Synthesis, docking study, and structure activity relationship of novel anti-tumor 1, 2, 4 triazole derivatives incorporating 2-(2, 3- dimethyl aminobenzoic acid) moiety

docking study, and structure activity relationship of novel derivatives incorporating Abstract A series of 1,2,4 triazole derivatives (H7-12) have been synthesized by reacting an excess of hydrazine hydrate with carbothioamide derivatives (H1-6). The final compounds (HB1-HB6) were synthesized by reacting the triazole derivatives with mefenamic acid using DCC as a coupling agent. The chemical structures were confirmed by FT-IR, 1 H, and 13 C-NMR spectra, and some physicochemical properties were determined. The cytotoxicity of the different compounds (HB1-HB6) was evaluated by the MTT assay against two human epithelial cancer cell lines, A549 lung carcinoma and Hep G2 hepatocyte carcinoma, and one normal human cell line WI-38 lung fibroblasts. The mode of cell killing (apoptosis versus necrosis), as well as the effect on cell cycle phases were evaluated via flow cytometry. Additionally, EGFR tyrosine kinase inhibition assay was performed. The results presented in the current study indicate that the six tested compounds exhibited cytotoxicity against both cancer cell lines, and the lowest IC 50 was achieved with compound HB5 against Hep G2 cancer cells which was found to be highly selective against cancer cells. HB5-treated Hep G2 cells were arrested at the S and G2/M cell cycle phases. Compound HB5 caused cell killing via apoptosis rather than necrosis, and this was achieved by inhibiting EGFR tyrosine kinase activity needed for cell proliferation, and cell cycle progression. In silico pre -ADMET studies confirmed all final compounds don’t cause CNS side effects, with little liver dysfunction effect.

which include chemotherapy, surgery, and radiation therapy (Debela et al. 2021). Today, several studies have been focusing on developing new therapies with limited side effects as compared to conventional medications (Iacopetta et al. 2020).
Epidermal growth factor has a role in cell growth stimulation and differentiation through binding to its receptor, the epidermal growth factor receptor (EGFR). EGFR is a transmembrane protein belonging to the ErbB family of receptors. The ErbB is a subfamily of tyrosine kinase receptors including EGFR (ErbB-1), HER2/neu (ErbB-2), HER3 (ErbB-3), and HER4 (ErbB-4). EGFR is often upregulated in several cancers such as breast cancer, non-small-cell lung cancer (NSCLC), hepatocellular carcinoma (HCC), and glioblastoma (Sigismund, Avanzato and Lanzetti 2018a;Arienti et al. 2019;Maennling et al. 2019;Liu et al. 2021). The binding of EGF to the extracellular domain of EGFR causes homodimerization or heterodimerization of ErbB family receptors, which results in phosphorylation of intracellular Tyr1173 and Tyr1068 residues of EGFR. This in turn initiates several signaling pathways such as, phosphoinositide-3-kinase (PI3K)/serine/threonine-specific protein kinase (Akt), RAS/mitogen-activated protein kinase (MAPK), and Janus kinase/signal transducer and stimulation of transcription (Jak/Stat) pathways. The activation of these pathways mediates cellular proliferation and transformation, which in turn may cause invasion, growth, and cancer metastasis (Gurdal et al. 2019;Belli et al. 2020).
Heterocyclic compounds are interesting structures for medicinal chemists due to their important chemical and biological properties. Although many heterocyclic compounds have been developed, efforts are still ongoing to produce a new heterocyclic ring system with important biological activity (Al-Bayati et al. 2021a). One interesting heterocyclic compound is triazole, which consists of a five-member ring with 3N atoms and the chemical formula C 2 H 3 N 3 . 1,2,4 triazole nucleus has the greater benefit for researchers because of its extended applications in pharmaceutical, agrochemical, and material sciences (Kaur et al. 2016). The 1, 2, 4-triazole rings are stable against metabolic degradations and show target selectivity as well as several pharmacological activities. The nitrogen atoms in the triazole moiety play an important role in binding the receptor by forming bonds via H-bond donors or acceptors (Chopra et al. 2018;Kerru et al. 2020;Al-Bayati et al. 2021b). Significant improvement in both pharmacokinetics and pharmacodynamics of ligand-containing triazole rings is expected due to a rise in water solubility owing to the polar characteristics (Al-Bayati et al. 2021b). Many studies have shown that this important pharmacophore nucleus has important role in different types of anti-cancer compounds such as kinase inhibitors, tankyrase inhibitors, methionine aminopeptidase inhibitors, tubulin modulators, aromatase, nucleoside-based anticancer agents, steroid sulfatase inhibitors, and metal complex based anti-cancer agents .
Additionally, one of the important medications based on triazole nucleus which possess anti-proliferative activity against different types of cancer are the non-steroidal anti-inflammatory drugs (NSAIDs) (Kuntala et al. 2021). Their ability to inhibit cyclooxygenase-2 (COX-2) was proposed to be a mediator for their anti-tumor activity, but higher doses were required. Moreover, studies have indicated that NSAIDs induce apoptosis in many types of cancer cell lines such as colon, stomach, and prostate (Woo et al. 2004;Kazberuk et al. 2020).
In this regard, the objective of this study was to design and synthesize some novel 1, 2, 4-triazole derivatives incorporating 2-(2, 3-dimethyl aminobenzoic acid) moiety. Different electron withdrawal and donating groups at the p-position of benzene moieties were chosen to improve the physicochemical properties and cytotoxicity of the new synthetic compounds.

