Synthesis and Evaluation of New Pyrazoline Derivatives as Potential Anticancer Agents in HepG-2 Cell Line

Cancer is a major public health concern worldwide. Adverse effects of cancer treatments still compromise patients’ quality of life. To identify new potential anticancer agents, a series of novel pyrazoline derivatives were synthesized and evaluated for cytotoxic effects on HepG-2 (human liver hepatocellular carcinoma cell line) and primary hepatocytes. Compound structures were confirmed by 1H-NMR, mass spectrometry, and infrared imaging. An in vitro assay demonstrated that several compounds exerted cytotoxicity in the micromolar range. Benzo[b]thiophen-2-yl-[5-(4-hydroxy-3,5-dimethoxy-phenyl)-3-(2-hydroxy-phenyl)-4,5-dihydo-pyrazol-1-yl]-methanone (b17) was the most effective anticancer agent against HepG-2 cells owing to its notable inhibitory effect on HepG-2 with an IC50 value of 3.57 µM when compared with cisplatin (IC50 = 8.45 µM) and low cytotoxicity against primary hepatocytes. Cell cycle analysis and apoptosis/necrosis evaluation using this compound revealed that b17 notably arrested HepG-2 cells in the G2/M phase and induced HepG-2 cells apoptosis. Our findings indicate that compound b17 may be a promising anticancer drug candidate.


Introduction
Worldwide, liver cancer is the third most common cause of cancer deaths. It is the fifth and seventh most common cancer in men and women, respectively [1]. Cancer is a complex disease caused by various factors, such as high stress, bad dietary habits, aging, and smoking; uncontrolled, rapid, and pathological proliferation of abnormally transformed cells is a direct cause of a large group of diseases [2][3][4]. Great progress has been made in medical treatments, but cancer is still a major cause of mortality. Resistance to chemotherapeutic agents, lack of selectivity, and serious adverse effects are the primary challenges in the fight against cancer [2][3][4][5][6]. Therefore, new anticancer agents are continually developed and tested to selectively destroy tumor cells or at least limit their proliferation.

Synthesis of New Pyrazoline Derivatives
The synthesis of new pyrazoline derivatives (b1-19) was carried out according to the steps shown in Scheme 1. First, chalcone derivatives (a1-5) were obtained via the base-catalyzed Claisen-Schmidt condensation with corresponding ketones and aldehydes. Chalcone ring-closure reactions were carried out with hydrazine or phenylhydrazine with tetrabutylammonium bromide (TBAB) as a catalyst to obtain compounds b1-4, b18, and b19. Next, compound b1 was treated with corresponding acyl chlorides or sulfonyl chlorides at 80 °C with ethanol as a solvent and pyridine as a catalyst to yield compounds b5-17. Compounds b1- 19 were elucidated by infrared (IR), 1 H-NMR, and mass spectrometry imaging. Thus, the synthetic procedure was shown to be versatile and applicable to the preparation of many derivatives. Compounds a1-5 are shown in Table 1; compounds b1-19 are shown in Table 2.

Synthesis of New Pyrazoline Derivatives
The synthesis of new pyrazoline derivatives (b1-19) was carried out according to the steps shown in Scheme 1. First, chalcone derivatives (a1-5) were obtained via the base-catalyzed Claisen-Schmidt condensation with corresponding ketones and aldehydes. Chalcone ring-closure reactions were carried out with hydrazine or phenylhydrazine with tetrabutylammonium bromide (TBAB) as a catalyst to obtain compounds b1-4, b18, and b19. Next, compound b1 was treated with corresponding acyl chlorides or sulfonyl chlorides at 80 • C with ethanol as a solvent and pyridine as a catalyst to yield compounds b5-17. Compounds b1- 19 were elucidated by infrared (IR), 1 H-NMR, and mass spectrometry imaging. Thus, the synthetic procedure was shown to be versatile and applicable to the preparation of many derivatives. Compounds a1-5 are shown in Table 1; compounds b1-19 are shown in Table 2.

