Novel [1,2,3]triazoles, [1,2,3]triazolo[4,5-d]Pyrimidines, and Some of Their Glycoside Derivatives: Synthesis and Molecular Modeling as Potential Apoptotic Antitumor Agents

Abstract Two new series of 4,5-difunctionalized 1-bromobenzyl[1,2,3]triazole (2a, 3a, 4a, 5a, and 6a) and 4,5-difunctionalized 1-(2-oxo-2-(p-tolylamino)ethyl-[1,2,3]triazole (2b, 3b, 4b, 5b, and 6b) were synthesized using related 1-(azidomethyl)-4-bromobenzene 1a and p-tolylcarbamoyl azide 1b respectively. The substituted [1,2,3]triazolo[4,5-d]pyrimidine-7-one derivatives (7a, 7b, 8a, and 8b) were synthesized by the reaction of [1,2,3]triazolo derivatives 2a and 2b with carbon disulfide in the presence of 10% sodium hydroxide/dimethylformamide and/or by the reaction with formic acid respectively. The S-glucoside derivatives (9–12) of newly synthesized [1,2,3]triazolo[4,5-d]pyrimidines were also synthesized. By using several spectroscopic methods, including IR, 1H NMR, 13C NMR, and elemental analysis, the chemical structures of the novel derivatives were confirmed. The synthetic compounds’ cytotoxicity and in vitro anticancer activity were examined vs. human breast carcinoma (MCF-7), human laryngeal carcinoma (HEP-2), and human colorectal carcinoma (HCT-116) cell lines. According to the findings, HEP-2 and HCT-116 cells are more sensitive to the tested compounds than the other cell lines. In the HEP-2 and MCF-7 cell lines, respectively, compounds 5b and 11 showed potential anticancer activity when compared to the effect of the commonly used anticancer medication, doxorubicin. The selectivity of compounds against the cancer cell line was confirmed by testing their cytotoxicity on VERO (African Green Monkey kidney) normal cells. The anticancer activity of those compounds is suggested to be due to nuclear damage generated by a high generation of reactive oxygen species (ROS). In addition, the induction of apoptosis by significantly upregulating the apoptotic genes PAR-4 and BAX while substantially downregulating the anti-apoptotic genes BCL-2 and BCL-xl. Molecular docking research was undertaken to predict the probable binding poses of the most effective drugs in the active site of CDK-2. The more active compounds (2a, 3a,b, 4a,b, 5a,b, 6b, 11, and 12) have been docked on the CDK-2 enzyme to demonstrate their mode of action as anticancer medicines. The compounds exert many interactions and showed high binding to the CDK-2 receptor. Finally, a hypothetical pharmacophore model was created using the Molecular Operating Environment (MOE) software and five compounds that are structurally similar to the synthesized ones with known anticancer action.

C NMR, and elemental analysis, the chemical structures of the novel derivatives were confirmed.The synthetic compounds' cytotoxicity and in vitro anticancer activity were examined vs. human breast carcinoma (MCF-7), human laryngeal carcinoma (HEP-2), and human colorectal carcinoma (HCT-116) cell lines.According to the findings, HEP-2 and HCT-116 cells are more sensitive to the tested compounds than the other cell lines.In the HEP-2 and MCF-7 cell lines, respectively, compounds 5b and 11 showed potential anticancer activity when compared to the effect of the commonly used anticancer medication, doxorubicin.The selectivity of compounds against the cancer cell line was confirmed by testing their cytotoxicity on VERO (African Green Monkey kidney) normal cells.The anticancer activity of those compounds is suggested to be due to nuclear damage generated by a high generation of reactive oxygen species (ROS).In addition, the induction of apoptosis by significantly upregulating the apoptotic genes PAR-4 and BAX while substantially downregulating the anti-apoptotic genes BCL-2 and BCL-xl.Molecular docking research was undertaken to predict the probable binding poses of the most effective drugs in the active site of CDK-2.The more active compounds (2a, 3a,b, 4a,b, 5a,b, 6b, 11, and 12) have been docked on the CDK-2 enzyme to demonstrate their mode of action as anticancer medicines.The compounds exert many interactions and showed high binding to the CDK-2 receptor.Finally, a hypothetical pharmacophore model was created using the Molecular Operating Environment (MOE) software and five compounds that are structurally similar to the synthesized ones with known anticancer action.