Material and methods
The chemical reagents and solvents were used without further purification from Sigma-Aldrich, Milano, Italy and Merck, Taufirchen, Germany. Mefenamic acid was purchased from Pioneer Company (Erbil, Iraq). Infrared spectra were measured using a Shimadzu model 8400s (Nakagyo, Japan) spectrophotometer on disk of KBr, (v = cm -1 ). Elemental microanalysis (CHNS) was done using a Euro EA300 elemental analyzer (Carlo Erba, Emmendingen, Germany). The proton ( 1 H) and carbon ( 13 C) NMR spectra were measured using the Inova model Ultra shield at 500, and 125 MHz respectively, δ= ppm was used to express the chemical shift. The solvent used was DMSO d6 .

Chemical synthesis
The intermediates and final compounds were synthesized according to Scheme 1.

Synthesis of methyl ester derivatives: compounds (3 and 4)
Few drops of conc. H 2 SO 4 were added to 10 mmol of benzoic acid (1) or p-nitro benzoic acid (2) in 30 mL MeOH, the mixture was then refluxed for 3h. The product was formed after neutralization of the reaction mixture with Na 2 CO 3 . Compound (3) was obtained by extracting the aqueous layer with chloroform, and dried using anhyd. MgSO 4 . While compound (4) was filtrated after precipitation and recrystallized with EtOH. The physical properties are mentioned in Table 1.

Synthesis of aryl hydrazide derivatives: compounds (5 and 6)
To 10 mmol of compound (3) or (4) in 15mL EtOH, 30 mmol of hydrazine hydrate was added. The mixture was refluxed for 4h, the solvent was then reduced using a rotary evaporator, and the residual was added to ice water. The formed precipitate was filtered, and recrystallized from EtOH (Shabeeb et al. 2019). The physical properties are mentioned in Table 1.

Synthesis of 1,2,4 triazole derivatives, compounds (H 7-12)
2 mmol of compounds (H1-6) was suspended in 10 mL of MeOH, and 10 mmol of hydrazine hydrate was added. A red mixture was formed and refluxed for 20h. The solvent was reduced with a rotary evaporator. 5 mL of Et 2 O were added to the residual and kept for 24h at a cold place. The precipitate was washed repeatedly with diethyl ether, and dried overnight at 25 ο C (Fattah 2014). The physical properties are mentioned in Table 1.

Synthesis of amide derivatives (compounds HB 1-6)
10 mmol of mefenamic acid was dissolved in (5mL dioxan and 10 mL THF) on ice bath, to this solution, 10 mmol of DMAP, and 10 mmol of the corresponding compounds (H7-12) were added, respectively. 10 mmol of N, N --dicyclohexylcarbodiimide (DCC) in 5 mL of dichloromethane CH 2 Cl 2 was added. The mixture was stirred at 0 ο C for 3 days. The product was filtered to remove DCU, and the solvent was reduced using a rotary evaporator. EtOAc (5mL) was added to the residual, and the mixture was washed with 10% HCl, 5%NaHCO 3 , and H 2 O, respectively. The organic layer was dried with anhyd. MgSO 4, and left to dry for 24h (Mahdi and Alsaad 2012). The physical properties are mentioned in Table 1.