MTT Assay
To identify the most promising antitumor agent among the synthesized pyrazoline derivatives (b1- 19), their cytotoxic effects were tested on HepG-2 cells; cisplatin was used as a positive control. We found that compounds b5, b9, and b14-18 displayed IC50 values lower than 50 µM against HepG-2 cells at 48 h. The most effective cytotoxic agent was compound b17, with an IC50 value of 3.57 µM at 48 h; cisplatin, the positive control, had an IC50 value of 8.45 µM at 48 h ( Table 3). The pyrazoline derivatives also displayed dose-dependent and time-dependent trends. We selected the three most effective compounds, b15-17, along with cisplatin to generate growth-inhibitory curves ( Figure 2).

MTT Assay
To identify the most promising antitumor agent among the synthesized pyrazoline derivatives (b1- 19), their cytotoxic effects were tested on HepG-2 cells; cisplatin was used as a positive control. We found that compounds b5, b9, and b14-18 displayed IC50 values lower than 50 µM against HepG-2 cells at 48 h. The most effective cytotoxic agent was compound b17, with an IC50 value of 3.57 µM at 48 h; cisplatin, the positive control, had an IC50 value of 8.45 µM at 48 h ( Table 3). The pyrazoline derivatives also displayed dose-dependent and time-dependent trends. We selected the three most effective compounds, b15-17, along with cisplatin to generate growth-inhibitory curves ( Figure 2).

MTT Assay
To identify the most promising antitumor agent among the synthesized pyrazoline derivatives (b1- 19), their cytotoxic effects were tested on HepG-2 cells; cisplatin was used as a positive control. We found that compounds b5, b9, and b14-18 displayed IC50 values lower than 50 µM against HepG-2 cells at 48 h. The most effective cytotoxic agent was compound b17, with an IC50 value of 3.57 µM at 48 h; cisplatin, the positive control, had an IC50 value of 8.45 µM at 48 h ( Table 3). The pyrazoline derivatives also displayed dose-dependent and time-dependent trends. We selected the three most effective compounds, b15-17, along with cisplatin to generate growth-inhibitory curves ( Figure 2).

MTT Assay
To identify the most promising antitumor agent among the synthesized pyrazoline derivatives (b1- 19), their cytotoxic effects were tested on HepG-2 cells; cisplatin was used as a positive control. We found that compounds b5, b9, and b14-18 displayed IC50 values lower than 50 µM against HepG-2 cells at 48 h. The most effective cytotoxic agent was compound b17, with an IC50 value of 3.57 µM at 48 h; cisplatin, the positive control, had an IC50 value of 8.45 µM at 48 h ( Table 3). The pyrazoline derivatives also displayed dose-dependent and time-dependent trends. We selected the three most effective compounds, b15-17, along with cisplatin to generate growth-inhibitory curves ( Figure 2).

MTT Assay
To identify the most promising antitumor agent among the synthesized pyrazoline derivatives (b1- 19), their cytotoxic effects were tested on HepG-2 cells; cisplatin was used as a positive control. We found that compounds b5, b9, and b14-18 displayed IC50 values lower than 50 µM against HepG-2 cells at 48 h. The most effective cytotoxic agent was compound b17, with an IC50 value of 3.57 µM at 48 h; cisplatin, the positive control, had an IC50 value of 8.45 µM at 48 h ( Table 3). The pyrazoline derivatives also displayed dose-dependent and time-dependent trends. We selected the three most effective compounds, b15-17, along with cisplatin to generate growth-inhibitory curves (Figure 2).

MTT Assay
To identify the most promising antitumor agent among the synthesized pyrazoline derivatives (b1- 19), their cytotoxic effects were tested on HepG-2 cells; cisplatin was used as a positive control. We found that compounds b5, b9, and b14-18 displayed IC50 values lower than 50 µM against HepG-2 cells at 48 h. The most effective cytotoxic agent was compound b17, with an IC50 value of 3.57 µM at 48 h; cisplatin, the positive control, had an IC50 value of 8.45 µM at 48 h ( Table 3). The pyrazoline derivatives also displayed dose-dependent and time-dependent trends. We selected the three most effective compounds, b15-17, along with cisplatin to generate growth-inhibitory curves (Figure 2).

