Evaluation of the anti-tumor activities of Sulfonylurea Derivatives

This study prepared 25 sulfonylurea compounds to evaluate anti-tumor activity. Through experimental investigations in MDA-MB-231 and MCF-7, i.e., cell lines of breast carcinoma of human, we have concluded that some compounds can significantly suppress breast carcinoma cells from growing and proliferating. Moreover, the compound M’s inhibitory effect on cells of breast carcinoma is concentration-dependent under a certain treatment time; and the inhibitory effect of the compound M on breast carcinoma cells is time-dependent under a certain concentration. In addition, we also found that the compound M can effectively suppress cells of breast carcinoma from migration and independent survival. The results can show the prospect of research and development of new breast carcinoma treatment drug.


Introduction
Carcinoma [1] has been found as a major disease that poses a serious threat to human life and health, i.e., malignant tumor [2] . Today, breast carcinoma [3] has become a malignant tumor threatening health of females, and the incidences among females are rising year by year. Although numerous treatment methods can be used for breast carcinoma treatment (e.g., surgery [4] , radiotherapy [5] , chemotherapy [6] , targeted drug therapy [7] ), breast carcinoma, in particular triple-negative breast carcinoma [8] , cannot be completely cured because of its strong metastatic ability, and the existing treatment methods have several defects that affect the treatment of breast carcinoma. Thus far, breast carcinoma remains a malignant tumor disease with the highest mortality rate worldwide [9] , and it significantly threatens the health of human beings, especially women. Accordingly, new drugs [10] are urgently needed for the treatment of breast carcinoma.
Sulfonylurea compounds have achieved wide applications as hypoglycemic [11] agents in medicine and as pesticides in agriculture [12] . Sulfonylurea drugs are found as the earliest but most widely applied oral hypoglycemic drugs, which fall into the first and second generations. As researchers have been investigating the hypoglycemic effect of sulfonylurea drugs, the third generation of sulfonylurea hypoglycemic [13] drugs has been developed. Glimepiride [14] has been found as the main representative of the third-generation sulfonylurea hypoglycemic agents, which shows the advantages of small dosage and the ability to significantly improve insulin resistance.
Moreover, Sulfonylurea compounds as herbicides have been investigated as early as the 1970s. After continuous research in the pesticide field, the sulfonylurea herbicides that have been extensively employed currently primarily consist of chlorsulfuron [15] , metsulfuron-methyl [16] , chlorimuron-methyl [17] , etc. As the hypoglycemic mechanism of sulfonylurea compounds has been recently studied in depth, the second-generation hypoglycemic drugs have been found with potential antitumor activity. Thus, the antitumor activity of sulfonylurea compounds has begun to come into the attention in carcinoma treatment. It has become a hot spot in the research of this compound over the past few years. Pooja Rathore designed and synthesized a series of pyrazoline-substituted benzenesulfonylurea derivatives, and tested 14 compounds in vitro for anti-tumor activity [18] . As indicated by the experimental results, these compounds exhibited high malignant tumor inhibitory activity. To be specific, 4 compounds showed broad-spectrum anti-tumor activity and had a strong inhibitory effect on lung carcinoma [19] , prostate carcinoma [20] , colon carcinoma [21] , kidney carcinoma [22] , ovarian carcinoma [23] and breast carcinoma [24] . A series of 4-phenoxyquinoline derivatives containing sulfonylurea groups were designed, synthesized and assessed for their c-Met kinase inhibitory activity and in vitro cytotoxicity. According to the experimental results, one compound exhibited good selectivity and significant anti-tumor activity against human lung carcinoma cell line HT460, human gastric carcinoma cell line MKN-45, and human colorectal carcinoma cell line HT-29 and the human breast carcinoma cell line MDA-MB-231 with IC 50 values of 0.055 μM, 0.064 μM, 0.16 μM and 0.49 μM, respectively [25] . According to these results, 25 sulfonylurea compounds were prepared to evaluate anti-tumor activity that was reported in the literatures. Moreover, an investigation was conducted on the effect of the new compounds on the migration ability of breast carcinoma cells and the effect on the cloning ability of breast carcinoma cells.
Material and experiment 1

. Cell Recovery
The steps of this method took the recovery of a tube of MCF-7 cells as an example.
A tube of MCF-7 cell cryopreservation tube was taken out of liquid nitrogen and quickly put into a 37-degree water bath with float to melt. After the cells completely thawed, they were aspirated with a pipette in the ultra-clean table. Then, they were added to a 15 mL centrifuge tube and pipetted evenly. The 15 mL centrifuge tube was put in the centrifuge at 1000 rpm for 4 min. After the centrifugation, 700 μL of fresh 10% serum DMEM medium was used to blow up the cell pellet, and it was pipetted evenly, and then the cell suspension was averaged. They were divided into 4 wells in a 12-well plate, and then medium was added to each well to make up to 800 μL. Last, the 12-well plate was put in a 37-degree incubator for culture.

Cell exchange
The steps of this method took the MCF-7 cells in a 12-well plate as an example.
When the cell culture medium in the 12-well plate was found to turn yellow, or the growth and proliferation speed of the cells were found to slow down, the medium should be changed. The steps for cell exchange are as follows. The cell culture solution was aspirated in the 12-well plate, and 800 μL of PBS adherently was added to the wall, the 12-well plate was gently shaken, and the PBS was quickly aspirated. 800 μL of fresh culture medium was added to each well, and the cell culture plate was put into the 37°C cell incubator to continue culturing.