Introduction
It is well established that anticancer medications are essential for cancer treatment; however, given the side effects and drug resistance related to the currently existing anticancer medications, it is urgent that new medications with minimal side effects and high efficacy be developed. 1][10][11] Furthermore, 1,2,3-triazole is a key component in the development of new anticancer drugs, and several of its derivatives are already being used in clinics or in clinical trials to combat cancer (Figure 1). 12Since hybrid molecules play a significant role in cancer control, so combining the 1,2,3-triazole framework along with other anticancer pharmacophores could be a useful therapeutic intervention for cancer treatment, particularly drug-resistant cancer. 136][17][18][19][20][21][22][23] Besides, the epidermal growth factor receptor (EGFR), one of the most wellknown oncogenic kinases, plays a crucial mediating function in cell proliferation, angiogenesis, apoptosis, and metastatic dissemination and is overexpressed in a range of human malignancies, including breast, colorectal, lung, prostate, ovary, and pancreatic cancers. 24Additionally, several of anti-EGFR medication recommendations were given because they were more effective and safe than conventional chemotherapy. 25Furthermore, compounds containing triazole scaffolds V, VI, and VII strongly inhibited the EGFR and showed outstanding in vitro cytotoxicity against some of human cancer cell lines (Figure 1). 26Likewise, heterocyclic thioglycoside analogs are known for their potential anticancer activity. 27,28Several heterocycle-glycoside hybrids are also important systems that have attracted a lot of biological interest.Glycosyl heterocyclic compounds, such as those with the markers V, VI, and VII, have shown anticancer efficacy and other crucial biological functions like antiviral and antibacterial capabilities. 29It has also been reported that triazole-glycoside V (Figure 1) exhibits encouraging anticancer and kinase inhibitory activities against EGFR. 26,30Encouraging by these features and our present research interest in developing new anticancer candidates, 20,[31][32][33][34][35][36][37][38][39][40] we have herein synthesized two series of triazoles, triazolopyrimidine, and their thioglycoside analogs and studied their cytotoxic action as well.
The infrared spectra of compound 2a showed the characteristic carbonyl amide group clearly at 1666 cm À1 , while compound 4a showed a peak at 1718 cm À1 of carbonyl ketone of an acetyl group.On the other hand, compound 5a showed a peak at 1748 cm À1 of the carbonyl ester group (Scheme 1).
The infrared spectra of the triazole 4b and 5b were proved by the presence of two bands of carbonyl groups, one of them at a high frequency ranging between 1722 cm À1 for the ketone group and 1752 cm À1 for the ester group, in addition to the presence of another band at a low frequency for the carbonyl amide group.On the other hand, the presence of the characteristic peaks of the cyano group at 2223 and 2225 cm À1 confirmed the structures of [1,2,3] triazole 6a and 6b, respectively (Scheme 1).
The hetero-cyclization of the substituted triazole compounds to their derived triazolopyrimidine products was confirmed by their spectral data since their IR and NMR spectra revealed the disappearance of the amino groups, and instead, two characteristic signals assigned to the NH groups are present.On the other hand, The IR spectra of 8a revealed one distinctive absorption band for C ¼ O at m ¼ 1665 cm À1 , whereas derivative 8b revealed two bands for the same functional group at ¼ 1723 and 1638 cm À1 .In addition to the additional aromatic, aliphatic, and NH protons for each derivative, The research was extended to investigate the reaction between the triazolopyrimidine derivative 7a and 2,3,4,6-tetra-O-acetyl-a-D-gluco-or 2,3,4,-tri-O-acetyl-a-D-xylopyranosyl bromide derivatives, which gave rise to the corresponding glycosyl derivatives of the triazolopyrimidine Scheme 1. Synthesis of novel 1,2,3-triazole derivatives nucleus 9 and 10, respectively.The attachment of the sugar part to the triazolopyrimidine system was confirmed by the IR and NMR spectra which showed the acetyl absorption bands in their IR spectra and the characteristic methyl acetyl signals in their NMR spectra.In addition, the acetyl carbonyls were also revealed in the corresponding 13 C NMR spectra.The nature of the attachment of the sugar part through the thio-linkage was achieved by the NMR spectra since the b-orientation of the glycosidic bond of the resulting glycoside 9 and 10 was proven by the doublet signal in the range d 5.89 ppm related to the anomeric proton of the sugar moiety with J coupling around 10.3 Hz and appearance of a peak at about d 90.9 ppm attributed to the anomeric-C 1 in 13 C NMR spectra.On the other hand, the low chemical shift value of the anomeric hydrogen (H-1) indicated the attachment of the sugar part to the triazolopyrimidine at the sulfur rather than the nitrogen atom.The chemical shift value of H-1 of the sugar part attached to the nitrogen atom is known to be at higher values (5.95-6.10ppm) due to the anisotropic deshielding effect of the adjacent C ¼ S group, which is not the case in the currently assigned structures.Deacetylation of the latter glycosides 9 and 10 lead to the derived free hydroxy analogs 11 and 12, respectively (Scheme 3).The structure elucidation of 11 and 12 depended on the appearance of characteristic peaks of hydroxyl groups of the deacetylated sugar part and the disappearance of absorption bands of acetyl carbonyl groups in their IR spectra.The 1 H-NMR of the later free hydroxyl glycosides also revealed the presence of the signals corresponding to the hydroxyl groups and the disappearance of the acetyl methyl signals present in their acetylated precursors, which confirms the assigned structures.

