Theoretical and molecular mechanistic investigations of novel (3-(furan-2-yl)pyrazol-4-yl) chalcones against lung carcinoma cell line (A549)

A new chalcone series has been developed that may be useful in the treatment of lung cancer. The new series was confirmed by the different spectral tools. MTT assay was used to detect the cytotoxic effect of the novel chalcones against lung cancer cell line (A549). Molecular docking studies were performed on the most two effective chalcones 7b and 7c. Different molecular techniques were utilized to study the activity and the effect of two chalcones 7b and 7c on apoptosis of A549 cell line. Supplementary Information The online version contains supplementary material available at 10.1007/s00210-022-02344-x.


Introduction
One of the most common cancers that occurred in the world was lung cancer. Smoking is approximately the main risk factor for lung cancer. There were two types of lung carcinoma, the detection of them was performed in literature (Tantraworasin et al. 2013). Undesired effects and risks of the disease can be overcome if the disease was early detected and diagnosed. If the disease was not cured early, it may invade and spread rapidly to the neighbouring tissue and other organs of the body. Chalcones, exist in a conjugated form, where the two rings (A and B) are linked by the keto-ethylenic system (Lemes et al. 2020). The biological action of these compounds is thought to be due to the conjugation of the double bond with the carbonyl group. Chalcones are a subject of ongoing research since they exhibit a wide range of biological activities including antibacterial (Asiri and Khan 2011;Mohamed et al. 2012), antimalarial (Li et al. 1995), antioxidant (Bandgar et al. 2009;Shenvi et al. 2013), antiviral (Onyilagha et al. 1997), anti-inflammatory (Hsieh et al. 2000;Bekhit and Abdel-Aziem 2004), analgesic (Heidari et al. 2009) antiplatelet (Lin et al. 2001), and anti-cancer agents (Sashidhara et al. 2010;Shenvi et al. 2013). Pyrazoles are nitrogen-containing heterocycles that play an important role in medicinal chemistry due to their wide range of biological applications, including anticancer (Sangani et al. 2014;Metwally et al. 2015;Alam et al. 2016), anti-inflammatory (Farghaly et al. 2000;Kendre et al. 2015), anticonvulsant (Kaushik et al. 2010), antioxidant (Viveka et al. 2015;Bellam et al. 2017;Sallam et al. 2020), and antimicrobial activities (Viveka et al. 2015;Kendre et al. 2015). In addition, 3-(furan-2-yl)-1H-pyrazoles (Andicsová et al. 2018;Ryan et al. 2020; Barus et al. 2021) have been reported to have biologically interesting applications. Based on these findings, and in continuation of our study interest in bioactive heterocycle production , 2021a, b, c, Sroor et al. 2019Fathi et al. 2021;Helmy et al. 2021;Ragheb et al. 2022;Salem et al. 2022;Waly Eldeen et al. 2022), we were inspired to synthesize the pyrazolyl-chalcones and evaluate their in vitro

Structural activity relationship
The structure can be seen as α,β-unsaturated enone group linked to two rings (A-ring and B-ring). A-ring represents the heteroaldehyde part and B-ring represents the acetyl part of chalcone, which is 3-(furan-2-yl)-5-methyl-1-(4nitrophenyl)-1H-pyrazole) group. In all compounds, B-ring and the enone group are fixed. The change is only in A-ring (Scheme 3).

Molecular docking
Our goal in this simulation study is to develop a promising irreversible inhibitor with large selectivity and binding affinity toward the active centers of the tested protein markers from the database. The compounds model designed in this study was primarily validated to check for the enhanced and promised model that can identify the active compounds in a virtual screening process against different protein marker sets. Current modelling studies on the most active two chalcones 7b and 7c were  focused on their inhibition and binding affinities toward the protein domains that help cancer cells to resist and propagate as (2w3l, 2c6o, 4kmn, 1m17, and 4wt2) active domains . In this work and related references (Stamos et al. 2002;Berman et al. 2003;Porter et al. 2009;Tantraworasin et al. 2013), it was observed that the same standard inhibitors co-crystallized with selected domains were utilized as reference ligands for comparison with our new prepared compounds. The tested two chalcones 7b and 7c indicated different binding modes into the target domains with variable energies of binding. For a visual representation of the degree of binding affinities of each chalcone to the active sites, authors were referred to energy readings of each compound respectively (Tables 3 and 4). On the other hand, the binding energies of standard inhibitor complexed