MTT Assay
To identify the most promising antitumor agent among the synthesized pyrazoline derivatives (b1- 19), their cytotoxic effects were tested on HepG-2 cells; cisplatin was used as a positive control. We found that compounds b5, b9, and b14-18 displayed IC50 values lower than 50 µM against HepG-2 cells at 48 h. The most effective cytotoxic agent was compound b17, with an IC50 value of 3.57 µM at 48 h; cisplatin, the positive control, had an IC50 value of 8.45 µM at 48 h ( Table 3). The pyrazoline derivatives also displayed dose-dependent and time-dependent trends. We selected the three most effective compounds, b15-17, along with cisplatin to generate growth-inhibitory curves ( Figure 2).

MTT Assay
To identify the most promising antitumor agent among the synthesized pyrazoline derivatives (b1- 19), their cytotoxic effects were tested on HepG-2 cells; cisplatin was used as a positive control. We found that compounds b5, b9, and b14-18 displayed IC50 values lower than 50 µM against HepG-2 cells at 48 h. The most effective cytotoxic agent was compound b17, with an IC50 value of 3.57 µM at 48 h; cisplatin, the positive control, had an IC50 value of 8.45 µM at 48 h ( Table 3). The pyrazoline derivatives also displayed dose-dependent and time-dependent trends. We selected the three most effective compounds, b15-17, along with cisplatin to generate growth-inhibitory curves ( Figure 2).

MTT Assay
To identify the most promising antitumor agent among the synthesized pyrazoline derivatives (b1- 19), their cytotoxic effects were tested on HepG-2 cells; cisplatin was used as a positive control. We found that compounds b5, b9, and b14-18 displayed IC50 values lower than 50 µM against HepG-2 cells at 48 h. The most effective cytotoxic agent was compound b17, with an IC50 value of 3.57 µM at 48 h; cisplatin, the positive control, had an IC50 value of 8.45 µM at 48 h ( Table 3). The pyrazoline derivatives also displayed dose-dependent and time-dependent trends. We selected the three most effective compounds, b15-17, along with cisplatin to generate growth-inhibitory curves (Figure 2).

MTT Assay
To identify the most promising antitumor agent among the synthesized pyrazoline derivatives (b1- 19), their cytotoxic effects were tested on HepG-2 cells; cisplatin was used as a positive control. We found that compounds b5, b9, and b14-18 displayed IC 50 values lower than 50 µM against HepG-2 cells at 48 h. The most effective cytotoxic agent was compound b17, with an IC 50 value of 3.57 µM at 48 h; cisplatin, the positive control, had an IC 50 value of 8.45 µM at 48 h ( Table 3). The pyrazoline derivatives also displayed dose-dependent and time-dependent trends. We selected the three most effective compounds, b15-17, along with cisplatin to generate growth-inhibitory curves ( Figure 2).

MTS Assay
According to the results of MTT (thiazolyl blue tetrazolium bromide) assay on HepG-2 cell line, the cytotoxicity of compounds b5, b9, and b14-18 were evaluated against primary hepatocytes using the MTS assay. (Isolated primary hepatocytes were unstable. Using the MTS assay, the solubilization steps were eliminated because the MTS formazan product is soluble in tissue culture medium. It can avoid further damage to cells.) Compounds b5, b9, and b14-18 showed lower cytotoxicological activity against primary hepatocytes. The IC50 value of compound b17 against primary hepatocytes was 33.47 µM at 48 h. In terms of their anticancer potential, compound b17 can be considered as the most promising anticancer agent against HepG-2 due to its low cytotoxicity against primary hepatocytes (Table 3).

Cell Cycle Analysis
To determine whether compound b17 would affect the cell cycle in HepG-2 cells, we examined cell cycle progression using flow cytometry (Figure 3). HepG-2 cells were treated with compound b17 at concentrations of 0.9 µM, 2.7 µM, and 4.5 µM for 24 h. A notable decrease in cells in the G1 and S phases was observed. At 4.5 µM, up to 83.01% of the cells were arrested in the G2/M phase (the data shown in Figure 3 is about living cells after exclusion of dead cells). These findings indicate that compound b17 may be a potent anticancer agent.

MTS Assay
According to the results of MTT (thiazolyl blue tetrazolium bromide) assay on HepG-2 cell line, the cytotoxicity of compounds b5, b9, and b14-18 were evaluated against primary hepatocytes using the MTS assay. (Isolated primary hepatocytes were unstable. Using the MTS assay, the solubilization steps were eliminated because the MTS formazan product is soluble in tissue culture medium. It can avoid further damage to cells.) Compounds b5, b9, and b14-18 showed lower cytotoxicological activity against primary hepatocytes. The IC 50 value of compound b17 against primary hepatocytes was 33.47 µM at 48 h. In terms of their anticancer potential, compound b17 can be considered as the most promising anticancer agent against HepG-2 due to its low cytotoxicity against primary hepatocytes (Table 3).