In vitro cytotoxicity activity
After performing single dose (100 lg/ml) testing for all synthesized triazole derivatives and triazolopyrimidine derivatives on three different cancer cells and VERO normal cell.Table 1 shows the % inhibition of the prepared derivatives on the used three cancer lines and the VERO normal line.All compounds exert mild to moderate effect (compared to inhibition% exerted by Dox) on MCF-7 cell line.On the other hand, compounds 3a, 4a, 5a, 5b, and 6b exert moderate to severe effect (compared to inhibition % exerted by Dox) on HEP-2 cell line.Compounds 3a, 4a, 8b, 9, and 11 exert moderate to severe effect on HCT cell line.
Compounds 3a, 4a, 5a, 5b, and 6b show anticancer activity on HEP-2 cell line with IC 50 ¼12, 16, 8, 4.8, and 17.5 lg/ml, respectively (Figure 2(A)).Compound 5b has the superior effect on HEP-2 cell line.Compounds 3a, 4a, 8b, 9, and 11 show a promising anticancer effect on HCT cell line with IC 50 ¼8, 6, 7, 6.5, and 5.5 lg/ml, respectively (Figure 2(B)).Compound 11 had a better effect on HCT cell line.The selectivity of compounds against cancer cell lines was confirmed by testing its cytotoxicity on VERO (African Green monkey kidney) normal cells.All the compounds produce mild to moderate effect on the VERO cell line except compound number 3b produce a moderate to strong effect after performing single dose (100 lg/ml) testing.Compounds 3a, 4a, 5a, 5b, 6b, 8b, 9, and 11 were found to be safe and selective as no detected IC 50 on normal cell line till 50 lg/ml (Figure 2(C)).Compound 5b was highly selective for the human laryngeal cancer cell line, and compound 11 was highly selective for the colon cancer cell line.By correlation of the afforded activity results against HEP-2 and HCT cell lines with the characteristic structural features of the most active compounds, it was found that the N-substituted-1,2,3-triazole derivatives with thioamide and acetyl (3a and 4a) incorporating the p-bromobenzyl substituent at N-1 showed higher activities than their structural analogs possessing the N-p-tolylacetamide substituent.For the activity of the triazolopyrimidine derivatives 8a and 8b, as well as the amino-substituted 1,2,3-triazole against HCT cancer cell line, the derivatives with N-p-tolylacetamido substituent showed higher activity hence, indicating the effect of the nature of the substituent at N-1 in the 1,2,3-triazole motif.Furthermore, the activity of the triazole derivative substituted with thioamide and amine functional groups at C-4 and C-5, respectively, and possessing N-p-tolylacetamide at N-1 accounts for the possible revealed selectivity as a result of the substituent.
On the other hand, the difference in activities recorded for the triazolopyrimidine glycosides 9-12 showed the influence of the nature of the sugar moieties attached to the heterocyclic core in such type of structures.Thus, the triazolopyrimidine thioglycosides with an acetylated glycopyranosyl moiety (gluco-and xylopyranosyl) resulted in higher activities against HCT cancer cell than their structural analogs with deprotected free hydroxyl groups in the sugar moieties.By observing the activity results of all tested compounds against the human cancer cell line, it was found that the glycosides 9 and 11 were the most active, which showed the effect of the acetylated glycopyranosyl moiety attached to the heterocycle structure.

