Design, Synthesis, Anticancer Activity and Molecular Docking of New 1,2,3-Triazole-Based Glycosides Bearing 1,3,4-Thiadiazolyl, Indolyl and Arylacetamide Scaffolds

New 1,3,4-thiadiazole thioglycosides linked to a substituted arylidine system were synthesized via heterocyclization via click 1,3-dipolar cycloaddition. The click strategy was used for the synthesis of new 1,3,4-thiadiazole and 1,2,3-triazole hybrid glycoside-based indolyl systems as novel hybrid molecules by reacting azide derivatives with the corresponding acetylated glycosyl terminal acetylenes. The cytotoxic activities of the compounds were studied against HCT-116 (human colorectal carcinoma) and MCF-7 (human breast adenocarcinoma) cell lines using the MTT assay. The results showed that the key thiadiazolethione compounds, the triazole glycosides linked to p-methoxyarylidine derivatives and the free hydroxyl glycoside had potent activity comparable to the reference drug, doxorubicin, against MCF-7 human cancer cells. Docking simulation studies were performed to check the binding patterns of the synthesized compounds. Enzyme inhibition assay studies were also conducted for the epidermal growth factor receptor (EGFR), and the results explained the activity of a number of derivatives.


Introduction
The heightened global burden of cancer in recent decades has driven extensive research to discover effective and selective anticancer candidates. Because of the resistance and severe toxicity associated with current drugs, there is a great demand to provide more efficient therapeutic strategies and molecular targets [1,2].
As a privileged scaffold, indole displays a promising and leading heterocyclic nucleus that provides it with a range of attractive biological activities, chiefly antitumor ones [20]. The indole derivatives VI and VII ( Figure 1) were found to exert their anticancer activities through the inhibition of cyclin-dependent kinases CDK-9 and CDK-4, respectively [21,22].
Recently, several pharmacophores containing glycoside fragments, e.g., I and V (Figure 1), have been developed and reported to have potential antitumor activity [23][24][25]. The epidermal growth factor receptor (EGFR) belongs to a fundamental subfamily of protein kinases that contains four members: EGFR (HER-1), HER-2, HER-3 and HER-4. EGFR is a crucial mediator performing a very vital role in the regulation of essential cellular processes involving proliferation, migration and survival [26]. The overexpression of EGFR is one of the most widely investigated for its role in the progression of many human solid tumors, such as breast cancer, non-small cell lung cancer and hepatocellular carcinoma [27]. In addition, the amplification of the HER-2 oncogene is detected in 15-30% of women suffering from breast cancer [28], which is regarded as the most serious The epidermal growth factor receptor (EGFR) belongs to a fundamental subfamily of protein kinases that contains four members: EGFR (HER-1), HER-2, HER-3 and HER-4. EGFR is a crucial mediator performing a very vital role in the regulation of essential cellular processes involving proliferation, migration and survival [26]. The overexpression of EGFR is one of the most widely investigated for its role in the progression of many human solid tumors, such as breast cancer, non-small cell lung cancer and hepatocellular carcinoma [27]. In addition, the amplification of the HER-2 oncogene is detected in 15-30% of women suffering from breast cancer [28], which is regarded as the most serious subtype [29,30]. Attributable to the essential role of overactive EGFR and HER-2 tyrosine kinases, the inhibition of these enzymes is a well-known effective target for anticancer therapies [31,32]. The most representative drugs in this class are afatinib, lapatinib, gefitinib and erlotinib, which have been used clinically for targeted anticancer therapies [33]. Lately, it has been observed that the triazole glycoside VIII and the indole analog IX (Figure 2) Molecules 2022, 27, 6960 3 of 23 demonstrate strong cytotoxic potency against different cancer cells through their EGFR inhibitory activity [19,34].
subtype [29,30]. Attributable to the essential role of overactive EGFR and HER-2 tyrosine kinases, the inhibition of these enzymes is a well-known effective target for anticancer therapies [31,32]. The most representative drugs in this class are afatinib, lapatinib, gefitinib and erlotinib, which have been used clinically for targeted anticancer therapies [33]. Lately, it has been observed that the triazole glycoside VIII and the indole analog IX ( Figure 2) demonstrate strong cytotoxic potency against different cancer cells through their EGFR inhibitory activity [19,34]. Molecular hybridization is an effective and simple tool to covalently merge two drug pharmacophores to obtain a single molecule [35]. Considering the facts discussed above regarding the biological significance of indole, thiadiazole, triazole and glycoside fragments ( Figure 2) and upon the continuation of our research in the discovery of potent anticancer targets [36][37][38][39][40][41], it was conceived that the joining of these pharmacophores into one nucleus through a fragment-based drug design approach would develop highly potent anticancer targets. Herein, a novel series of glycosides incorporated into indolethiadiazole-triazole bases or substituted arylacetamino-triazole hybrids were designed, synthesized and evaluated for their cytotoxic activities. A kinase inhibition assessment against EGFR WT , EGFR T790M and HER-2 was performed for the promising analogs to detect their mechanism of action. Furthermore, the apoptotic activity of the most potent derivatives was estimated by cell cycle analysis. Molecular docking at the binding sites of EGFR WT , EGFR T790M and HER-2 receptors was carried out in order to gain insight into new anticancer leads.