Conc. µg/ml Surviving Fraction
with each domain were demonstrated in Table 2. In general, the two chalcones showed enhanced binding affinities against all tested protein markers relative to standard inhibitors. In a specific way, chalcone 7c was the most promising compound and illustrated strong affinities toward 1m17 domain as showed in Fig. 6. The amino acids blocked by our target new chalcone 7c were MET A:769, LEU A:820, LEU A:694, LYS A:721, VAL A:702, PHE A:698, and ASP A:83. In addition, the chalcone 7c achieved the best affinity with Gibbs free energy (− 24.5 kcal/mol) that proposed a high binding affinity more  Regarding chalcone 7b, it proposed a high binding affinity toward 1m17 domain with binding affinity (− 23.99 kcal/ mol) relative to standard inhibitor (− 23.7 kcal/mol). Related to chalcone 7b, it achieved the best and comparable affinity toward 2c6o with Gibbs free energy (− 23.87 kcal/mol) relative to 1m17 domain. As shown in Fig. 6, the amino acids included in the binding process of chalcone 7b against 1m17 protein were ASP A:776, CYS A:773, LEU A:694, LEU A:768, ALA A:719, THR A:760, LYS A:721, and MET A742. As exposed in Fig. 3, chalcone 7b inhibited the active site of 2c6o through In addition, 7b illustrated a promising and similar affinities toward 4wt2 domain (− 22.42 kcal/mol) and 4kmn (-22.12 kcal/mol) in comparison to positive control inhibitor (− 14.3, and − 14.4 kcal/mol) respectively, As illustrated in Fig. 5, the amino acids of 4wt2 involved in the interactions with chalcone 7b were LYS A:70, MET A:6, ILE A:61, and TRP A:323, where the amino acid included in the blocking process of chalcone 7b against 4kmn domain were GLU A:306, ASP A:297, ARG A:308, CYS A:309, ASP A:314, GLU A:319, and TRP A:328. Additionally, chalcone 7b illustrated good binding affinity toward 2w3l protein with binding energy (− 19.4 kcal/mol) in comparison to co-crystallized standard inhibitor (− 18.3 kcal/mol). As exposed in Fig. 2, the amino acid of 2w3l domain that inhibited by chalcone 7b were ASN A:102, ARG A:105, LEU A:96, ALA A:108, and MET A:74. Concerning both chalcone 7c and 7b, the best binding affinity was achieved toward the 1m17 domain with binding energy (− 24.53, and − 23.99 kcal/mol) respectively, relative to co-crystallized standard inhibitor (− 23.7 kcal/ mol). With respect to 4wt2 domain, chalcone 7b ensured better binding affinity (− 22.42 kcal/mol) than chalcone 4c (− 21.86 kcal/mol). In addition, the two chalcones 7b and 7c proved the best binding affinity toward 4wt2 domain in comparison to standard inhibitor ligand (− 14.3 kcal/mol). It was clearly noted that chalcone 7b still offered the higher binding affinities more than chalcone 7c regarding the three domains 4kmn, 2w3l, and 2c6o with interaction force (− 22.12, − 19.43, − 23.8 kcal/mol) respectively, for chalcone  Tables 3 and 4. Finally, the authors suggested and concluded that theoretical studies proved the new additions and modifications performed in chalcones 7b and 7c enhanced their binding affinities toward active sites of protein sets and thus improving the inhibitory effect and death of cancer cells.

