The synthesis of the compounds was described in a previous study and the purity of the compounds was assessed on the basis of standard melting point (21).

Physicochemical parameters have a vital role in the modulation of the bioactivity of chemical entities. Molinspiration (www.molinpiration.com), web-based software was used to obtain various parameters, including octanol/water partition coefficient (MiLogP), topological polar surface area (TPSA) and drug likeness. The MiLogP is calculated using the method developed by Molinspiration by summing the fragment-based contributions and correction factors. The method is robust and is able to process practically all organic and most organometallic molecules. TPSA is calculated based on the methodology published by Ertl et al (24) as a sum of the fragment-based contributions (25) in which O- and N-centered polar fragments are considered and the surface areas that are occupied by oxygen and nitrogen atoms and by hydrogen atoms attached to them are calculated. TPSA has been demonstrated to be a useful factor for characterizing drug absorption, including intestinal absorption, bioavailability, Caco-2 permeability and blood brain barrier penetration. Therefore, TPSA is associated with the hydrogen bonding potential of a compound. The compounds were evaluated on the basis of these parameters.

Docking calculations were performed using Docking Server (26). Gasteiger partial charges were added to the ligand atoms. Non-polar hydrogen atoms were merged and rotatable bonds were defined. Docking calculations were carried out on compounds (ligands) using the EGFR kinase domain model (Protein code: 1M17.pdb; rcsb.org/3d-view/1M17). Essential hydrogen atoms, Kollman united atom type charges and solvation parameters were added with the aid of AutoDock tools (27). Affinity (grid) maps of 60×60×60 Å grid points and 0.375 Å spacing were generated using the Autogrid program. AutoDock parameter set- and distance-dependent dielectric functions were used in the calculation of van der Waals and the electrostatic forces, respectively. Docking simulations were performed using the Lamarckian genetic algorithm and the Solis-Wets local search method (28). Initial position, orientation and torsions of the ligand molecules were set randomly. All rotatable torsions were released during docking. Each docking experiment was derived from 10 different runs that were set to terminate following a maximum of 250,000 energy evaluations. The population size was set to 150. During the search, a translational step of 0.2 Å and quaternion, and torsion steps of 5 were applied.

Three breast cancer cell lines, specifically, the highly metastatic MDA-MB-231, HER2-positive BT-474 and ER-positive MCF7 cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM), RPMI (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) and α-minimal essential medium (Sigma-Aldrich, Merck KGaA), for the MDA-MB-231, HER2-positive BT-474 and ER-positive MCF7 cells, respectively. Cells were cultured at 37°C with 5% CO₂ and 100% humidity. The medium was supplemented with 10% fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences, Logan, UT, USA), 100 U/ml penicillin and 100 µg/ml streptomycin.

MTT (Sigma-Aldrich; Merck KGaA) assay was used to evaluate the effect of compounds (1a-1d) on cell proliferation capacity. Cells were cultured in a 96-well plate at a density of 7×10³ cells/well and in a volume of 200 µl. Stock solutions of the compounds were prepared in dimethyl sulfoxide (DMSO). The cells were then treated with compound 1d (0, 10, 25 and 50 nM). The final concentration of the solvent in the medium was 0.5%. At appropriate time intervals, the medium was removed and replaced with 200 µl 0.5 mg/ml MTT in the growth medium and then the plates were transferred to a 37°C incubator for 3 h. Then the medium was removed and the purple formazan crystals were dissolved in DMSO (200 µl/well). Absorbance was determined on an ELISA plate reader (Biotek Instruments, Inc., Winooski, VT, USA) with a test wavelength of 570 nm and a reference wavelength of 630 nm to obtain the sample signal optical density (OD; 570–630 nm).

Kinase activity was determined using Kinase-Glo Plus luminescence kinase assay kit (Promega Corporation, Madison, WI, USA) by calculating the amount of adenosine triphosphate (ATP) remaining in the kinase reactionsolution. The luminescent signal is correlated with the residual amount present and it was inversely associated with kinase activity. The tested compounds were diluted to 100 mM in 10% DMSO, then 5 ml the dilution was added to a 50 ml reaction. All of the enzymatic reactions were performed at 30°C for 40 min using a 50 ml reaction volume containing 10 mM MgCl₂, 40 mM Tris, (pH 7.4), 0.1 mg/ml bovine serum albumin, 0.2 mg/ml poly (Glu, Tyr) substrate, 10 mM ATP and EGFR. The plate was incubated for 5 min at room temperature then 50 ml Kinase-GloPlus Luminescence kinase assay regent was added to each reaction. ADP-Glo assay kit (Promega Corporation) is a protein kinase assays used to determine IC50 values in which adenosine diphosphate (ADP) generation was measured which leads to an increase in the luminescence signal. The reaction mixture was incubated in a 96-well plate at 30°C for 30 min, following the incubation period 25 ml ADP-Glo reagent was added to terminate the assay. The 96-well plate agitated for 30 min at ambient temperature and incubated, then 50 ml kinase detection reagentwas added. The 96-well plate was read using the ADP-Glo Luminescence reader. All the assay components were added to the blank control except the substrate. The blank control value was used to correct the activity for each protein kinase target is determined.

Cells at a density of 1×10⁵ cells/well were cultured in 96-well plates in medium supplemented with 10% FBS for 24 h and was followed by treatment with compound 1d (0, 10, 25 or 50 nM). After 48 h, cells were collected by 300–350 × g for 5 min at room temperature, rinsed with PBS, fixed in 4% paraformaldehyde for 30 min at room temperature and then rinsed again with PBS to remove the fixing agent. The fixed cells were resuspended in PBS that already containing 5 µg/ml Hoechst 33258 and incubated at room temperature for 15 min in the dark room. The cells were examined to record the percentages of apoptotic cells by determining the nuclear condensation and chromatin fragmentation via fluorescence microscopy. A total of 250 nuclei from random microscopic fields were examined for the quantification of the apoptotic rate.