Effect on oxidative stress
The tested compounds significantly increased the oxidative stress in HEP-2 and HCT-116 cells The tested 5b and 11 compounds significantly increased the NOx level (P¼ 0.032, 0.026) compared to untreated cells, accompanied by a significant reduction in the GSH content (P ¼ 0.01, 0.02) in HEP-2 and HCT-116 cells, respectively (Figure 3) compared to untreated cells.
Nuclear damage was discovered to be caused by the increased generation of reactive oxygen species (ROS) following treatment with these compounds.That may lead to increase expression of apoptotic proteins.These results illustrate that triazole and its fused pyrimidine derivatives have strong anticancer potential.In this regard, we offer details on the president and anticancer properties of derivatives of [1,2,3]triazolo [4,5-d]pyrimidine.Previous investigations on these compounds have concentrated on their antioxidant rather than their prooxidant capabilities because the majority of these synthetic compounds are known to impact the redox state of the cell. 47

Effect on apoptotic genes expression
The tested compounds significantly increased the apoptotic gene expression.Further investigation by using real-time PCR analysis showed that 5b and 11 induced apoptotic effect via significant downregulation of anti-apoptotic BCL-2 (P ¼ 0.027, 0.02) and insignificant decrease of BCL-xl (P ¼ 0.1, 0.07) and significant upregulation by a two-fold increase of apoptotic genes PAR-4 (P ¼ 0.04, 0.02) and BAX (P ¼ 0.017, 0.03) on HEP-2 and HCT cells, respectively.
Our results indicated that 5b and 11 modulate the gene expressions of the BAX, BCL-2, BCLxl and PAR-4.As shown in Figure 4, 5b and 11 significantly upregulate apoptotic genes and downregulate antiapoptotic gene expression relatives to the control.By calculating and studying the apoptotic index (BAX/BCL-2 ratio), compound 5b showed a fourfold increase relative to the control and compound 11 showed significantly the highest BAX/BCL-2 ratio in comparison to control (seven-fold increase).
VERO: normal African Green monkey kidney cell line, HCT: human colorectal carcinoma cell line, HEP-2: human laryngeal carcinoma cell line, MCF-7: human breast carcinoma cell line.Values are the means ± SD of three independent experiments performed in triplicates.Red: strong effect, blue: moderate activity, and Yellow: weak activity S (2): Cytotoxicity and IC 50 values of standard Doxorubicin on A: HEP-2 cell line, B: HCT cell line, and C: MCF-7 cell line after 48h.Values are the means ± SD of three independent experiments performed in triplicates.