The effect of chalcones 7b and 7c on genes expression
The expression level of the apoptotic genes (Bax and P53) and the anti-apoptotic genes (Bcl2 and CDK4) was studied in A549treated cells. A549 cells were treated with the IC 50 concentration of compounds 7b and 7c (20.05 and 13.86 µg/mL respectively) for 48 h and then subjected to real-time PCR. The untreated A549 cells were used as a negative control. As seen from Table 5 and Fig. 7, for compound 7c-treated cells, the apoptotic genes P53 and Bax were upregulated with fold change equalled 2.32 and 4.78 respectively relative to the negative control. Regarding the anti-apoptotic genes Bcl2 and CDK4, the expression level was downregulated by about a half as compared to the negative control (fold change = 0.61 and 0.54 respectively). A comparable result was seen for compound 7b-treated A549 cells. It was found that the expression level of P53 and Bax was increased almost twofold to that of compound 7c treated cells (fold change = 4.69 and 7.93 respectively). The expression level of Bcl2 and CDK4 was significantly decreased with fold change equalled 0.44 and 0.39 respectively. It was notable that compound 7b enhanced the expression of the apoptotic genes and lowered the expression of the anti-apoptotic genes more than compound 7c. It was reported that 3,4-disubstituted pyrazole analogues, and 3-(imidazol-2-yl)-4-[2(pyridin-3-yl)-vinyl]pyrazoles have been acted as cyclin-dependent kinase (CDK) inhibitors and showed anticancer activity against various cancer cell lines (Hawash et al. 2017). So that the downregulation of CDK4 may be due to the effect of the pyrazole moiety of our compounds. It was found that the substituted chalcone-pyrazole hybrid (2-((3-(4-Methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl) methylene)-2H-indene-1,3-dione) showed promising activity against MCF-7, HepG2, and HCT116 cancer cell lines, and induced apoptosis in breast cancer cells through the upregulation of tumor suppressor genep53 (Gao et al. 2020). So this hybridization showed enhanced activity, not a deleterious one.

ELISA assay for chalcones 7b and 7c
Apoptosis is a programmed cell death that occurred inside the cells under physiological and pathological conditions. There are two pathways of the apoptosis process, mitochondrial (intrinsic) pathway and death receptor (extrinsic) pathway (Fulda and Debatin 2006).Caspase-8 is activated in the extrinsic pathway upon stimulation of  death receptor which results in the activation of effector caspase-3 (Fulda and Debatin 2006). While, in the intrinsic pathway, caspase-9 participates in the formation of the cytochrome c/Apaf-1/caspase-9 apoptosome complex results in the activation of caspase-3 (Saelens et al. 2004). Caspase-3 is responsible for the breakdown of proteins and DNA resulting in cell death (Degterev et al. 2003). The activity of caspase-3, caspase-8, and caspase-9 in A549 cells after 48 h of treatment with the IC 50 concentration of compounds 7b and 7c was determined using the sandwiched ELISA assay (Table 6). It was found that compounds 7b and 7c increased the expression level of the studied caspase-3, caspase-8, and caspase-9. For caspase-3, both compounds enhanced greatly the activity (495.4 and 377.2 pg/mL for compounds 7b and 7c respectively) relative to the control (53.69 pg/mL), while the concentration of caspase-8 was slightly increased in response to compounds 7b and 7c (1.575and 1.042 pg/mL respectively) relative to the negative control (0.375 pg/mL). Compounds 7b and 7c upregulated the expression level of caspase-9 moderately (15.76and 11.2 pg/mL respectively) as compared to the negative control (1.49 pg/mL). It was noticed that compound 7b had a higher effect on the activity of the studied caspases than compound 7c. So from these results, compounds 7b and 7c induced both extrinsic and intrinsic pathways of apoptosis.