Chemistry
The synthetic strategy for the preparation of the targeted glycosides involved two different pathways to obtain two types of hybrid compounds; the first represents new Molecular hybridization is an effective and simple tool to covalently merge two drug pharmacophores to obtain a single molecule [35]. Considering the facts discussed above regarding the biological significance of indole, thiadiazole, triazole and glycoside fragments ( Figure 2) and upon the continuation of our research in the discovery of potent anticancer targets [36][37][38][39][40][41], it was conceived that the joining of these pharmacophores into one nucleus through a fragment-based drug design approach would develop highly potent anticancer targets. Herein, a novel series of glycosides incorporated into indole-thiadiazoletriazole bases or substituted arylacetamino-triazole hybrids were designed, synthesized and evaluated for their cytotoxic activities. A kinase inhibition assessment against EGFR WT , EGFR T790M and HER-2 was performed for the promising analogs to detect their mechanism of action. Furthermore, the apoptotic activity of the most potent derivatives was estimated by cell cycle analysis. Molecular docking at the binding sites of EGFR WT , EGFR T790M and HER-2 receptors was carried out in order to gain insight into new anticancer leads.

Chemistry
The synthetic strategy for the preparation of the targeted glycosides involved two different pathways to obtain two types of hybrid compounds; the first represents new glycosides of indole-thiadiazole-triazole hybrid bases, and the other is a group of substituted arylacetamino-triazole glycosides. The starting 1,3,4-thiadiazolylindole derivative 1 was prepared as previously reported [42]. The alkylation of the functionalized 1,3,4thiadiazolylindole compound 1 with 1,2-dibromoethane afforded the alkylated derivative 2, which contains an S-substituted bromoethyl side chain, followed by azidation with sodium azide, leading to the formation of the corresponding functionalized azide derivative 3. The infrared spectra of the azide product 3 displayed the distinctive bands of the azide group, and its corresponding 1 H NMR spectrum revealed the triplet signals of methylene groups in the side-chain substituent attached to the thiadiazole ring.
The synthesized azide 3 was reacted with the propargyl glycosides of the glucoand xylopyranosyl moieties 4a,b under click conditions by a Cu-catalyzed cycloaddition reaction (CuCAAC) and resulted in the formation of the 1,2,3-triazole glycosides 5 and 6, respectively. Sodium ascorbate and copper sulfate were used for the generation of the required Cu(I) catalyst by the in situ reduction of Cu(II) in the reaction medium. Upon testing a number of solvent systems, the THF-H 2 O mixture (2:1) was determined to be the best medium and resulted in the formation of the targeted 1,2,3-triazole products. The other tested media systems in the click reaction were a t BuOH/H 2 O mixture (2:1) and a mixture of H 2 O/ t BuOH/CH 2 Cl 2 (1:2:8), which, after 2 days and 55 h with stirring at 60 • C, respectively, had yields lower than 52%. In the structures of the latter glycosides, the glycosyl moiety was attached to C 4 of the triazole system as analogs of modified Cnucleosides. The IR spectra of compounds 5 and 6 demonstrated characteristic bands related to the carbonyl frequency in the acetate group at their known assigned values. Their 1 H NMR spectra exhibited signals attributed to the sugar fragments, which were assigned to the formed β-confirmation of the triazole-sugar linkage, as indicated by the coupling constant of the anomeric proton. A deprotection reaction of the acetylated glycosides 5 and 6 was achieved under the action of a saturated ammonia solution in methanol and afforded the triazole glycosides 7 and 8 with free hydroxyl groups, which was in accord with their spectral data (Scheme 1). The click synthetic approach was also applied for two other functionalized azide compounds, 9a,b, incorporating a substituted aryl acetamide system. Accordingly, in this way, the reaction was performed to realize the linkage of the terminal acetylenic sugar with 9a,b to obtain the 1,2,3-triazole glycosides 11-14 (Scheme 2). Scheme 1. Synthesis of indolyl-thiadiazolyl-1,2,3-triazolyl hybrid glycosides. The click synthetic approach was also applied for two other functionalized azide compounds, 9a,b, incorporating a substituted aryl acetamide system. Accordingly, in this way, the reaction was performed to realize the linkage of the terminal acetylenic sugar with 9a,b to obtain the 1,2,3-triazole glycosides 11-14 (Scheme 2).

