Synthesis, cytotoxic assessment, and molecular docking studies of 2,6-diaryl-substituted pyridine and 3,4- dihydropyrimidine-2(1H)-one scaffolds

Cancer is one of the main global health problems. In order to develop novel antitumor agents, we synthesized 3,4-dihydropyrimidine-2(1H)-one (DHPM) and 2,6-diaryl-substituted pyridine derivatives as potential antitumor structures and evaluated their cytotoxic effects against several cancer cell lines. An easy and convenient method is reported for the synthesis of these derivatives, employing cobalt ferrite (CoFe 2 O 4 @SiO 2 -SO 3 H) magnetic nanoparticles under microwave irradiation and solvent-free conditions. The structural characteristics of the prepared nanocatalyst were investigated by FTIR, XRD, SEM, and TGA techniques. In vitro cytotoxic effects of the synthesized products were assessed against the human breast adenocarcinoma cell line (MCF-7), gastric adenocarcinoma (AGS), and human embryonic kidney (HEK293) cells via MTT assay. The results indicated that compound 4r (DHPM derivative) was the most toxic molecule against the MCF-7 cell line (IC 50 of 0.17 μg/mL). Moreover, compounds 4j and 4r (DHPM derivatives) showed excellent cytotoxic activities against the AGS cell line, with an IC 50 of 4.90 and 4.97 μg/mL, respectively. Although they are pyridine derivatives, compounds 5g and 5m were more active against the MCF-7 cell line. Results showed that the candidate compounds exhibited low cytotoxicity against HEK293 cells. The kinesin Eg5 inhibitory potential of the candidate compounds was evaluated by molecular docking. The docking results showed that, among the pyridine derivatives, compound 5m had the most free energy of binding (–9.52 kcal/mol) and lowest Ki (0.105 μM), and among the pyrimidine derivatives, compound 4r had the most free energy of binding (–7.67 kcal/mol) and lowest Ki (2.39 μM). Ligand-enzyme affinity maps showed that compounds 4r and 5m had the potential to interact with the Eg5 binding site via H-bond interactions to GLU116 and GLY117 residues. The results of our study strongly suggest that DHPM and pyridine derivatives inhibit important tumorigenic features of breast and gastric cancer cells. Our results may be helpful in the further design of DHPMs and pyridine derivatives as potential anticancer agents.


Introduction
A common approach in cancer chemotherapy is the development of drugs that interrupt the mitosis phase of cell division. The mitotic spindle is an important target in cancer chemotherapy, and a fundamental spindle motor protein is kinesin Eg5, which is considered as an important therapeutic target due to its specific role during mitosis in assembly [1,2]. * Correspondence: aliramazani@gmail.com This work is licensed under a Creative Commons Attribution 4.0 International License.
In recent years, 3,4-dihydropyrimidine-2(1H)-ones (DHPMs) have been identified as compounds that specifically inhibit Eg5 function. Since mitotic kinesins are exclusively involved in the formation of the mitotic spindle, the inhibition of Eg5 by DHPMs is considered an attractive approach to cancer treatment [3]. It was reported that monastrol has an antitumor effect on various cancer cell types, such as breast, renal, and glioma cell lines. Unlike taxol, this compound, as an antimitotic agent, has not showed neuronal cytotoxicity [4,5].
Most cancer drugs usually show high toxicity and/or multidrug resistance of tumors. Hence, the development of novel bioactive compounds with anticancer effects is a very important issue [17,18]. Much research has been conducted on pyrimidine and pyridine derivatives; however, the simple synthesis and cytotoxic activity profiles of 2,6-diaryl-substituted pyridine and DHPMs on the human breast adenocarcinoma cell line (MCF-7), gastric adenocarcinoma (AGS), and human embryonic kidney (HEK293) cells has not been explored thus far.
This paper reports the simple and efficient synthesis, cytotoxic assessment, and molecular docking studies of 2,6-diaryl-substituted pyridine and DHPM derivative scaffolds.
In light of the importance of DHPM derivatives, several improved methods have been reported for the preparation of these compounds using various catalysts [19][20][21][22]. Although multiple methods have been reported for preparing functionalized pyridine derivatives [23][24][25][26], most require multiple steps, a large excess of expensive reagents, long reaction times, toxic solvents, and forceful reaction conditions.
In view of these reports, and in continuation of our previous work towards the development of efficient and environmentally benign heterogeneous catalysts [27][28][29][30], we synthesized some DHPM/pyridine derivatives in the presence SO 3 H-substituted silica-coated cobalt-based (CoFe 2 O 4 @SiO 2 -SO 3 H) magnetic nanoparticles (MNPs) as a highly efficient catalyst under solvent-free conditions with microwave irradiation (Schemes 1 and 2) to evaluate their cytotoxic effects on MCF-7, AGS, and HEK293 cells. The CoFe 2 O 4 @SiO 2 -SO 3 H MNPs were synthesized as highly efficient magnetic nanocatalysts and employed for the synthesis of DHPM and pyridine derivatives under solvent-free conditions with microwave irradiation (Schemes 1 and 2). Finally, the screened derivatives were subjected to molecular docking simulations to explore their binding potential toward the Eg5 enzyme as a validated cancer target for these compounds.  Figure 1. Two characteristic bands at 3300 and 592 cm −1 were related to the O-H stretching of the sulfonic acid functional group and M-O vibration, respectively [31]. The bands that appeared at 1218 and 1124 cm −1 belonged to the sulfonyl group that overlapped with a stronger peak of Si-O at 1091 cm −1 . The peaks at 1061, 1073, and 474 cm −1 confirmed the presence of Si-O-Si moiety in the nanocatalyst structure [32,33]. The presence of peaks at ranges of 592-629 cm −1 and 1091-1218 cm −1 can be considered as similarities of the 3 stages (a, b, and c) and 2 stages (b and c), respectively. The peaks that appeared at 1218 and 1124 cm −1 , which overlapped with the stronger peak of Si-O at 1091 cm −1 , were the differences of stages a and c.
The TGA curve of CoFe 2 O 4 @SiO 2 -SO 3 H MNPs demonstrated the weight losses of the surface hydroxyl groups and physically adsorbed solvent below 150°C ( Figure 2). Moreover, weight loss in the range of 600-800°C was attributed to the weight loss of the SO 3 H groups in the nanocatalyst structure [34].
The crystallinity and the average diameter of the CoFe 2 O 4 @SiO 2 -SO 3 H MNPs were obtained from the analysis of the XRD pattern. The XRD pattern of the CoFe 2 O 4 @SiO 2 -SO 3 H MNPs is presented in Figure  3, where it can be seen that the