Flow-cytometric analysis of cell cycle and apoptosis
As shown from the above molecular studies, compound 7b demonstrated higher activity than compound 7c, suggesting that the thiophene was better than the pyridine ring. So, the flow cytometric analysis was done on the most active compound 7b. The cell cycle is regulated by several proteins called cyclins and their associated serine/threonine cyclindependent kinases (CDKs) (Baker and Reddy 2012). Among these CDKs, CDK4 mediates the transition of cells from the G1 to S phase. As mentioned in the RT-PCR section, that compound 7b inhibited the expression of CDK4. This result suggested the accumulation of cells at the G1 phase which was already happened in our flow cytometric analysis of the cell cycle. As shown in Fig. 8 and Table 7, compound 7b caused cell cycle arrest at G0/G1 and S phases. Regarding the flowcytometric analysis of apoptosis, it was noticed that compound 7b induced apoptosis in A549-treated cells. Figure 8 and Table 8 indicated that the percentage of early apoptotic cells was 3.69% relative to the untreated control cells (0.34%). The percentage of late apoptotic cells was significantly increased (21.24%) as compared to the control cells (0.11%). Also, it was found that compound 7b induced necrosis process by a percentage of 17.23% relative to the control cells (1.26%). By comparing our results with the results of other different chalcone hybrids from literature, it was found that the chalcone-pyridine hybrid (E)-1-(2,6-

Fig. 7
Bar graph represents the relative expression levels of apoptotic genes (BAX and P53) and anti-apoptotic genes (Bcl2 and CDK4) as measured by qPCR. A549 cells treated with compound 7b or compound 7c or left untreated were lysed and the extracted total RNA was reverse transcribed into cDNA. The fold expression change was determined by the 2 −ΔΔCt method after normalization to the expression level of β-actin as a reference gene

Anticancer test
In vitro inhibition activity of the newly synthesized series was investigated on cell viability according to the literature review described by Mohamed et al. (Mohamed et al. 2021b). In a brief explanation of MTT assay employed herein. In vitro, toxicological MTT Kit has been used to perform an (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) to measure the cytotoxicity effect of a novel (3-(Furan-2-yl)pyrazol-4-yl) chalcones against A549 cell line. Lung carcinoma cell line A549 was seeded in microplate where the prepared chalcones solution were added to triplicates well. Incubation of plates was done at 37 °C for 48 h. The fresh medium was replaced by the old one. Lung cells treated with different range concentration of our series [100, 50, 25, 12.5, 6.25, 3.125, and 1.56 mg/mL] were incubated, also untreated cells were used as a reference negative control. One percent antibiotic mixture was used, and after incubation. The positive control utilized here was 5-FU (100 mg/mL).

Molecular docking
A trial was performed to establish 3D model structures of chalcones 7b and 7c with different anti-apoptotic protein markers by the same proposal as illustrated by Mohamed et al. (Mohamed et al. 2021b). Based on the crystal structure of selected protein sets under the code access (2w3l, 2c6o, 4kmn, 1m17, and 4wt2), the amino acid sequences were collected from the PDB protein data bank (http:// www. RCSB. org/). The 3D structure was established using homology steps implemented by MOE 2010 program. Stable binding energies were achieved by constructing the models and subjecting them to energy minimization 0.05 rms gradient. The final chalcones structures were docked with the active sites of the above-mentioned protein sets. The two synthesized chalcones 7b and 7c were sketched using ChemDraw builder and were converted to their perspective 3D form. Subsequently, the energy of compounds was minimized up to 0.05 using the MMFF94x force field. All compounds were docked into the active pocket of all protein groups and conformations of each compound were generated with a docking score. Each compound was analyzed and visualized as a 3D representation according to the BIOVIA Discovery Studio program.

ELISA assay
Elisa assay was performed to detect the concentration of human caspase-3, caspase-8, and caspase-9 in A549 cell culture lysates. The instructions were followed up according to the manuscript instructions described in the following kits: DRG® human Caspase-8 ELISA Kit, Catalogue # (EIA-4863), DRG® human Caspase-9 ELISA Kit, Catalogue # (EIA-4860), and Invitrogen human Caspase-3 Elisa Kit, Catalogue # KHO1091, respectively. All reagents, samples, and standards were prepared as described in the above ELISA kits. Briefly, the procedure was described as followed; the microwell strips were washed twice with wash buffer. A total of 100 μl of standard or samples were added to each well and incubated for 2 h at room temperature (18 to 25 °C). The microwell strips were emptied and washed three times with wash buffer. A total of 100 μl of prepared antibodyanti-rabbit-IgG-HRP) were added to each well for 1 h at room temperature. The step of washing was repeated three times and then, 100 μl of prepared TMB (tetramethyl-benzidine) substrate solution was pipetted to all wells and incubated for 10 min at room temperature. For complete inactivation of the enzyme, 100 μl of stop solution was added to each well. The absorbance of each microwell was read immediately on a spectrophotometer at a wavelength of 450 nm. The concentrations of the unknown samples and controls were determined from the standard curve which was plotted using curve fitting software.