Cytotoxic Screening
The synthesized compounds 1-3, 5-9 and 11-18 were all examined in vitro for thei cytotoxic activities against HCT-116 and MCF-7 human cancer cells using the MTT assay and doxorubicin was applied as a reference drug. All compounds suppressed both cance cells in a dose-dependent manner, and the results are depicted in Table 1 and 1 H NMR of the formed substituted glycosyl heterocycles showed all of the hydrogens in the formed compounds at their characteristic regions, revealing the β-configuration of the attachment of the sugar part. The latter triazolyl-glycosides 11-14 were hydrolyzed to afford the deacetylated derivatives 15-18, respectively, by using a methanolic ammonia solution (Scheme 2). The absorption bands characterized the hydroxyl groups of the afforded deacetylated glycosidic compounds at their characteristic regions, in addition to the disappearance of the acetyl-carbonyl absorption bands. Furthermore, the NMR spectra indicated the deacetylation process and revealed the existence of hydroxyl proton signals and the disappearance of the signals of the methyl protons of the acetyl groups. The purity of the compounds was revealed by the adequate results of the applied spectral and analytical analyses, such as 1 H and 13 C NMR, IR and elemental analysis, for the structural confirmation of the synthesized compounds, in addition to the TLC technique, and the proportions of the afforded products indicated a sufficient degree of purity.

Cytotoxic Screening
The synthesized compounds 1-3, 5-9 and 11-18 were all examined in vitro for their cytotoxic activities against HCT-116 and MCF-7 human cancer cells using the MTT assay, and doxorubicin was applied as a reference drug. All compounds suppressed both cancer cells in a dose-dependent manner, and the results are depicted in Table 1 and  15 The compound concentration that inhibits the proliferation of cells by 50%; SD: mean of the standard deviation; each value is the mean of three values.
Finally, combined with our previous study [16], it can be concluded that the promising cytotoxic activity is attributed to the existence of the 1,2,3-triazole-deacetylated N-glycoside hybrid. Furthermore, the incorporation of indole and 1,3,4-thiadiazole scaffolds provided an opportunity for more potency, and they could be selected as leads for the discovery of anticancer agents in the future.

In Vitro Inhibitory Assessment of EGFR WT , EGFR T790M and HER-2
Regarding the previous cytotoxic potency, the triazole-based glycosides 7, 8, 11, 12 and 15 were chosen for further elucidation of their EGFR WT , EGFR T790M and HER-2 inhibitory activities using erlotinib as a reference drug. As illustrated in Table 2, compounds 7, 8 and 15 displayed the highest potential inhibitory activities, with IC 50 ranges of 0.21-0.38, 1.50-1.95 and 2.18-2.77 µM against EGFR WT , EGFR T790M and HER-2, respectively, but they were slightly less than that of erlotinib (IC 50 = 0.13, 0.62 and 1.40 µM). However, the remaining derivatives 11 and 12 exhibited a drastic decrease in the suppression effect (IC 50 = 6.45 and 12.70 µM against EGFR WT ; 11.80 and 15.22 µM against EGFR T790M ; and 8.60 and 18. 40 µM against HER-2, respectively), which might be ascribed to a poor fit within the EGFR WT , EGFR T790M and HER-2 binding pockets.