Cell Passaging
The steps of this method took the passage of MCF-7 cells in a 12-well plate as an example.
When the cells in the 12-well plate grew to about 80% of the area of the well plate, cell passaging operations would be required. The culture medium was aspirated in the 12-well plate, and 800 μL of PBS was added to each well. After the 12-well plate was gently shaken, the PBS was quickly aspirated. 200 μL of 0.25% trypsin was added to each well. When the cells were found to be rounded to large cells falling off, 800 μL of medium was added to each well to stop the digestion, and it was pipetted evenly to form a cell suspension. Next, 700 μL of fresh culture medium was added to each well of the new 12-well plate, and then about 100 μL of cell suspension was added to each well. The 12-well plate was shaken gently and carefully placed in a 37°C cell culture incubator for culture.

Cell cryopreservation
The steps of this method took a tube of MCF-7 cells cryopreserved as an example.
The old medium was aspirated in the 12-well plate. 800 μL of PBS was added to each well. The well was washed twice, and the PBS was aspirated. 200 μL of 0.25% trypsin was added to each well for digestion. The 12-well plate was placed under a microscope for observation. When many cells fell off, 800 μL of fresh medium was added to stop the digestion and remove the cells, and it was pipetted evenly to form a cell suspension. The 2-well cell suspension was transferred in the 12-well plate to a 15 mL centrifuge tube. Then, the centrifugation was made at 1000 rpm for 4 min.
After the centrifugation, the supernatant was aspirated and discarded, and 900 μL of fresh medium was added to blow up the cell pellet and pipette evenly. 100 μL of DMSO was added and mixed quickly. The cell suspension was transferred to the cryopreservation tube and marked. The cryopreservation tube was put into the gradient freezer box, and then the gradient freezer box was placed in the -80°C refrigerator overnight. The next day, the cryopreservation tube was transferred in the gradient cryopreservation box.

Cell count
Take out the 12-well plate to be counted from the incubator, place it in the ultra-clean table and wash one well of the cells with 800 μL PBS twice. 200 μL of 0.25% trypsin was added for digestion, after terminating the digestion. 10 μL of cell suspension was taken out and placed in a 1.5 mL EP tube, and 90 μL of PBS was added and mixed well. Take a clean cell counting plate, place a cover glass on it, take 10 μL of the cell diluent in the 1.5 mL EP tube and slowly add it from the side of the cover glass to

Compound preparation
An appropriate amount of the new compounds was weighed into a 1.5 mL enzyme-free sterile EP tube, and the name of the compounds was marked. According to the relative molecular mass of the compounds, the volume of DMSO was calculated, and then the DMSO was added to make the final concentration 10 mM. A pipette was used to add the corresponding volume of DMSO and mix evenly, so the compound could be completely dissolved. Put the 1.5 mL EP tube into a 95°C metal bath and heat it for 10 min. Lastly, the compounds solution was stored in the refrigerator-20°C.

Cell viability assay experiment . MTT method to determine cell viability experiment
The growth of the cells was observed in the 12-well plate, one of the wells was digested when it grew to an appropriate density. It was transferred to a 15 mL centrifuge tube and diluted to a concentration of 50,000 cells/mL. The 96-well plate was taken out, and 100 μL volume of cell suspension was added to each well of the 96-well plate. Then, it was placed in a 37°C incubator overnight. The medium was changed by aspirating the medium in each well, a new control medium or medium with the corresponding compound concentration was added and placed in a 37°C incubator for cultivation. The medium was changed every day. After the compound was treated for the corresponding time, 15 μL of 5 mg/mL MTT solution was added to each well and incubated for 4 h in a 37-degree cell culture incubator in the dark.
The 96-well plate was taken out, and the culture medium was aspirated in each well under dark conditions. Then, 150 μL of DMSO was added to each well to dissolve formazan. At last, it was shaken at a medium speed for 10 min on a shaker. Finally, the absorbance of each well of a 96-well plate was detected at a wavelength of 490 nm.

Cell Scratch Repair Experiment
In the cell scratch repair experiment, the selected cell line was the human breast The specific operation is presented below. The prepared compound mother liquor was taken out and left to melt at the ambient temperature. The compound was dissolved in an appropriate amount of medium to a final concentration of 50 μM, and it was treated in the human breast carcinoma cell MCF-7 cell line. The respective compound was provided with 5 replicate wells. After 72 h, the microplate reader was 490 nm.
The absorbance was measured at the wavelength, and the growth inhibition rate of the 25 compounds on the MCF-7 cell line was obtained. Fig. 1 presents the results.        with the culture medium to make the final concentration of 100 μM. It is noteworthy that a serum-free medium was used to avoid the effects of cell proliferation. Next, the cells were cultured in a 6-well plate. A scratch test was performed after the cells were overgrown. Immediately after scratching, a picture was taken under the microscope, during which the cell scratch width was 0 h. After that, the prepared 100 μM culture medium was added for treatment and placed in a cell incubator for cultivation. After the compound treatment was taken. In this experiment, a control was set, the compound-free serum-free medium was added, and the other group was the compound M-added experimental group. Fig. 7~10 present the scratch test results of the control and the experimental group. The cell scratch experiment was repeated three times to count and analyze the cell migration distance.       1000 cells without any compound treatment were added to three wells of 6-well plate.
1000 cells with the treatment of 100 μM sulfonylurea compound M for 24 h were added to three wells, and another 6-well plate was taken. The cells were placed in a cell incubator and incubated. They were observed once every three days until the clones were formed.
After the clones were formed, the cells were fixed, stained and washed with water. In addition, the 6-well plate was placed in a ventilated place for 30 min before taking pictures and counting the clones. Fig. 13 presents the experimental results.