Catalytic effect of CoFe 2 O 4 @SiO 2 -SO 3 H for the synthesis of DHPM and 2-amino-4,6diarylnicotinonitrile derivatives
After characterization of the catalyst, its catalytic effect on the synthesis of DHPMs was evaluated. To optimize the reaction conditions, the reaction of 4-chlorobenzaldehyde, ethyl acetoacetate, and urea was evaluated under different microwave powers in the presence of various amounts of CoFe 2 O 4 @SiO 2 -SO 3 H as a model reaction (Tables 1 and 2). In the absence of a nanocatalyst, only trace amounts of the products could be obtained (Table   1).   Reaction conditions: 4-Chlorobenzaldehyde 1 (1 mmol), urea 2 (2 mmol), ethyl acetoacetate 3 (1.5 mmol), and CoFe2O4@SiO2-SO3H (0.015 g), with microwave irradiation under solvent-free conditions.
After reaction optimization, the reactions of ethyl acetoacetate with different aldehydes (1a-1r) and urea were performed in the presence of CoFe 2 O 4 @SiO 2 -SO 3 H MNPs under microwave irradiation within 3-5 min (Table 3).
In order to investigate catalyst reusability, after completion of the reaction, the catalyst was separated by an external magnet and then washed with ethanol. The CoFe 2 O 4 @SiO 2 -SO 3 H MNPs could be reused at least 5 times (Table 5).

Cytotoxicity
Two human cancer model cell lines (MCF-7 and AGS) were treated with various concentrations (0.  µg/mL) of pyrimidine and pyridine derivatives. Interestingly, the primary screenings exhibited high cytotoxic effects against AGS and MCF-7 cells with fewer side effects (low cytotoxic effects) on normal cells (HEK293).
Cell viabilities were recorded after treatment for 24 and 48 h. As shown in Figure (Tables 6 and 7). As shown in Table 7, it was found that, with the  presence of an electronegative substituent at the ortho position, the cytotoxic activity had increased. In other words, in pyridine derivatives, the cytotoxic activity against the MCF-7 cell increased with the transfer of a substituent from para to ortho positions. Among the pyridine derivatives, the strongest and weakest cytotoxic effects belonged to 5g (2-F derivative) and 5j (4-Br derivative), respectively. It was shown that the cytotoxic effect decreased by a greater amount with the para substituent in the brominated compounds.

Molecular docking
Recent drug discovery methods have relied heavily on the crystallographic 3D structural data of biomolecular compounds. Numerous crystallographic records in the Protein Data Bank (PDB) have facilitated the effective-    structure with PDB code 2IEH was selected as the most appropriate crystallographic structure for further modeling studies. Docking validation results are illustrated in Table 8. Moreover, Figure 7 shows the docked MOY and the cocrystallized one in almost the same positions among the receptors (RMSD = 0.61 ?) that confirmed validation of the docking protocol.