Detection of Apoptosis and Cell Cycle Analysis
The annexin V/PI binding assay was carried out on derivatives 7 and 15, which showed the most potent cytotoxicity and kinase inhibitory activity, to examine their apoptotic effects on the MCF-7 cancer cells. These cells were stained with two dyes, annexin V/propidium iodide (PI), after treating them with derivatives 7 and 15 at their IC 50 concentrations of 0.5 and 0.6 µM for 24 h through the flow cytometry method [43]. It was observed that there was an increase in the percentages of late apoptosis caused by the Molecules 2022, 27, 6960 8 of 23 tested compounds 7 and 15 from 0.28% (control DMSO/MCF-7 cells) to 18.81% and 25.48%, respectively. Furthermore, the screened derivatives exhibited early apoptotic effects of 4.39% and 2.69% compared to 0.46% in untreated MCF-7 cells, with necrosis percentages of 11.41% and 14.28%, respectively, versus 1.25% produced by the DMSO control (Table 3) ( Figures S4 and S5, Supplementary Data). The proportion of late apoptosis obtained with compounds 7 and 15 was higher than that of the early phase, leading to the induction of apoptosis and confirming the effectiveness of these derivatives in cancer therapy. Moreover, the cessation of cell division at cell cycle checkpoints is considered one of the main roles of anticancer therapeutics [44]. The data presented in Figures 3 and S5 demonstrate that there was cell accumulation at sub-G1 and S phases, with percentages of 34.61% and 43.51% in MCF-7 cells treated with compound 7 and 42.45% and 52.08% in those treated with compound 15, compared to 1.99% and 33.85% in the untreated MCF-7 cells, respectively. The previous findings clearly show that derivatives 7 and 15 triggered a significant cell cycle arrest at sub-G1 and S phases compared with the untreated MCF-7 cells.

Molecular Docking Study
Docking simulations for targets 7, 8 and 15 were performed using Molecular Operating Environment (MOE-Dock) software version 2014.0901 [45,46] to understand their binding modes within the EGFR WT , EGFR T790M and HER-2 active sites and to interpret their promising in vitro inhibitory activities. The Protein Data Bank files (PDB codes: 1M17, 3UG2 and 3RCD) were chosen and downloaded for the corresponding EGFR WT , EGFR T790M and HER-2 enzymes, respectively [46][47][48]. The native ligands erlotinib, gefitinib and TAK-285 complexed with the co-crystallized structures of these enzymes were redocked to validate the docking processes and afforded energy scores of −12.80, −11.64 and −12.33 kcal/mol, with root-mean-square deviation (RMDS) values of 0.88, 0.75 and 1.23 Å, respectively.
As previously reported [47][48][49], erlotinib and gefitinib, bearing quinazoline scaffolds, formed H-bonds between the nitrogen at p-1 and the key amino acids Met769 and Met793 within the active sites of EGFR WT and EGFR T790M , respectively. In addition, the N-1 of pyrrolo [3,2-d]pyrimidine in the ligand TAK-285 displayed hydrogen bonding with Met801 within the binding site of HER-2.
As illustrated in the docking of our targets (Figures 4-6), the hydroxyl groups of the glycoside scaffolds formed H-bonds with the backbones of Met769, Met793 and Met801 within the EGFR WT , EGFR T790M and HER-2 binding pockets. Moreover, the glycoside moieties improved fixation within EGFR WT through the formation of more H-bonding with Gln767 (in 8 and 15) and Thr766 (in 15) and within EGFR T790M through H-bonding with Gln791.
demonstrate that there was cell accumulation at sub-G1 and S phases, with percentag of 34.61 % and 43.51 % in MCF-7 cells treated with compound 7 and 42.45 % and 52.08 in those treated with compound 15, compared to 1.99 % and 33.85 % in the untrea MCF-7 cells, respectively. The previous findings clearly show that derivatives 7 and triggered a significant cell cycle arrest at sub-G1 and S phases compared with the u treated MCF-7 cells.     By focusing on Figure 6, it can be noted that 15 has an improved fit within the active site of HER-2 through an additional arene-arene interaction between the p-tolyl moiety and the essential amino acid Phe864.