Molecular docking study
In addition to biological investigation, a molecular docking study was conducted to make predictions about the probable binding poses of the most effective drugs in the active site of CDK-2.For this purpose, the protein data bank file (PDB: 2A4L) was chosen and obtained from the protein data bank (https://www.rcsb.org/structure/2A4L).The molecular operating environment (MOE) software 2015.10 was used for all docking methods.The docking procedure was initially validated by re-docking the co-crystallized ligand (RRC) in the enzyme binding pocket with an energy score (S) ¼ 6.57 kcal/mol and a root mean standard deviation (RMSD) of 1.85, resulting in an arene-H interaction with Leu 134 residue (Figure 7).The ligand was removed from the complex, then the docking of compounds (2a, 3a,b, 4a,b, 5a,b, 6b, 11, and 12) was performed, and the data obtained were recorded in Table 2.As shown in Figures 5-7, the compounds had good binding to CDK-2 active sites, with a different number of H-bonds and H-pi bonds interactions.The docking energy score of compounds 11 and 12 (docking score (S) ¼ (À8.35 and À7.28) kcal/mol, respectively) is greater than the co-crystallized RRC docking score (-6.57kcal/mol) and can establish different H-bonds with Asp 145, Thr 14 and Lys 33 amino acids.The derivatives 5a and 5b have the same energy score, S¼ À6.44 kcal/mol, which is close to that of the RRC ligand.Compounds 2a, 3a,b, 4a,b, and 6b showed a docking score slightly less than that of the co-crystallized RRC.

Conclusion
The newly synthesized functionalized 1,2,3-triazoles were useful key structures for heterocyclization processes resulting in new triazolopyrimidines and their derived thioglycosides products.The afforded bioactivity results revealed the effect of the nature of substituents for a number of the synthesized derivatives, especially against human Hep2 and HCT cancer cell lines.Moreover, the most susceptible tested cancer cells were Hep2 and HCT by the tested compounds.Interestingly, most of the highly active derivatives against the cancer cell lines were found safe for the human normal cell line in the current investigation, which indicates a degree of the desired selectivity to the cancer cells.The more active compounds (2a, 3a,b, 4a,b, 5a,b, 6b, 11, and 12) have been docked on the CDK-2 enzyme in an attempt to demonstrate their mode of action as anticancer medicines.The compounds exert many interactions and showed high binding to the CDK-2 receptor.

Creating Pharmacophores
The goal of this technique is to create and test a pharmacophore model (hypothesis) developed from CDK-2 inhibitors as anticancer medicines. 48For 3D pharmacophore-based applications, three phases are often used: To begin, 3D structures of training set compounds with known biological activity must be generated.Second, the pharmacophoric characteristics are assigned.Finally, a technique for conducting conformational searching of databases for novel structures matching the given pharmacophoric properties 49 is used.These processes will be detailed for several previously reported anticancer compounds (I-V) (Figure 8), as well as the produced compounds (2a, 3-5(a,b), 6b, 11, and 12) utilizing the Molecular Operating Environment (MOE) 2015.10.The software expresses the degree of mapping of a given compound to a produced hypothetical model in terms of rmsd (the root of the mean square distance) between the query characteristics, and their matching ligand-target points, 50 which relates to the activity of the molecule.
Hydrogen bond acceptors (Acc), hydrogen bond donors (Don), charged or ionizable groups (Cat and Ani), hydrophobic (Hyd), metal ligator (ML), and/or aromatic rings (Aro) are traditional pharmacophoric properties, as are geometrical limitations such as distances, angles, and dihedral angles.The initial pharmacophoric inquiry was carried out, and it consists of four  characteristics, as shown in Table 3 and visually in Figure 9.A test set search applying this modified consensus query yielded six hits: 4b, 5a,b, 6b, 11, and 12 (Figure 10).
Table 3 shows the results, which show that the lower the rmsd values, the stronger the inhibitory activity.The most active compounds were superimpositions of compound 11 with rmsd values of 0.4876.Compounds 4b and 12 also demonstrated strong anticancer activity, with rmsd values of 0.7552 and 0.9361, respectively.Compound 6b was chosen as an example of a practically unfortunate matching compound owing to its high rmsd value.