Total proteins were extracted using a RIPA buffer (Beyotime Institute of Biotechnology, Haimen, China). A bicinchoninic protein assay kit was used to quantify protein concentrations. Then, the protein samples were separated by 12% SDS-PAGE and transferred to polyvinylodene difluoride membranes. The membranes were blocked with 5% skim milk powder overnight at 4°C. Primary antibodies against β-catenin (~90 kDa) and β-actin were purchased from Santa Cruz Biotechnology, Inc., (anti-β-catenin; cat. no. sc-7963; 1:200; anti-β-actin, cat. no. sc-130301; 1:10,000) and incubated overnight at 4°C. The secondary antibody, goat anti-mouse immunoglobulin (Ig) G-horseradish peroxidase, was purchased from Santa Cruz Biotechnology, Inc., (cat. no. sc-2005; 1:5,000) and incubated at 37°C for 1 h. Finally, the bands were visualized using an enhanced chemiluminescence kit (Solarbio, Beijing, China) and analyzed by Image pro plus (version 9.3; Media Cybernetics, Inc., Rockville, MD, USA).

All experiments were done in triplicate or more. One-way analysis of variance was used to estimate overall significance followed by post-hoc Tukey's tests corrected for multiple comparisons. Data are presented as the mean ± standard deviation of the mean. P<0.05 was considered to indicate a statistically significant difference. Statistical analyses were performed with GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA, USA).

In order to find a probable candidate for an anti-breast cancer agent as shown in Fig. 1, four previously reported 1,3,5-triazine compounds were selected for the present study. The molecular properties of the compounds are presented in Table I, where the molecules were assessed on the parameters of the Lipinski's rule of five (29). The molecules demonstrated no violations of the rules and proved to be effective drug candidate to be act as anticancer agent.

As the drug likeness profile of the 1,3,5-triazine derivatives was good, docking analysis with EGFR-TK protein domain was performed. The results have been presented in Table II. It has been demonstrated that all compounds exhibited good inhibition constants against the target protein ranging from 0.44 nM to 3.10 µM and engaging the key active site as shown in Fig. 2. The molecules also demonstrated considerable interaction with key catalytic residues, including Asn818, Lys721, Leu694, Val702, Met 742 and other residues as exhibited in Table II. Particularly, in the case of compound 1a, as presented in Fig. 3, it had an inhibitory constant (Ki) of 3.10 µM with polar interaction with Lys721 and Asn818. It also had interaction with Leu694, Val702 and Leu678. Compound 1b exhibited marked improvement in the Ki value as a result of improved binding contacts with the target protein, including additional residues Arg817, Met442 and halogen binding with Glu738, Asp831, and Lys721, Fig. 4. Fig. 5 demonstrated that compound 1c exhibited 1.80 µM via interacting with Thr766, Leu694 and Met742. Compound 1d had the most potent activity, where the compound exhibited Ki value of 0.44 nM with improved contacts in the 3D protein structure (Fig. 6). The docking interactions were further demonstrated to be in agreement with the 2D H-plot, as shown in Fig. 2.

As a result of the excellent drug likeness profile and considerable docking affinity of the 1,3,5-triazine derivatives, these derivatives were synthesized and tested for inhibitory activity against EGFR-TK. The results are presented in Table III.

It has been demonstrated that synthesized derivatives exhibit excellent inhibitory activity compared with dacomitinib as a standard in the present study. For instance, compound 1d was demonstrated to exhibit the most potent activity among all the tested analogues. The lowest activity against EGFR was reported by compound 1a. The other compounds demonstrated mild to moderate activity. These results further confirmed the EGFR-TK inhibitory effect of 1,3,5-triazines as determined in docking experiments.

Subsequent to determining the inhibitory activity, compound 1d was further analyzed for anticancer activity in breast cancer cell lines, specifically, the highly metastatic MDA-MB231, HER2-positive BT-474 and ER-positive MCF7 cell lines. The compound was synthesized as described elsewhere and characterized using the melting point as compared with the reported value (21). As presented in Fig. 7, it has been demonstrated that the cellular viability of all the cells were decreased in a significant manner (P<0.05). Out of the tested cells, compound 1d causes inhibition of the BT-474 and least activity against MCF-7 at 50 nM. The inhibitory activity of compound 1d was revealed to be concentration dependent with maximum at 50 nM and minimum at 10 nM. Whereas 25 nM demonstrated considerable effects on the viability of cells.

The effect of compound 1d was further evaluated by measuring the apoptosis of the all three tested breast cancer cells. The cells were treated with the vehicle (0 nM) and different concentrations (10, 25 or 50 nM) of the compound 1d for 48 h. The apoptotic rates were determined via the staining of all three cells treated with compound 1d or the control cells with Hoechst 33258. As shown in Fig. 8, compound 1d increased in apoptosis in all three cells. At the highestconcentration of compound 1d (50 nM), the level of apoptosis in all the cells were demonstrated to be increased significantly (P<0.05; Fig. 8).

The results suggested that compound 1d causes significant increases in the apoptotic rates. The effect of compound 1d on the degradation of β-catenin was also evaluated. The total protein was isolated and the expression levels of β-catenin were determined by immunoblot analysis using β-actin as a loading control. As shown in Fig. 9, it has compound 1d decreased the protein expression of β-catenin in MDA-MB-231 compared with the control. These results indicated that compound 1d caused a significant decline in the protein expression of β-catenin expression in the MDA-MB-231 cells (P<0.05). Thus, compound 1d may exertan anticancer effect via inhibition of the β-catenin signaling pathway.