Docking study
The chemical structure of the designed compounds was compared with crystal ligands to choose the molecular targets. A protein data bank (PDB) (https://www.rcsb. org/) was used to select the target site, additionally, the main requirements for binding with essential amino acids in the target site were determined.

Method of the docking study
Molecular Operating Environment 19.0901 software was used to predict the binding modes of the designed compounds inside target sites of EGFR tyrosine kinase. The ligand-binding sites were created from a co-crystallized structure within PDB (ID: 1M17) (https://www.rcsb.org).
Initially, water molecules were reduced from the complex. The clean protein options and protein report and utility were used to correct unfilled valence atoms and crystallographic disorders. The protein energy was reduced using MMFF94 force fields, while fixed atom constraint was applied to obtain the protein rigid binding site. 2D structures were generated using ChemBioDraw Ultra16.0 and saved in MDL-SD form. The 3D structures were protonated after opening in MOE, 0.05 RMSD kcal/mol was applied to minimize the energy, and the docking protocol was used for docking the minimized structures. CDOC-KER protocol was used to accomplish the process of molecular docking. The ligands were permitted to be flexible while the receptor was held rigid. Thirty conformation poses were used in the placement process, scored by London DG, and the best 10 docking scores (DG) of fitted poses with the active site at EGFR tyrosine kinase (scored by GBVI/WSA) were used and 3D perspective was done by Discovery Studio 2019 Client software.
These methods were also used to anticipate the binding profile, ideal orientation of each docking pose, affinity, and binding free energy (∆G) of the prepared compounds with EGFR tyrosine kinase.

In silico ADMET and drug-likeness prediction
In-silico study ADMET along with drug-likeness prediction assist the drug discovery and development process. Here, Pre-ADME online software was utilized for estimating absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiling of the tested compounds. These descriptors involve human gastrointestinal absorption, bioavailability, penetration to the blood-brain barrier, binding to plasma protein, inhibition of liver enzymes, and carcinogenesis. The drug-likeness of the derivatives were predicted following Lipinski's Rule. The measured parameters were number of H-bond donors and acceptors, clog p, and Topological Polar Surface Area (TPSA). The study was done using Chem. Informatics on the web (http://www.molinspiration.com).

Biological study Cell culture
Human A549 lung cancer cell line was grown in Ham's F-12K medium (Thermo Scientific, USA) supplemented with fetal bovine serum (10%) (FBS; Gibco, USA), human Hep G2 hepatocyte carcinoma cell line and human WI-38 cell line (lung fibroblasts) were grown in Eagle's minimum essential medium (LONZA, Switzerland), to which 10% FBS was added. Trypsin-EDTA (Millipore-Merck, USA) was used during subcultu res. Growth of cells was established at 37 °C in 5% CO 2 and 95% air.

In vitro MTT cell proliferation assay
The MTT assay was used to evaluate the proliferation of control and treated cells (Mosmann 1983). 96-well plates were used throughout the experiment. 30,000 cells were added to each well containing the appropriate media and grown for 24 h. The stock solutions of each of the tested compounds were prepared in DMSO. Eight concentrations (100, 30, 10, 3, 1, 0.3, 0.1, and 0.03 µM) were prepared in the growth media for each compound and added to the cells for 48 h. MTT salt (Freshly prepared; 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) (5 mg/ml; Sigma) was then added to each well to obtain 0.5 μg/μL as a final concentration. 200 μL of DMSO and isopropanol mixture 1:1 was added to each well and incubated for 30-45 min. Cell proliferation was detected by measuring the absorbance of each well at 590 nm using Multiskan EX (Thermo Scientific, USA) MicroPlate Reader. The experiment was performed three times in triplicates.