Docking analysis
The estimated free binding energies, the populations in the optimum clusters, and the calculated inhibition constants (k i ) of the synthesized compounds extracted from the docking studies are summarized in Table 9,  where it can be seen that compounds 4r and 5m showed the highest free energies of binding, which indicated their probable binding ability to Eg5 residues. These products had the highest values in their groups, because there were 2 kinds of molecules. Structure binding explorations proved that H-bonds and hydrophobic contacts were responsible for such interactions. Compound 4r interacted with Glu116 to ensure the stability of the α -helix [46]. Compound 5m exhibited hydrogen bonds with Glu118 and Gly117 (Figures 8 and 9).
To get more data, a 2D schematic presentation of the ligand-receptor interactions for the 3 compounds, with high energy and good cytotoxic activity, is displayed in Figures 10-12. According to the schematic binding representations, H-bond interaction could also be detected between the Glu116 and NH (ring) of compound   4r, whereas H-bond donor interaction between Glu118 and NH (ring) was recorded for compound 5m. The docking analysis revealed that all of the compounds interacted with Eg5 in an appropriate manner, with the dominance of a H-bond donor group in the DHPM ring.
In past reports, the existence of some drawbacks, such as harsh conditions, toxic organic solvents, long reaction times, high costs, difficult reuse, low product yields, and eco-unfriendly catalysts have limited the use of these methods [19][20][21][22][23][24][25][26]. Therefore, this paper reports a novel, simple, and efficient strategy for the preparation of pyrimidine and pyridine derivatives with anticarcinogenic potential, without solvent, under microwave irradiation, and using magnetic cobalt ferrite nanoparticles as the catalyst. Based on the literature, thio-derivatives of monastrol (pyrimidine scaffold) have displayed relevant antiproliferative effects against melanoma (UACC.62), kidney (786-0), breast (MCF-7), ovarian (OVCAR03), and particularly colon (HT-29) cancer cell lines [5], while the results herein strongly suggest that DHPMs inhibit important tumorigenic features of AGS cancer cells, leading them to death. Among the pyridine derivatives, a series of 2-amino-3-cyano-6-(1H-indol-3-yl)-4-phenyl pyridine derivatives have shown cytotoxic activity against four human cell lines (A549, H460, HT-29, and SMMC-7721) that form preliminary structure-activity relationships, and the introduction of halogen groups into the benzene ring was essential for their cytotoxic activity [11]. To the best of our knowledge, the cytotoxic assessment of five derivatives, 5c, 5f, 5g, 5l, and 5m, has not been previously reported, and among them, compounds 5g and 5m exhibited good cytotoxic activity against the MCF-7 cell line. Affinity maps of the ligand-enzyme showed that derivatives with halogen groups in the benzene ring had the potentiality to interact with the Eg5 binding site via H-bond interactions. Based on the reports and results herein, hydrogen bond formation and hydrophobic interactions were the key factors affecting the inhibitory effect of these compounds [1].

Chemicals
All chemical materials, solvents, salts, and other chemical materials were commercially purchased from Sigma-Aldrich (St. Louis, MO, USA), Fluka (Dorset, England, UK), and Merck (Darmstadt, Germany). Thin-layer chromatography (TLC) was used to follow the reactions.

Synthesis of CoFe 2 O 4 @SiO 2 -SO 3 H
This catalyst was prepared in three steps according to method presented in a previous report [36].