Cell Cycle Analysis
To determine whether compound b17 would affect the cell cycle in HepG-2 cells, we examined cell cycle progression using flow cytometry (Figure 3). HepG-2 cells were treated with compound b17 at concentrations of 0.9 µM, 2.7 µM, and 4.5 µM for 24 h. A notable decrease in cells in the G 1 and S phases was observed. At 4.5 µM, up to 83.01% of the cells were arrested in the G 2 /M phase (the data shown in Figure 3 is about living cells after exclusion of dead cells). These findings indicate that compound b17 may be a potent anticancer agent.

Annexin-V Assay
A biparametric cytofluorimetric analysis was performed to determine the mode of cell death induced by compound b17 using propidium iodide (PI) and fluorescent immunolabeling of the protein annexin V. HepG-2 cells were treated with compound b17 at concentrations of 0.9 µM, 2.7 µM, and 4.5 µM for 12 h. The percentage of cell apoptosis was 0.3% at 0 µM for compound b17, and 7.6%, 8.7%, and 10.2% at 0.9 µM, 2.7 µM, and 4.5 µM, respectively ( Figure 4). Thus, we conclude that compound b17 can induce apoptosis.

Annexin-V Assay
A biparametric cytofluorimetric analysis was performed to determine the mode of cell death induced by compound b17 using propidium iodide (PI) and fluorescent immunolabeling of the protein annexin V. HepG-2 cells were treated with compound b17 at concentrations of 0.9 µM, 2.7 µM, and 4.5 µM for 12 h. The percentage of cell apoptosis was 0.3% at 0 µM for compound b17, and 7.6%, 8.7%, and 10.2% at 0.9 µM, 2.7 µM, and 4.5 µM, respectively ( Figure 4). Thus, we conclude that compound b17 can induce apoptosis.

Chemical Reagents and Equipment
All reagents were purchased from commercial suppliers. Melting points were determined using an Electrothermal 9100 melting point apparatus (Weiss-Gallenkamp, Loughborough, UK). IR spectra were recorded on a Bruker Tensor 27 Fourier IR spectrometer (Bruker, Karlsruhe, Germany). 1 H-NMR spectra were recorded on a Bruker Avance 300 spectrometer (Bruker) using tetramethylsilane (TMS) as the internal standard. Mass spectra were recorded on a SCIEX Triple Quad 6500+ LC/MS/MS system (SCIEX, Los Angeles, CA, USA). HRMS (high-resolution mass spectrometry) was performed on a Thermo Scientific Q Exactive (Thermo, Waltham, MA, USA).

Chemical Reagents and Equipment
All reagents were purchased from commercial suppliers. Melting points were determined using an Electrothermal 9100 melting point apparatus (Weiss-Gallenkamp, Loughborough, UK). IR spectra were recorded on a Bruker Tensor 27 Fourier IR spectrometer (Bruker, Karlsruhe, Germany). 1 H-NMR spectra were recorded on a Bruker Avance 300 spectrometer (Bruker) using tetramethylsilane (TMS) as the internal standard. Mass spectra were recorded on a SCIEX Triple Quad 6500+ LC/MS/MS system (SCIEX, Los Angeles, CA, USA). HRMS (high-resolution mass spectrometry) was performed on a Thermo Scientific Q Exactive (Thermo, Waltham, MA, USA).

General Procedures for the Synthesis of Compounds a1-5
Compounds a1-4 were obtained by a mixture of substituted phenylethanone (0.01 mol), substituted benzaldehyde (0.012 mol), and piperidine (1 mL) as a catalyst, and stirred at 160 • C for 20 min. Next, 30% aqueous sodium hydroxide solution (30 mL) was added and stirred for 15 min [27]. Compound a5 was prepared by appropriate substituted phenylethanone (0.01 mol), substituted benzaldehyde (0.012 mol), and 30% aqueous sodium hydroxide solution (10 mL) in ethanol (30 mL), and stirred at 30 • C for 24 h. The progress of the reaction was checked by thin-layer chromatography (TLC). Upon completion, the reaction mixture was poured onto crushed ice, followed by neutralization with HCl [28]. The precipitated solid was filtered, washed with water, and dried by vacuum pump. The product was finally crystallized from ethanol.