General
The melting points were recorded through a Reichert Thermovar apparatus an uncorrected. The infrared spectra were recorded using a Perkin-Elmer (model 1720) spectrometer. Nuclear magnetic resonance investigations were performed on a B AC-300 or DPX-300 spectrometer (400 MHz 1 H) (100 MHz 13 C). The values of (chemical shifts) are reported relative to TMS as a standard reference. The J values pling constants) are provided in Hz. The progress of the reaction was checked guided by TLC using aluminum silica gel 60 F245. IR, 1 H NMR, 13 C NMR and elem analyses were measured at the Micro Analytical Center at the Faculty of Science, University, Egypt.
Within EGFR T790M ( Figure 5), arene-cation interactions were illustrated between Lys745 and the indole moiety in 8 and p-tolyl in 15. However, the triazole nitrogen and oxymethyl oxygen in 7 served as H-bond acceptors with the side chain of Ser719 (distance: 3.04 and 3.24 Å, respectively).
By focusing on Figure 6, it can be noted that 15 has an improved fit within the active site of HER-2 through an additional arene-arene interaction between the p-tolyl moiety and the essential amino acid Phe864.
The superimposition patterns in Figure S6 (in Supplementary Data) demonstrate that the screened derivatives 7, 8 and 15 are well accommodated, along with the native ligands, erlotinib, gefitinib and TAK-285, within the binding pockets of EGFR WT , EGFR T790M and HER-2 through similar H-bonding with the key amino acids Met769, Met793 and Met801, respectively. In general, these results highlight targets 7, 8 and 15 as excellent leads for the discovery of promising anticancer agents through the suppression of EGFR WT , EGFR T790M and HER-2 kinases.

General
The melting points were recorded through a Reichert Thermovar apparatus and are uncorrected. The infrared spectra were recorded using a Perkin-Elmer (model 1720) FTIR spectrometer. Nuclear magnetic resonance investigations were performed on a Bruker AC-300 or DPX-300 spectrometer (400 MHz 1 H) (100 MHz 13 C). The values of δ ppm (chemical shifts) are reported relative to TMS as a standard reference. The J values (coupling constants) are provided in Hz. The progress of the reaction was checked and guided by TLC using aluminum silica gel 60 F245. IR, 1 H NMR, 13 C NMR and elemental analyses were measured at the Micro Analytical Center at the Faculty of Science, Cairo University, Egypt. To a well-stirred mixture of the acetylated acetylenic gluco-or xylopyranosyl sugar (2 mmol) and the azidothio thiadiazole derivative 3 (2 mmol) in THF-H 2 O (2:1; 25 mL) was added CuSO 4 ·5H 2 O (0.4 mmol), followed by sodium ascorbate (0.4 mmol) and a few drops of diisopropylethylamine at 0 • C. The reaction mixture was stirred at room temperature for 2 h and then at 60 • C for 6 h (TLC, petroleum ether-EtOAc, 3:1). Ethyl acetate (30 mL) was added, and the organic layer was separated, washed with water (2 × 40 mL) and dried. The residue was purified by column chromatography (petroleum ether-EtOAc, 4:1) to afford the acetylated glycosyl triazole compounds 5 and 6, respectively.

Synthesis of Deacetylated O-Glycosides (7,8)
The acetylated 1,2,3-triazole glycoside 5 or 6 (5 mmol) was added to a saturated methanolic ammonia solution (15 mL) at 0 • C and stirred for 20 min, followed by stirring at room temperature for 7 h. After the completion of the deacetylation process (TLC, petroleum ether-hexane, 2:1), the solvent was evaporated under reduced pressure at 40 • C to afford a yellowish residue. Trituration with cold diethyl ether (20 mL) upon stirring afforded a solid, which was filtered off, dried and crystallized from ethanol to yield compound 7 or 8, respectively.

In Vitro Cytotoxic Activity
HCT-116 (human colorectal carcinoma) and MCF-7 (human breast adenocarcinoma) cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA) and maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U mL −1 penicillin and 100 U mL −1 streptomycin. The cells were grown at 37 • C in a humidified atmosphere of 5% CO 2 .

MTT Cytotoxicity Assay
Cytotoxicity activity against HCT-116 and MCF-7 was estimated by the 3-[4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. This test is based on MTT cleavage by mitochondrial dehydrogenases from viable cells [50][51][52]. Cells were placed in a 96-well sterile microplate (5 × 10 4 cells well −1 ) and incubated at 37 • C in serum-free media containing dimethyl sulfoxide (DMSO) and either a series of various concentrations of each compound or doxorubicin (positive control) for 48 h before the MTT assay. After incubation, the media were removed, and 40 µL of MTT (2.5 mg mL −1 ) was added to each well. Incubation was resumed for an additional 4 h. The purple formazan dye crystals were solubilized with 200 µL DMSO. Absorbance was measured at 590 nm in a Spectra Max Paradigm Multi-Mode microplate reader (Molecular Devices, LLC, San Jose, CA, USA). Relative cell viability was expressed as the mean percentage of viable cells compared to the untreated control cells.

