Anticancer activity of novel 3‐(furan‐2‐yl)pyrazolyl and 3‐(thiophen‐2‐yl)pyrazolyl hybrid chalcones: Synthesis and in vitro studies

Twelve novel chalcone derivatives were prepared using the Claisen–Schmidt condensation reaction. The reaction of 4‐acetyl‐5‐furan/thiophene‐pyrazole derivatives 5 with the corresponding aldehydes 6 afforded the targeted chalcone derivatives 7a–l in good yields. The newly synthesized chalcones were fully characterized by spectrometric and elemental analyses. The in vitro anticancer activities of the novel compounds 7a–l were evaluated against four human cancer cell lines: HepG2 (human hepatocellular carcinoma), MCF7 (human Caucasian breast adenocarcinoma), A549 (lung carcinoma), and BJ1 (normal skin fibroblasts). Compound 7g emerged as the most promising compound, with IC50 = 27.7 µg/ml against A549 cells compared to the reference drug doxorubicin (IC50 = 28.3 µg/ml), and IC50 = 26.6 µg/ml against HepG2 cells compared to the reference drug doxorubicin (IC50 = 21.6 µg/ml). The gene expression and DNA damage values and the DNA fragmentation percentages for compound 7g were determined on the lung and liver cell lines. The expression levels of the AMY2A and FOXG1 genes increased significantly (p < 0.01) in the negative samples of lung cancer cells compared with treated cells. Also, the expression values of the PKM and PSPH genes improved significantly (p < 0.01) in the negative samples compared with treated samples of liver cancer cells. The DNA damage values increased significantly (p < 0.01) in treated lung cell line samples (7g) and the positive control. The results showed a significant decrease (p < 0.05) in DNA damage values in the negative samples of liver cancer cells compared to those treated with 7g. However, the DNA fragmentation values increased significantly (p < 0.01) in the treated lung and liver cell line samples compared with the negative control.

Based on these facts and in continuation of our research interest in the preparation of bioactive heterocycles, [35][36][37][38][39][40][41][42][43][44] we aimed to synthesize pyrazolyl-chalcones and test their biological activity in vitro as anticancer agents against four human cancer cell lines: HepG2 (human hepatocellular carcinoma), MCF7 (human Caucasian breast adenocarcinoma), A549 (lung carcinoma), and BJ1 (normal skin fibroblast). The gene expression, DNA damage values, and DNA fragmentation percentages for the most promising compounds have been studied on lung and liver cell lines.
Cancer and mortality rates continue to increase rapidly worldwide. Due to the rapid and adaptive nature of cancer development, the resistance and side effects associated with the use of existing drugs pose challenges in the search for additional drugs with the aim of offering more effective therapeutic treatments. Therefore, new therapeutic drugs are always needed to overcome the side effects associated with existing drugs.

| Gene expression analysis of lung cancer-and liver cancer-related genes
Gene expression in the lung cell line Gene expression analysis in lung cancer cell lines was performed using lung cancer-related genes, namely, amylase alpha 2A (AMY2A) and forkhead box G1 (FOXG1) genes. The results revealed that the expression levels of AMY2A and FOXG1 genes increased significantly (p < 0.01) in the negative samples of the lung cancer cell lines compared with the treated cell lines (Figure 1a,b). In contrast, the expression values of AMY2A and

DNA damage in the liver cell line
The DNA damage in liver cancer cell lines was determined using the comet assay as shown in   fragmentation values were observed in 7g, much more than those in the positive liver cancer cell lines (+ve).
Cells were cultured for 10 days, and then seeded at a con- conditions. [51] The absorbance was then measured using a microplate multi-well   [52] and liver (ASNS and ACLY genes, Saur et al.) [53] cancer-related genes were designed and are listed in Table 7. At the end of each qPCR, a melting curve analysis was performed at 95°C to check the quality of the used primers. The relative quantification of the target to the reference was performed using the 2 −ΔΔCT method (Yang et al.). [54] 4.2.3 | DNA damage assessment using the comet assay The DNA damage using the comet assay was determined using lung and liver cancer cell lines according to Olive et al. [55] After the trypsin treatment to produce a single-cell suspension, approxi-

| DNA fragmentation assay
The DNA fragmentation assay in lung and liver cancer cell lines was performed in accordance with the method of Yawata et al., [57] with some modifications. Briefly, after 24 h of exposure of lung and liver cancer cell lines to the tested substances in different Petri dishes (60 × 15 mm, Greiner), the cells were trypsinized, suspended, homogenized in 1 ml of medium, and centrifuged (10 min at 800 rpm). Low-molecular-weight genomic DNA was extracted as described in Yawata et al. [57] Approximately 1 × 10 6 cells were plated and treated with the tested substances in various treatments. All the cells (including floating cells) were harvested T A B L E 7 Primer sequences used for qRT-PCR of the lung and liver cancer cell lines by trypsinization and washed with Dulbecco's phosphate-buffered saline.
Lysates were vortexed and cleared by centrifugation at 10,000g for 20 min. Fragmented DNA in the supernatant was extracted with an equal volume of a neutral phenol/chloroform/isoamyl alcohol mixture (25:24:1) and analyzed electrophoretically on 2% agarose gels containing 0.1 μg/ml ethidium bromide.

| Statistical Analysis
The obtained data were analyzed using one-way analysis of variance using the software of the Statistical Analysis System (SAS, 1982). The 2 −ΔΔCT method was used for relative quantification in qPCR data analysis and to calculate the relative gene expression. Significant differences between treatments (groups) were determined using the least significant digit multiple-range test (hoc test) at a level of p < 0.05. Data are expressed as mean ± standard error of the mean (SEM); there were at least four replicates.