Cell cycle analysis
Hep G2 cells were treated with the IC 50 concentration of drug HB5 (2.87 µM) for 48 h (see Table 3). Control and treated cells were trypsinized and the cell suspension was fixed in EtOH solution (70%) at 4 °C for 120 min and then centrifuged at 800× g in a low-temperature centrifuge (Eppendorf, USA) for 5 min. The cell pellet was washed twice with phosphate buffer solution (PBS), centrifuged, and suspended in 0.5 mL PBS containing 20 µg/mL propidium iodide (PI) and 100 µg/mL Ribonuclease A. After incubation for 30 min at 37 °C in the dark, cells were centrifuged, and the cell pellet was suspended in PBS (Cell Cycle Assay Kit; Elabscience Biotechnology, USA). The fluorescence of the PI-DNA complex was detected using Epics XL-MCL Flow Cytometer (Beckman Coulter) and the Flowing software (version 2.5.1, Turku Centre for Biotechnology, Turku, Finland) was used to analyze the cell distribution at different stages of the cell cycle: sub-G1; G1; S; and G2/M (Nunez 2001;Pozarowski and Darzynkiewicz 2004).

Detection of apoptosis/necrosis
Hep G2 cells were treated with the IC 50 concentration of drug HB5 (2.87 µM) for 48 h. Apoptosis (early and late)/ necrosis induction was detected in control and treated cells by phosphatidylserine translocation to the cell surface using annexin V-FITC apoptosis detection kit (Elabscience Biotechnology, USA).

Quantitative reverse transcriptionpoly merase chain reaction (qRT-PCR)
The quantity of BAX, Bcl-2, p53, and caspase 3 mRNA in control and HB5 (at the IC 50 concentration)-treated Hep G2 cells were as sessed by qRT-PCR. Total RNA from vehicletreated control (0.01% DMSO) and HB5-tre ated Hep G2 cells were extracted according to the manufacturer's instructi ons (RNeasy mini kit, Qiagen, Germany). After RNA extraction, cDNA was prepared using the Revert Aid First Strand cDNA Synthesis kit (Thermo Scientific, USA). Amplification of target cDNA for apoptosis markers and GAPDH [as a normalization (housekeeping) gene] was done using one-step RT-PCR SYBR Green kit Master Mix (Bio-Rad Laboratories, USA) on Rotor-Gene Q real-time PCR thermal cycler instrument. cDNA (2 μL ali quots) was mixed with forward primer (1 μL), re verse primer (1 μL) ( Table 2), master mixture (10 µL) and the reaction volume was completed to (20 μL) with nuclease-free water. All experiments were performed in triplicate.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitory assay
The in-vitro inhibitory activity of compound HB5 and erlotinib [standard receptor-tyrosine kinase inhibitor (TKI)] against EGFR tyrosine kinase was done using EG FR Kinase Assay Kit (BPS Bioscience, USA). Briefly, EGFR and its substrate were incubated either with HB5 or erlotinib (1000, 300, 100, 30, 10, 3, 1, and 0.3 nM) in the enzymatic buffer at 30 °C for 40 min to initiate the enzymatic reaction. Detection reagent (Kinase-Glo MAX; Promega, USA) was added to terminate the reaction, followed by incubation for 15 min at 25 °C. The Table 2. The sequence of qRT-PCR primers, forward (F) and reverse (R), used in the current study.

Statistical analysis
Results are recorded as mean ± SEM. GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA) was used for statistical analysis. The student's t-test was used to determine significance between means, p-value < 0.05 was considered significant.

Docking study
The binding form of the reference (Erlotinib) displayed an energy binding of -6.55 kcal/ mol against EGFR tyrosine kinase. The quinazoline ring formed pi-Alkyl interactions with amino acids Val702, Leu694, Leu820, Ala719, and one H-bonding with Met769 with a distance of 2.66 °A. The 3-ethynyl phenyl moiety interacted with Lys721 and Leu764 by pi-alkyl interactions as shown in Fig. 1. The energy of binding (∆G) of the tested compounds (HB1-6), the number of H-bonds, and pi interactions are displayed in Table 3. The 2D docking of compounds (HB1-6) against EGFR tyrosine kinase is shown in Fig. 2, where H-bonds are represented in green lines and the pi interactions are represented in purple, orange, and pink lines. Additionally, each of the compounds (HB1-6) were superimposed with erlotinib and both were docked inside the target site of EGFR tyrosine kinase (Fig. 2). The superimposition shows that the fittest compounds were compound HB4 and HB5.
The 4H-1,2,4-triazole ring of compound HB1 generated pi interactions with the essential amino acids Leu820, Ala719, and Val702 of EGFR tyrosine kinase, while the phenyl moiety formed other pi interactions with Ala719, Lys721, and Val702, and the phenylamino moiety formed pi interaction with leu694. The binding features of compound HB2 to EGFR tyrosine kinase was through 4H-1,2,4-triazole ring which showed pi interactions with the essential amino acids Leu820, Ala719, and Val702 of EGFR tyrosine kinase, while the phenyl moiety formed another sulfur-pi interaction with Met742, and pi-alkyl interaction with Lys721. The 4-chloro phenylamino moiety formed pi interaction with Leu694, and the (2,3-dimethyl phenyl) amino moiety formed one H-bond with Asp831 (2.45 °A), and pi-pi interaction with Phe699, while the benzamide moiety interacted with Cys773 by sulfur-pi interaction.