Chemistry
All melting points were measured on Electro thermal IA 9000 series digital melting point apparatus.The IR spectra were recorded in potassium bromide disks on a PyeUnicam SP 3300 and Shimadzu FT IR 8101 PC infrared spectrophotometer.The NMR Spectra were recorded at 270 MHz on a Varian Mercury VX-300 NMR spectrometer. 1 H NMR (300 MHz) and 13 C NMR (75.5 MHz) were run in deuterated chloroform (CDCl 3 ) or dimethylsulfoxide (DMSO-d 6 ).Chemical shifts were related to that of the solvent.Mass Spectra were recorded on a Shimadzu GCMS-QP1000 EX mass spectrometer at 70 eV.Elemental analyses were carried out at the Micro analytical Centre of Cairo University, Giza, Egypt.All reactions were followed by (TLC) using Silica gel, Aluminum Sheets 60 F 254 , (Merck).The anticancer screening occurred in Cancer
To the residue, water (20 ml) was added, and the solution was acidified with 4 N acetic acid at pH ¼ 5.The precipitated solid was filtered and crystallized from ethanol to afford compounds 2a, 2b -6a, and 6b, respectively.

Synthesis of compounds 7a and 7b
To a solution of the carboxamide derivative 2a or 2b (10 mmol) in dimethyl formamide (30 ml), sodium hydroxide (12 ml, 10%) was added dropwise at 0 C, the reaction mixture was stirred at 0 C for one hour.Carbon disulfide (14 mmol) was added, and the temperature of the reaction was raised up to room temperature and then refluxed in a water bath for 8 h.The solvent was reduced under vacuum, and the residue was dissolved in ice water and then acidified with diluted hydrochloric acid to afford compounds 7a and 7b.

Synthesis of compounds 8a and 8b
The triazole derivative 2a or 2b (10 mmol) was refluxed in 20 ml of formic acid for 8h, then cooled and placed on crushed ice while stirring for 1 h.Compounds 8a and 8b were produced from ethanol by crystallizing the precipitated solid and filtering it off. 3-(4-Bromobenzyl)-

Synthesis of compounds 9 and 10
To a solution of the compound 7a or 7b (5 mmol) in aqueous potassium hydroxide [(0.56 g, 10 mmol in distilled water (16 ml)] was added a solution of 2,3,4,6-tetra-O-acetyl-a-D-galactopyranosyl bromide, or 2,3,4-tri-O-acetyl-a-D-xylopyranosyl bromide (5 mmol) in acetone (20 ml).The reaction mixture was stirred at room temperature for 10-12 h (TLC).The solvent was evaporated under reduced pressure at 40 C, and the residue was washed with distilled water to remove potassium bromide formed.The product was dried, and crystallized from ethanol to give compounds 9 and 10, respectively.

Human cancer cell line
In this study, a panel of human cancer cell lines was tested for their chemosensitivity to [1,2,3]triazole and [1,2,3]triazolo [4,5-d]pyrimidine derivatives as well as their glycoside derivatives: normal African Green monkey kidney cell line (VERO), human colorectal carcinoma cell line (HCT), human breast carcinoma cell line (MCF-7), human laryngeal carcinoma cell line (HEP-2), were obtained frozen in liquid nitrogen (-180 C) from American Type Culture Collection (ATCC; Washington, DC, USA) and were maintained at National Cancer Institute as monolayer cultures in RPMI-1640 supplemented with 10% FBS and 1% penicillin-streptomycin.