Experimental procedure for the synthesis of DHPM and 2-amino-4,6-diarylnicotinonitrile derivatives
After characterization of the catalyst, its catalytic effect on the formation of the DHPMs and 2-amino-4,6diarylnicotinonitrile derivatives was evaluated.
In the first step, in order to optimize the reaction conditions, the reaction of 4-chlorobenzaldehyde, ethyl acetoacetate, and urea under different microwave powers in the absence and presence of various amounts of CoFe 2 O 4 @silica sulfuric acid were performed as a model reaction (Tables 1 and 2). The progress of the reaction was monitored via TLC after every minute (EtOAc and n -hexane, 6:10). After completion of the reaction, the solid product was filtered and the pure product was obtained by recrystallization from hot ethanol. The CoFe 2 O 4 @SiO 2 -SO 3 H nanocatalyst was subsequently washed with ethanol and then dried for use in the next catalytic cycles.
For optimization of the catalyst amount, different quantities of catalyst were evaluated. As can be seen in Table 1, in the absence of a nanocatalyst, only trace amounts of the products could be achieved. When the reaction was performed in the presence of 0.015 g of the nanocatalyst, an excellent yield of products was achieved ( Table 1, Table 3). The yields of all of the reactions were good to excellent. It was found that aldehydes with electron-donating groups represented lower reactivity when compared with aldehydes with electron-withdrawing groups.
Next, the 2-amino-4,6-diarylnicotinonitrile derivatives were synthesized using CoFe 2 O 4 @SiO 2 -SO 3 H as a nanocatalyst under microwave radiation according to the method presented in a previous project [36]. In a 5-mL microwave reaction vessel, a mixture of ammonium acetate 4 (1.5 mmol), malononitrile 3 (1 mmol), acetophenone 2 (1 mmol), an aromatic aldehyde (1a-1m) (1 mmol), and CoFe 2 O 4 @silica sulfuric acid (0.012 g) was reacted for 1-2 min. The reaction progress was elucidated via TLC after every minute ( n -hexane and EtOAc, 10:6). It was found that aldehydes with electron-withdrawing groups in the ortho and meta positions reacted slower than those in the para position. This catalyst was separated from the mixture using an external magnet and washed for the recycling tests. All of the products were known compounds that were characterized by melting point, IR, and 1 H NMR and 13 C NMR spectra.

Structural characterization
Proton NMR spectra were recorded on a Bruker DRX-250 Avance spectrometer (Billerica, MA, USA) at 250.13 MHz. Proton chemical shifts were reported in ppm ( δ) relative to the internal standard tetramethylsilane

Cell culture and cell proliferation assay via MTT
In this research, the effects of synthesized compounds on the viability of MCF-7, AGS, and HEK cells were evaluated at various concentrations (0.39-200 µg/mL) utilizing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [47]. After thawing, the cells were cultured in RPMI 1640 medium containing 10% FBS, penicillin (100 U/mL), and streptomycin (100 mg/mL) at 37°C in a humidified 5% CO 2 incubator. The cells were seeded in a 96-well plate at a density of 5000 cells per well (the cells were stained with trypan blue and counted with a hemocytometer). The cells were incubated overnight at 37°C before cell viability testing. A stock suspension of each of the synthesized compounds at 50 mg/mL in distilled water was prepared. After that, fresh suspensions of different concentrations of the synthesized compounds (2-fold serial dilutions from 0.39 to 200 µg/mL) were made using serial dilutions of the stock suspensions of the synthesized compounds in the RPMI 1640 medium immediately before use. Next, 200 µL of suspension (different concentrations of synthesized compounds) was added to each well of the micropentaplicates. The cells were incubated for 24 and 48 h under the same conditions. Wells without any synthesized compounds served as a negative control. The experiments were performed in pentaplicate for each concentration. To assess cell survival, 100 µL of MTT solution (2 mg/mL in PBS) was added to each well and incubated for 3 h at 37°C to produce insoluble formazan.
Next, 100 µL of DMSO was added to dissolve the formazan crystals, and the absorbance was measured using an Infinite M200 ELISA plate reader (Tecan, Männedorf, Switzerland) at 570 nm, with 630 nm as a reference wave length. The percentage of cell viability was calculated via the formula (A test /A control ) ×100, in which A test is the average absorbance of the treated cells and A control is the average absorbance of the negative control [47].
All crystallographic water molecules and cognate ligands (MOY) were eliminated from the original receptor structure for the provision of a target protein as a model (PDB code: 2IEH). All of the procedures for the target/ligand preparation were conducted according to previous reports [54]. LIGPLOT was used for the schematic 2D representations of the ligand-receptor interactions [55].

Analysis of statistical data
Cytotoxicity assessment of the cell lines were performed in pentaplicate, and the results were computed as mean ±SD. The experimental results were analyzed using one-way ANOVA analysis in SPSS 16.0 for Windows (SPSS Inc., Chicago, IL, USA) and P <0.05 was considered significant. The IC 50 values were calculated by nonlinear regression analysis using Finney probit analysis in SPSS.

Conclusions
In the present work, a very facile and efficient method was reported for the synthesis of pyrimidine and pyridine derivatives in the presence of CoFe 2 @SiO 2 -SO 3 H as a recoverable catalyst under microwave irradiation and solvent-free conditions. The results revealed that the synthesized DHPM derivatives had the potentiality to bind to the Eg5 binding site (4r and 5m) via H-bond interactions and hydrophobic contacts with GLU116, GLU118, and GLY117 residues. Biological assessments showed that the candidate molecules exhibited appropriate cytotoxic effects against MCF-7 and AGS cells with low cytotoxicity to normal embryonic kidney (HEK293) cells. Such advantages afforded the great potential of these compounds to be modified and developed towards more selective anticancer agents.