In silico ADMET and Drug likeness prediction
The ADMET properties of drugs are usually predicted by intestinal absorption, activity against colon cancer cell line (Caco-2), P-glycoprotein binding, and skin permeability levels (Han et al. 2019). The data displayed in Table 4 showed that among the tested compounds, HB1 had the best ADMET properties. Additionally, the results indicated the inability of all compounds to penetrate the blood-brain barrier (BBB), this in turn indicate that central nervous system (CNS) side effects are not expected  upon administration. The metabolism profile was predicted based on the CYP models, in particular (CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4), the results showed no inhibitory effect against most of the tested liver enzymes. Moreover, the toxicity of the tested compounds was predicted based on the AMES test, and the data displayed in Table 4 showed carcinogenicity profile of most derivatives close to zero. The drug-likeness study indicated that all of the synthetic compounds (HB1-6) have most of Lipinski's Rule criteria for oral administration. In addition, it is known that for good bioavailability after oral administration the value of TPSA must not exceed 140 A° (Abbas et al. 2022), thus all tested compounds have good bioavailability, as shown in Table 5.

The newly synthesized 1,2,4 triazole derivatives (HB1-6) are cytotoxic to cancer cells
The cytotoxic effects of the 6 compounds (HB1-HB6) were evaluated against Hep G2 and A549 human cancer cell lines using MTT assay (Suppl. material 1: Fig. S1). The IC 50 values are shown in Table 6. The results revealed that all tested compounds exhibited cytotoxicity against Hep G2 hepatocyte carcinoma, and A549 lung cancer cell lines, 4 compounds (HB2, HB4, HB5, and HB6) were more cytotoxic to Hep G2 than to A459 cancer cells (lower IC 50 ; Table 6), while compounds HB1 and HB3 were more cytotoxic against A549 lung cancer cells. The lowest IC 50 value achieved was with compound HB5 against Hep G2 hepatocyte carcinoma (2.87 µM; Table 6); thus, this compound was selected for further investigation at the IC 50 concentration. The results of the MTT assay confirmed the superimposition results shown in Fig. 2, where the most cytotoxic compounds (HB5 and HB4) were the best superimposed (fittest) with erlotinib.

Compound HB5-treated Hep G2 hepatocyte carcinoma cells are arrested at S and G2/M cell cycle phases
The effect of 48 h treatment of compound HB5 on Hep G2 cell cycle progression was evaluated by staining cells with PI followed by flow cytometry (Fig. 3). Data shown in Table 7 indicate that treating Hep G2 cells with the IC 50 concentration of compound HB5 resulted in cell cycle arrest in the S and G2/M phases. The percentage of cells arrested in these phases increased by 50% when compared to control Hep G2 cells (Table 7).

Cytotoxicity of compound HB5 is attributed to apoptosis not necrosis
To investigate the mode of cell death (apoptosis versus necrosis) caused by 48h of compound HB5 treatment, Hep G2 cells were treated, stained with PI and annexin V-FITC, and analyzed by flow cytometry (Fig. 4A). As shown in Fig. 4A, Table 8, compound HB5 induced early apoptosis in treated cells as compared to control untreated cells. Additionally, the level (fold change) of mRNA of apoptosis markers such as, p53, BAX (pro-apoptosis), Bcl-2 (anti-apoptosis), and caspase-3 was measured by qRT-PCR. , and late apoptotic cells (top right; annexin V positive, PI negative); B Total RNA was extracted from control and HB5-treated cells, reverse transcribed, and assayed for p53, BAX, Bcl-2, and caspase-3 gene expression by qRT-PCR. Data are presented as mean ± SEM of three independent experiments of the fold change in the ratio of relative mRNA levels of target gene/GAPDH (housekeeping gene). Fold change in control cells was set at 1 arbitrary unit. *p < 0.05, **p < 0.01 as compared to control cells.
The results shown in Fig. 4B indicate that the fold change of p53, BAX, and caspase-3 mRNA (normalized over GAPDH) increased, while the that of Bcl-2 mRNA decreased in HB5treated Hep G2 cell line as compared to vehicle-treated control cells (set as 1 arbitrary unit) confirming induction of apoptosis in HB5-treated Hep G2 cells.

