Synthesis, biological evaluation, and in silico studies of new CDK2 inhibitors based on pyrazolo[3,4-d]pyrimidine and pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine scaffold with apoptotic activity

Abstract Cyclin-dependent kinase inhibition is considered a promising target for cancer treatment for its crucial role in cell cycle regulation. Pyrazolo pyrimidine derivatives were well established for their antitumor activity via CDK2 inhibition. In this research, new series of pyrazolopyrimidine derivatives (4–15) was designed and synthesised as novel CDK2 inhibitors. The anti-proliferative activities against MCF-7, HCT-116, and HepG-2 were used to evaluate their anticancer activity as novel CDK2 inhibitors. Most of the compounds showed superior cytotoxic activity against MCF-7 and HCT-116 compared to Sorafenib. Only compounds 8, 14, and 15 showed potent activity against HepG-2. The CDK2/cyclin A2 enzyme inhibitory activity was tested for all synthesised compounds. Compound 15 showed the most significant inhibitory activity with IC50 0.061 ± 0.003 µM. It exerted remarkable alteration in Pre G1 and S phase cell cycle progression and caused apoptosis in HCT cells. In addition, the normal cell line cytotoxicity for compound 15 was assigned revealing low cytotoxic results in normal cells rather than cancer cells. Molecular docking was achieved on the designed compounds and confirmed the two essential hydrogen binding with Leu83 in CDK2 active site. In silico ADMET studies and drug-likeness showed proper pharmacokinetic properties which helped in structure requirements prediction for the observed antitumor activity.


Introduction
Protein kinases represent a large group of structurally related enzymes that are essential and regulate cell cycle progression involved in cell division [1][2][3] . Cyclin-dependent kinases (CDKs) are serine-threonine kinases responsible for cell cycle regulation and cell differentiation 2 . Cyclin-dependent kinases (CDK) are mainly responsible for the phosphorylation process of proteins [4][5][6] . Cyclin is the regulatory protein bound by CDK leading to ATP binding region modification 2 . CDKs in absence of cyclin have less activity where the activation loop (known as T-loop) blocks the cleft, and the key amino acid residues are not optimally positioned for ATP binding 2 . CDK2 has a catalytic effect in cyclin-dependent protein kinase complex 7,8 . Protein phosphorylation has a critical role in cellular function regulation. This essential role during the cell cycle could be altered in tumour cells [7][8][9][10][11] . Alteration in kinases may lead to the development of many diseases, including cancer. Hence, the control of CDK dependent cell cycle is essential for tumour progression management. Where overexpression of CDK enzymes occurs in cancer 2 . As uncontrolled CDK2 activation in human cancer is associated with overexpression of cyclins A and E in many human cancers 12 . Thus, CDKs are considered critical targets for the development of novel anticancer drugs 9,10 .
The aim of this research was based on the previously reported work with a rationale for anticancer drug discovery research, to design and synthesise new pyrazolo [3,4-d]pyrimidine and its glycosyl amino derivatives as well. In addition to the thioxo-pyrazolo [4,3-e] [1,2,4]triazolo [1,5-c]pyrimidine compounds and the thioglycoside derivatives, based on pyrazolo [3,4-d]pyrimidine scaffold to develop novel CDK2 inhibitors 34,35 . The new derivatives were evaluated for their in vitro anti-proliferative activity against three cell lines: breast cancer (MCF-7), hepatocellular carcinoma (HepG-2), and colorectal carcinoma (HCT-116) cell lines. The potential inhibition of the most potent promising compounds on CDK2/Cyclin A was also tested. Also, molecular docking studies were applied to investigate the binding mode of the promising compounds by calculating their binding energies and visualising their orientations with respect to the active site of CDK-2 protein compared to the Roscovitine ligand. The computational results were in agreement with the reported observations.
The two essential hydrogen bonds in the phosphate binding region with Leu83,1 HBD by -NH of the 6-substituted amino moiety, 1 HBA through N7 in Roscovitine, were replaced by N arylglycyl moiety in all the presented compounds.
The N5 pyrazolopyrimidine substitution by amino group (in compound 4) or N-glycosyl (in compounds 5 and 6) replaced the N butanol at position 2 of Roscovitine.
The pyrazolopyrimidine hydrophobic binding with Ala31, Val18, and Leu134 was maintained as in lead compound and the triazole ring fusion was considered in compounds 7-15 with hydrophobic binding with Val18.
In compounds 7-13, an additional hydrogen bonding by the thio group was achieved as in compound 8 with Asp145. And a hydrogen bond for compound 13 via the acetyloxy moiety with Thr14 was observed.
The hydrophobic binding with Ile10 by the benzyl group in Roscovitine and para bromophenyl group in compound VII was maintained by the isosteric replacement by the para fluorophenyl group in all the new compounds. In addition to the hydrogen binding via the para fluoro group with Lys89 in compounds 8, 9, 11, and 13. As in reported compound VII where it was published that substitution of the phenyl ring with halogen atom is preferable to the un-substituted ring for its cytotoxic effect, with either bromo, fluoro, or chloro group 25 . While a halogen hydrogen bond formation by compound 11 with Glu8 and compounds 14 and 15 with Asp145 was observed too.
The thioglycoside group at position 2 in compounds 14 and 15 showed also additional Carbon-Hydrogen binding with Glu8.

