. After completion of the reaction, a solution of 10% HCl was added slowly to the mixture to adjust the pH to 7. The mixture was then extracted with DCM (12 mL × 3). The combined organic layer was dried over anhydrous Na 2 SO 4 and evaporated in a vacuum to give a crude residue which was purified by column chromatography (cyclohexane: ethyl acetate = 15:1-1:1) to yield compounds 3a-3c. were dissolved in anhydrous DMF (2 mL), followed by the addition of the bromide 5 (1.45 mmol). The reaction mixture was stirred at 110 • C. After completion of the reaction, the reaction mixture was diluted with DCM (20 mL) and washed with water (10 mL) and saturated brine (10 mL). The organic layer was dried over anhydrous Na 2 SO 4 and evaporated in a vacuum to give a crude residue which was purified by column chromatography 10, and 20 µM) for 24 and 48 h, respectively. Images were captured at the identified time point (0, 24, and 48 h) and processed with the ImageJ software.

Analysis of Cell Invasion
For the assay, 24-well transwell chambers coated with 45 µL matrigel were used. In brief, 6 × 10 4 MDA-MB-231 cells per well were seeded in the upper part of the insert filled with 200 µL of DMEM without FBS, and for the lower part, the chamber was filled with 600 µL DMEM containing 10% FBS. The cells were allowed to grow for 48 h followed by treatment with different concentrations of compound 9c (5, 10, and 20 µM). The invaded cells were fixed with paraformaldehyde for 2 h and stained with crystal violet solution (0.2%) for 1 h, and non-invading cells in the top chambers of the transwell plates were scraped away by cotton swabs. Every insert was imaged at 10× magnification and analyzed using ImageJ software. 4.2.5. Apoptosis Analysis and Morphology Observation 5 × 10 5 MDA-MB-231 cells per well were seeded in a 6-well plate for 12 h and were treated with different concentrations of compound 9c (5,10,20,40, and 80 µM) for 48 h. After treatment, the cells were harvested, washed twice with ice-cold PBS and resuspended in 500 µL of 1× Annexin V binding buffer and then mixed with 5 µL of Annexin V-FITC and 5 µL of PI solution at room temperature in the dark for 10 min. Then, one part of the cells was immediately analyzed with a flow cytometer. The other part was washed twice with PBS, and the cells were photo-graphed by fluorescence microscope.

Intracellular ROS Level Detection
In 6-well plates, 2 × 10 5 MDA-MB-231 cells were plated and treated with different concentrations (0, 10, 20, 30, and 40 µM) of compound 9c for 48 h. ROS assay kit (Beyotime) was used to measure intracellular ROS level. DCFH-DA diluted with serum-free medium with 1:1000 was added into the cells at 37 • C for 20 min. The cells were collected after washing three times with serum-free medium. Images were acquired using an inverted fluorescence microscope, and the fluorescence intensity was detected at an excitation wavelength of 488 nm and an emission wavelength of 525 nm with microplate reader.

Western Blotting
For Western blotting analyses, the collected cells were rinsed in PBS and lysed in sodium dodecyl sulfate (SDS) lysis buffer. Equal amounts of protein were separated on 10% SDS-polyacrylamide gels, transferred to polyvinylidene fluoride (PVDF) membranes, and blocked with 5% nonfat dry milk in tris-buffered saline and tween 20 (TBST) for 1 h. The membranes were then incubated with primary antibodies (caspase-3, Bcl-2 and β-actin) overnight at 4 • C. After being washed three times with TBST for 10 min, the membranes were incubated with appropriate secondary antibodies in TBST for 1 h. After several washes of TBST, the blots were developed by enhanced chemiluminescence (ECL) solution.

Conclusions
A novel series of coumarin sulfonamides and amides derivatives were designed and synthesized. Compound 9c was found to be the most potent derivative, which displayed favorable antiproliferative activities toward MDA-MB-231 cells and inhibited migration and invasion and induced apoptosis in MDA-MB-231 cells. Furthermore, it was verified that compound 9c promoted MDA-MB-231 cell apoptosis by increasing intracellular ROS levels and upregulating the expression of caspase-3. This work presents information that is helpful for the design and synthesis of new coumarin derivatives as potential antitumor drug candidates.