General Procedures for the Synthesis of Compounds b1-19
Hydrazine hydrate (0.04 mol, 1.96 g) was added to a solution of compound a1 (0.01 mol, 3.00 g) in ethanol (10 mL). The mixture was refluxed under stirring for 4 h, and the reaction mixture was subsequently poured onto crushed ice; the precipitate was filtered out, and product b1 was later crystallized from ethanol. Compounds b2-4 were synthesized following this procedure. Compounds b5-17 were prepared by treating compound b1 (0.01 mol, 3.14 g) with corresponding substituted benzoyl chloride or substituted benzenesulfonyl chloride (0.015 mol) at 80 • C in ethanol as solvent with pyridine as catalyst for 1 h to yield the final N-substituted targeted compounds. The N-phenyl-substituted pyrazolines b18 and b19 were prepared by direct cyclization of a1 and a5, respectively, with phenylhydrazine in the presence of TBAB as a catalyst [29]. The concentrations of compounds were prepared in seven different concentrations (2.5 µM, 5 µM, 10 µM, 20 µM, 40 µM, 80 µM, 160 µM). Cells were seeded into 96-well plates at a density of 26,000 cells/well. The plates were incubated for 24 h prior to any treatment. At the end of this period, the medium was removed and 100 µL of different concentrations of pyrazoline derivatives were added to wells for 48 h. Following the exposure period, 20 µL of the combined MTS/PMS solution was added to cells and incubated for a further 2 h at 37 • C, then the absorbance was recorded at 490 nm using an ELISA Plate Reader (ELISA, Emeryville, CA, USA). The values of IC 50 , the effective concentration at which 50% of the primary hepatocytes were inhibited, were calculated to evaluate the cytotoxic activities.

Cell Cycle Analysis
Based on the results of the cytotoxicity assay, compound b17 was selected for further mechanistic study with HepG-2 cells. For flow cytometry analysis of DNA content, approximately 1.5 × 10 5 HepG-2 cells/well in exponential growth mode were plated in 6-well plates and allowed to adhere, then treated with different concentrations of compound b17 for 24 h. After incubation, the cells were collected, centrifuged, and fixed with ice-cold ethanol (70%). The cells were treated with RNase A and stained with PI. Samples were analyzed on a BD Accuri C6 flow cytometer (BD, New York, NY, USA). DNA histograms were analyzed using ModFit for Windows.

Annexin-V Assay
Approximately 1.5 × 10 5 HepG-2 cells/well were plated in 6-well plates and allowed to adhere. After 24 h, the medium was replaced with fresh culture medium containing compound b17 at final concentrations of 0 µM, 0.9 µM, 2.7 µM, and 4.5 µM. Cells were harvested after 12 h. The cells were trypsinized, washed in phosphate-buffered saline (PBS), and centrifuged at 1000 rpm for 5 min. The cells were then resuspended in 200 µL of staining solution (containing 10 µL PI and 5 µL Annexin V-PE in binding buffer), mixed gently, and incubated for 15 min at 25 • C in the dark. The samples were later analyzed with a BD Accuri™ C6 flow cytometer.

Conclusions
In the present study, we synthesized 19 new pyrazoline derivatives and investigated their antiproliferative effects on HepG-2 cells. We found that compound b17 was the most effective anticancer agent following a 48 h exposure with an IC 50 value of 3.57 µM compared with the cisplatin value of 8.45 µM. Compound b17 was therefore selected for cell cycle analysis and apoptosis/necrosis evaluation. We observed that HepG-2 cells treated with compound b17 could be arrested in the G 2 /M phase. In addition, compound b17 can be regarded as a potent inducer of apoptosis in the cells. These results provide an important foundation for further development of compound b17 as a potent antitumor agent. We also found that compounds with a heterocyclic ring, such as b15 and b16, exhibited better pharmacological activity than most other pyrazoline derivatives synthesized in this study. Compounds b15 and b16 will be tested further to improve characterization of their antitumor activity.