Chemistry
5-Amino-1-(4-fluorophenyl)glycyl)-1H-pyrazole-4-carbonitrile 2, was prepared by the reaction of 2-((4-fluorophenyl)amino)acetohydrazide 1 with ethoxymethylene malononitrile in ethanol under reflux. The isolated brown solid 2 was then treated with triethoxyorthoformate in acetic anhydride to afford ethyl-N-(4-cyano-1-((4fluorophenyl)glycyl)-1H-pyrazol-5-yl)formimidate 3. The key starting material pyrazolopyrimidine 4 was obtained by treatment of the formimidate derivative 3 with hydrazine hydrate in ethanol. Subsequent reaction of pyrazolopyrimidine 4 with sugar aldoses namely; D-glucose and D-xylose, in the presence of a catalytic amount of acetic acid, afforded the amino-sugar products 5 and 6 respectively (Scheme 1). The structures of this set of novel amino sugars were confirmed by their spectral and elemental analysis data (see Experimental Section). 1 H NMR spectra of the aminosugar compounds 5 and 6 showed a signal attributed to H-1 at 5.13 and 5.10 ppm confirming the sp 3 hybridisation of its corresponding carbon (C-1) and the cyclic form of the sugar moiety which agrees well with the reported results for such type of compounds following such mode of preparation 36 .
Adding a carbon disulphide to pyrazolopyrimidine 4 in the presence of potassium hydroxide in ethanol afforded the corresponding triazolomercapto derivative 7. The 1 H NMR spectra of compound 7 revealed the disappearance of the NH 2 and NH signals and instead another broad singlet at 11.66 ppm for the NH in the thione-thiol system appeared. The latter was utilised for the preparation of a series of acyclic nucleoside analogs via reaction with acyclic oxygenated halides. Thus, reaction with chloroacetonitrile, 2-chloro-1,1-dimethoxyethane, 2-chloropropane-1,2-diol, and 2-chloroethan-2-ol by stirring or by reflux in ethanol afforded the thioderivatives 8-10 and 12, respectively in good yields. In addition, the hydroxyl compounds 10 and 12 were acetylated with acetic anhydride to yield the corresponding O-acetylated acyclic analogs 11 and 13, respectively (Scheme 2). The 1 H NMR spectra of the resulted acetylated derivatives showed the signals of the acetyl-methyl groups in addition to the disappearance of the hydroxyl signals which was also confirmed by their IR spectra. The latter showed the carbonyl bands of the ester groups and the disappearance of the characteristic bands of the hydroxyl groups (see Experimental Section).

CDK2/cyclin A2 inhibitory activity
All the synthesised compounds 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 were tested for their in vitro CDK2/cyclin A2 assays using Promega Kinase-Glo Plus luminescence kinase assay 39 . The assay depends on kinase reaction and ADP measurement, ADP is first transferred to ATP and then converted to light. The produced luminescent signal is directly proportional to the amount of ATP and inversely proportional to the kinase activity. The results of the enzymatic inhibitory activity of the tested compounds against CDK2/cyclin A2 were presented in Table 2. All the tested compounds showed a good inhibitory effect with IC 50 values ranging from 0.061 ± 0.003 to 2.050 ± 0.05 mM compared to Sorafenib IC 50 : 0.184 ± 0.01 mM and Dinaciclib IC 50 : 0.029 ± 0.002. Results revealed that compounds 15, 11, 14, and 13 showed the most powerful inhibitory activity with IC 50 values of 0.061 ± 0.003, 0.089 ± 0.005, 0.118 ± 0.007, and 0.13 ± 0.007 mM, respectively, compared to control drug Sorafenib and/or Dinaciclib. Where compound 15 showed the superior activity ( Figure 5).