Flow-cytometric analysis of cell cycle and apoptosis
10 6 of A549 cells were cultured in 60-mm Petri dishes for 24 h and then treated with compound 7batIC 50 concentrationfor 48 h. The untreated A549 cells were used as a negative control. After 48 h of incubation, A549 cells were centrifuged at1000 rpm for 5 min at 4 °C. The cells pellet were washed in phosphate-buffered saline (PBS) and then centrifuged at 1000 rpm for another 5 min. After discarding the supeR-NAtant, the cells were collected in a single cell suspension and fixed in 70% ethanol on ice overnight. After the fixation step, cells were washed with 1 ml 1 × PBS. Finally, the cells pellet was incubated with a 200 µl 1 × propidium iodide (PI) mixture at room temperature in the dark for 30 min. Then, cells were subjected to an Epics XL-MCL flow cytometer (Beckman Coulter, Miami, FL) for DNA content analysis. Cells distribution at different phases was analysed by Multicycle software (Phoenix Flow Systems, San Diego, CA). Annexin V-FITC kit catalogue number (#K101-25) was used to detect the percentage of the apoptotic cells. About 10 6 of A549 cells were collected by centrifugation. Then, cells were washed in 500 µl of 1 × PBS. The cells were collected by centrifugation and then resuspended in the annexin V incubation reagent. The annexin V incubation reagent comprising of 10 μL binding buffer (10X) + 10 μL propidium iodide + 1 μL annexin V-FITC + 79 μL deionized water. The cells in 100 μL annexin V incubation reagent was incubated in dark at room temperature for 15 min ). Finally, the percentage of apoptotic cells was analyzed by flow cytometry using FITC signal detector (usually FL-1) and PI staining by the phycoerythrin emission signal detector (usually FL-2).

Conclusion
From the outcome of our investigations, it was possible to conclude that both chalcones 7b and 7c offered better and enhanced cytotoxic effects toward the lung cancer cell line (A549). Molecular modelling data revealed promising binding affinities of our new chalcones 7b and 7c toward antiapoptotic protein markers of the following domains (2w3l, 2c6o, 4kmn, 1m17, and 4wt2). On the other molecular side, compounds 7b and 7c enhanced the relative expression of P53 and Bax (apoptotic genes). On the other hand, they downregulated the anti-apoptotic genes Bcl2 and CDK4. The activity of caspase-3, caspase-8, and caspase-9 was significantly increased by the studied compounds. So, the results suggested that compounds 7b and 7c induced the extrinsic and intrinsic pathways of apoptosis in A549 cells. The flowcytometric analysis demonstrated that compound 7b arrested the cell cycle at G0/G1 and S phases and induced apoptosis and necrosis by a percentage of 24.93% and 17.23% respectively. The SAR revealed that compound 7bwith thiophene moiety was more active than compound 7c with pyridine ring. At the end of our studies, the authors proposed that both theoretical and experimental studies completed each other and confirmed the enhanced and effective role of novel two chalcones 7b and 7c on apoptosis of lung cancer cell line (A549). The two chalcones exerted both intrinsic and extrinsic pathways of apoptosis to die cancer cells and prevent its growth.

Author contribution
The authors declare that all data were generated in-house and that no paper mill was used. MFM and NSI shared in conceptualization, methodology, writing the experimental part related to biochemistry and writing-original Draft. AAS and OAA shared in revision. IAA and HMH shared in conceptualization, writing-review & editing. All authors read and approved the manuscript, and all data were generated in-house and that no paper mill was used.
Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).
Data availability Data available on request.

Declarations
Ethics approval Not applicable.

Consent to participate Not applicable.
Consent for publication Not applicable.

Competing interests
The authors declare no competing interests.
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