Cytotoxicity assay
Cytotoxicity was determined using sulforhodamine-B (SRB) method. 51Cells were seeded in 96well microtiter plates at a concentration of 3 Â 10 3 cells/well.They were left to attach for 24 h before incubation with drugs.The cells were treated for 48 h with a single dose (100 lg/ml) of all the compounds, and for IC 50, the cells were treated with different concentrations (0, 5, 12.5, 25 and 50 lg/ml) of compounds 3a, 4a, 5a, 5b, and 6b on HEP-2 cell line and 3a, 4a, 8b, 9, and 11 on HCT cell line.The optical density (O.D) of each well was measured spectrophotometrically at 570 nm using an ELISA microplate reader (TECAN Sunrise TM, Germany).The mean values were estimated as a percentage of cell viability as follows: O.D (treated cells)/O.D (control cells) Â 100.The IC 50 value (the concentration that produces 50% inhibition of cell growth) of each drug was calculated using dose-response curve-fitting models (Graph-Pad Prism software, version 5).
Preparation of cell-free media and cell lysate Cells of HEP-2 and HCT-116 cell lines were cultured in T75 flasks, left for 24 h, and then treated with IC 50 concentration of the compounds 5b and 11, respectively, for 48 h.The medium was collected and used for the determination of NOx level.Cell pellets were prepared by removing the cells from the flasks by trypsinization and used to determine glutathione content and realtime PCR.The treated and control cell pellet were collected, washed, and suspended in cold lysis buffer, then sonicated and centrifuged, and the clear supernatant was taken into another Eppendorf.
Determination of non-protein reduced thiols content (glutathione content) Reduced glutathione (GSH) in cell lysate was determined according to the method of Ellman [48]; it is based on the reduction of Ellman's reagent [5,5 0 -dithio-bis-(2-nitrobenzoic acid)] by SH groups to form 1 mole of 2-nitro-5-mercaptobenzoic acid per mole of SH.The optical density was measured at 412 nm against a reagent blank, and the results were expressed as lmol/mg protein.
Determination of total nitrate/nitrite (NOx) Total nitrate/nitrite (NOx) was measured in cell culture media as a stable end product, nitrite, according to the method of Miranda. 52The assay is based on the reduction of nitrate by vanadium trichloride combined with detection by the acidic Griess reaction.The diazotization of sulfanilic acid with nitrite at acidic pH is subsequent coupling with N-(10-naphthyl) ethylenediamine to an intensely colored product determined spectrophotometrically at 540 nm and expressed as nmol/mg protein.

Determination of mRNA expression of apoptotic genes
The expression of Bax, BCL-2, PAR-4, and BCL-xl genes [S1] in cells will be quantified using quantitative real-time PCR.Total RNA was extracted from the control and treated with TRIzol Reagent (Invitrogen, Carlsbad, CA), and the quality and the quantity of the RNA was determined using nanodrop (Thermo Fisher, UK).Single-stranded RNA was converted into complementary DNA using the cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA).Thermal cycling was commenced using a thermocycler (Biometra, Germany) according to the following conditions: 25 C for 10 minutes, 37 C for 120 minutes, 85 C for 5 minutes, and 4 C for 1. Real-time PCR analysis was conducted using the thermo-cycler Step One TM (Applied Biosystems).Each RT-reaction served as a template in a 20 lL PCR reaction containing 0.2 lmol/L of each primer and SYBR green master mix (Thermo Fisher Scientific, UK).Real-time PCR reactions were performed at 50 C for 2 minutes, 95 C for 10 minutes, followed by 45 cycles at 95 C for 15 minutes, and 56 C for 1 minute.The mRNA levels of these genes were normalized to GAPDH (DCT).The DCT was calibrated against an average of the control samples.