Statistical Analysis
All experiments were conducted in triplicate and repeated on three different days. All values are reported as mean ± SD. IC 50 values were determined by SPSS Inc. probit analysis (IBM Corp., Armonk, NY, USA).

EGFR WT , EGFR T790M and HER-2 Inhibition Assessment
The promising cytotoxic targets 7, 8, 11, 12 and 15 were assessed for their in vitro inhibitory activity against EGFR WT , EGFR T790M and HER-2 kinases. Erlotinib was used as a reference following the previously described method [53]. A homogeneous time-resolved fluorescence (HTRF) assay was applied in this test (Sigma). Firstly, EGFR WT , EGFR T790M and HER-2 and their substrates were incubated with the tested compounds in enzymatic buffer for 5 min. ATP (1.65 µM) was added to the reaction mixture to start the enzymatic reaction. The assay was conducted for 30 min at room temperature. The reaction was stopped by the addition of detection reagents that contained EDTA. The detection step continued for 1 h, and then the IC 50 values were determined using GraphPad Prism 5.0. Three independent experiments were performed for each concentration.

Detection of Apoptosis and Cell Cycle Analysis
Apoptosis investigation and cell cycle analysis were performed by flow cytometry [43,44]. MCF-7 cells were seeded at 8 × 10 4 and incubated at 37 • C in 5% CO 2 overnight. After treatment with the tested compounds 7 and 15 for 24 h, cell pellets were collected and centrifuged (300 g, 5 min). For cell cycle analysis, cell pellets were fixed with 70% ethanol on ice for 15 min and collected again. The pellets were incubated with propidium iodide (PI) staining solution at room temperature for 1 h and analyzed with a Gallios flow cytometer (Beckman Coulter, Brea, CA, USA). Apoptosis detection was carried out by the FITC AnnexinV/PI commercial kit (Becton Dickenson, Franklin Lakes, NJ, USA) following the manufacturer's protocol. The samples were analyzed by fluorescence-activated cell sorting (FACS) with a Gallios flow cytometer (Beckman Coulter, Brea, CA, USA) within 1 h after staining. Data were analyzed using Kaluza v 1.2 (Beckman Coulter).

Molecular Docking Study
The interactions of the newly synthesized targets 7, 8 and 15, illustrating the highest EGFR WT , EGFR T790M and HER-2 inhibitory activities, were examined and docked within the active sites of the target enzymes to study their binding modes and orientations using PDB IDs: 1M17, 3UG2 and 3RCD, respectively [47,49], using MOE-Dock software version 2014.0901 [45,46]. The 2D structures of the newly synthesized triazole-based glycosides 7, 8 and 15 were drawn using Chem. Draw. The protonated 3D structure was employed using standard bond lengths and angles. Then, geometry optimization and energy minimization were applied to obtain the Conf Search module in MOE, and the MOE file was then saved for the subsequent docking process. The co-crystallized structures of EGFR WT , EGFR T790M and HER-2 with their ligands, erlotinib, gefitinib and TAK-285, were downloaded (PDB codes: 1M17, 3UG2 and 3RCD, respectively) from the Protein Data Bank. All minimizations were performed using MOE until reaching an RMSD gradient of 0.05 kcal·mol −1 Å −1 with an MMFF94x force field, and the partial charges were automatically calculated. The preparation of the enzyme structure was performed for molecular docking using the Protonate 3D protocol with the default options in MOE. The London dG scoring function and Triangle Matcher placement method were used in the docking protocol. First, validation of the docking processes was established by docking the native ligands, followed by docking derivatives 7, 8 and 15 within the ATP-binding sites of EGFR WT , EGFR T790M and HER-2 after the elimination of the co-crystallized ligands.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules27206960/s1, include spectral and biological data of synthesized compound. Figure S1. Dose dependent cytotoxic activity of the synthesized derivatives against HCT-116 cancer cells according to the MTT assay. Figure S2. Dose dependent cytotoxic activity of the synthesized derivatives against MCF-7 cancer cells according to the MTT assay. Figure  S3. IC50 (µM) data of the screened targets in comparison with doxorubicin against human HCT-116 and MCF7 cancer cell lines according to the MTT assay. Figure S4. Apoptotic activity of the promising derivatives 7 and 15. Figure S5. Cell cycle analysis in MCF-7 cells treated with the promising derivatives 7 and 15. Figure

Conflicts of Interest:
The authors declare no conflict of interest.