Compound HB5 inhibited Hep G2 cell proliferation and induced apoptosis by inhibiting EGFR tyrosine kinase
EGFR is one of the most frequently mutated genes in solid epithelial cancers, either through overexpression of the EGFR protein or a kinase-activating mutation (Sigismund et al. 2018b). In addition, the results of the molecular docking studies of compounds HB1-6 indicated that these compounds might be able to target EGFR tyrosine kinase. After analyzing the results of the EGFR kinase inhibition assay, it was found that compound HB5 exhibited inhibitory activity against EGFR tyrosine kinase (EC 50 = 38.3 nM), which was comparable to the EC 50 concentration of the standard chemotherapeutic drug erlotinib (EC 50 = 9.6 nM) (Suppl. material 2: Fig. S2).
All tested compounds have the same pattern of pharmacophoric queries of EGFR tyrosine kinase inhibitors, however, compound HB5 has the highest fitting with these queries. Compound HB5 showed  93.55 ± 0.29 5.69 ± 0.30 0.62 ± 0.05 0.14 ± 0.03 HB5-treated Hep G2 28.21 ± 1.80**** 70.44 ± 1.80 **** 1.21 ± 0.20 0.14 ± 0.01 ****p< 0.0001 as compared to control cells. Figure 5. The synthesized compounds predicted feature as EGFR tyrosine kinase inhibitors. the lowest ∆G score (-8.32 Kcal/mol; Table 3) in the interaction with EGFR tyrosine kinase, which indicates the stability of interaction between HB5 and the "Hit" amino acids in the target site of EGFR tyrosine kinase. This in turn indicates high selectivity and activity against the EGFR tyrosine kinase target site, confirmed by docking studies and superimposition as well as the EGFR inhibitory assay, which explains the ability of compound HB5 to cause the strongest cytotoxicity, i.e., lowest IC 50 value (Table 6).

Structure-Activity Relationship (SAR) of compounds HB1-HB6
The SAR of the compounds (HB1-6) revealed several common findings (Fig. 5). For anti-cancer activity, the fragment binding to the hydrophilic linker is essential to be an aryl or heteroaryl (Gaber et al. 2021). Accordingly, the compounds which have 4-nitrophenyl moiety (compounds HB4, HB5, and HB6) showed good activity nearly equal with those compounds containing 4-phenyl moiety (compounds HB1, HB2, and HB3). Additionally, compounds that contain chloro (Cl) groups in hydrophobic tail, showed the best activity against targeted cells according to the MTT assay (compounds HB2 and HB5), moreover, the 4-nitrophenyl ring attached to central aromatic is more preferable than non-derivatization. Thus, for the tested compounds (HB1-HB6), chloro derivative in the hydrophobic tail (HB5) is better than methoxy(HB6), and phenyl derivative (HB4).

Conclusion
A series of 1,2,4 triazole derivatives (HB1-6) have been successfully synthesized, and their chemical structures were confirmed using FT-IR, 1 H, and 13 CNMR spectra and elemental microanalysis. After performing molecular docking studies and MTT assay for the tested compounds, the results showed a high correlation between the expected results from the molecular modeling and the wet-lab biological evaluations, which indicated high selectivity against the EGFR tyrosine kinase target site. Additionally, in silico pre-ADMET study showed that all tested compounds have no CNS side effects, and carcinogenicity score close to zero. Finally, compound HB5 showed the strongest cytotoxicity and highest selectivity against cancer cells through its stable interaction with EGFR tyrosine kinase leading to inhibition of its activating, and thus inducing cancer cell apoptosis.