Molecular docking study
The Molecular Operating Environment (MOE) version 2015.10 software was used for molecular modeling investigations.The structures of the most powerful compounds were drawn in Chem Draws 18.0 and stored as MDL molfiles.Before molecular docking, the following processes must be completed: a) converting ligands' 2D structures to their 3D forms; b) adding and removing polar hydrogen atoms; and c) minimalizing energy using the MMFF94x force field until an RMSD (Root-mean-square deviation) of atomic position gradient of 0.01 Kcal mol À1 A o-1 was attained and preserved as moe.
The R-Roscovitine (RRC) crystal structure with cyclin-dependent kinase-2 active site (PDB: ID 2A4L) was retrieved from the protein data bank.3D protonation, in which hydrogen atoms are added to the enzyme's conventional shape, was used to prepare it for docking.The structures of the selected compounds (2a, 3a,b, 4a,b, 5a,b, 6b, 11, and 12) were docked into the CDK-2 receptor through MOE-Dock using the triangle matcher placement method, London dG scoring function, and force field refinement was accomplished on the top 5 poses per each compound.To validate the docking process, RRC was re-docked with the 2A4L active site.Interactions of amino acids, affinity by bond strength, and the lengths of hydrogen bonds were illustrated in Table 2.

Designing pharmacophores
The processes for pharmacophore construction 50 are as follows.First, the MOE 2015.10 software used flexible alignment for compounds (I-V) (Figure 8) as a training set.The data is contained in the flexible alignment output (S: The alignment score of the configuration).Lower S values are supposed to imply better alignments.Second, paste the alignment structure with the lowest S value into the MOE window.Then, using the Pharmacophore Query Editor, generate a pharmacophore query for the compounds in the alignment training set.The resulting model is then run through the systemic test set (2a, 3-5(a,b), 6b, 11, and 12) with the Pharmacophore Search.Following that, the software uses the Pharmacophore Preprocessor to produce annotations for all compound conformations in the test set database using the presently specified PCH-All (Polarity-Charge-Hydrophobicity) pharmacophore scheme.Then.Modify the query using the consensus query approach and research the database.Finally, regarding the rmsd value, the program represents the degree of mapping for a given compound to a developed hypothetical model as displayed in Table 3.

Figure 3 .
Figure 3.Effect of treatment of control and compounds 5b and 11 on oxidative stress and antioxidants in HEP-2 and HCT-116 cell lines.GSH(A), NOx level (B).Data were expressed as means ± SD of three independent experiments.The statistical significance of the results was analyzed using the unpaired t-test.a: significantly different from the control group at P < 0.05.

Figure 4 .
Figure 4. Effect of treatment of control and Sample 5b and 11 on mRNA expression of BAX, BCL-2, BAX/BCL-2 ratio, BCL-xl, PAR-4 on HEP-2 and HCT-116 cells.BAX expression (A), BCL-2 expression (B), BAX/BCL-2 ratio BCL-xl (C), BCL-xl (D), and PAR-4 (E).Results are expressed as means ± SD of two independent experiments performed in duplicate.The statistical significance of the results was analyzed using the unpaired t test.(a) Significantly different from the control group at P ˂ 0.05.

Figure 5 .
Figure 5. 2D and 3D interaction of 2a, 3a,b, and 4a in the active site of CDK-2 (PDB: 2A4L).Hydrogen bonds are displayed in cyan and H-pi-bonds are in dark pink.

Figure 6 .
Figure 6.2D and 3D interaction of 4b, 5a,b, and 6b in the active site of CDK-2 (PDB: 2A4L).Hydrogen bonds are displayed in cyan and H-pi-bonds are in dark pink.

Figure 7 .
Figure 7. 2D and 3D interaction of 11, 12, and RRC in the active site of CDK-2 (PDB: 2A4L).Hydrogen bonds are displayed in cyan and H-pi-bonds are in dark pink.

Table 3 .
Pharmacophoric and structure features of the training inhibitors and rmsd values of the hit set.