In vitro cytotoxicity against normal cells WI-38
The cytotoxic effect of the most active compound 15 against WI-38 normal lung cells was in vitro assessed (Table 3) against Staurosporine as a reference standard. Compound 15 showed low cytotoxicity against normal cells compared to the reference standard with IC 50 values of 69.60 ± 2.47 and 57.57 ± 1.42 mM for the most active compound 15 and reference standard, respectively. This data indicated that the synthesised compounds, more specifically that compound 15 exhibit low cytotoxicity against normal cells than cancer cell lines.

Flow cytometry cell cycle analysis
Compound 15 showed the most potent enzymatic inhibitory activity against CDK2/cyclin A2 and potent anti-proliferative activity against the three selected cancer cell lines. Furthermore, compound 15 was selected for flow cytometry assay to evaluate cell cycle analysis against HCT-116 cells. The cell cycle analysis is used to investigate the mechanism of action of the new compounds 40 . Table 4 presents the results showing Pre-G1 and cell growth arrest in the S phase. Results were compared to normal control cells, in Dip Pre-G1with an increase from 1.85 to 32.73% for compound 15 ( Figure 6).

Flow cytometric analysis of apoptosis
Flow cytometric analysis of apoptosis was performed on compound 15 to determine the potentiality of apoptosis induction against the HCT-116 cell line 41 . Table 5 and Figure 7 showed that compound 15 induced apoptosis by 32.73% (9.26 and 19.12 at early and late apoptosis, respectively), which was 17.69 times more than the standard control (1.85%).

Molecular docking
A molecular docking study using the C-Docker protocol in Discovery Studio 4.0 Software was performed on all the synthesised compounds. The compounds were prepared and then docked into the binding site of the CDK2 enzyme. An analysis study was applied to determine the binding modes of the designed compounds and to interpret the biological results. Hence, more explanation about the binding poses and interactions with the key amino acids in the binding site was noticed. X-ray crystallographic structure of CDK2 being complexed with Roscovitine (PDB ID: 2A4L) was downloaded from Protein Data Bank 9,12 . It was known from the previous literature that there are two essential hydrogen bonds with Leu83 at the active site besides hydrophobic interactions 9,12 . Validation was confirmed by re-docking of Roscovitine in the active site of CDK2 with RMSD value ¼ 0.5 A . Superimposition of co-crystallised ligand and ligand after docking indicates the perfect superimposition at the binding site ( Figure  8). The best pose out of ten for each compound was selected showing maximum similarity to the binding mode compared to the ligand. The presented docking study showed comparable binding modes to the lead compound for the docked molecules.
The interaction energy with the essential amino acids of the most biologically active synthesised compounds at the active site is demonstrated in Figure 9(A,B). Results showed comparable binding interaction energy to the lead compound Roscovitine (E ¼ À55.75 kcal/mol), ranging from 60.89 to 50.85 kcal/mol). Compound 15 revealed a binding score ¼ À59.85 kcal/mol with a similar binding mode.
All the docked molecules revealed a hydrogen bond with Leu83. Most of the compounds formed 1 HBD through -NH of glycyl group where binding distance range was observed to be within (2.02-2.10 Å) and 1 HBA through the oxygen atom of the carbonyl group of the glycyl moiety with a binding distance range of (2.20-2.74 Å) ( Figure 9(A,B)). Compounds 4,5,6,10, and 12 showed different binding modes with either two hydrogen bonds with Leu83 as in compounds 4 and 6, or only one hydrogen bond as in compounds 5, 10, and 12. The sugar moiety in compounds 5 and 6 was responsible for the HBD interaction with Leu83 (Supplementary Material).
All the tested compounds showed binding via the parafluorophenyl group except compounds 4, 5, 7, 8, and 10. Either by a hydrogen bond with Lys89 as in compounds 9, 11, and 13 or by halogen hydrogen binding with Asp145 as in compounds 14 and 15 or extra binding with Glu81 as in compound 11. Compounds 6 and 12 undergo one halogen hydrogen bond with Glu81 and His84 via the parafluoro group, respectively. Compounds 7 and 11 showed only 1 1HBD with Leu83 (compound 11 showed better interaction energy after docking than compound 7). Also, compound 11 (the diacetoxy derivative) and compound 13 (the monoacetoxy derivative) showed an extra hydrogen bond with Thr14 via the acetyloxy group. Compounds 14 and 15 showed additional HBD with Glu81 via the -NH group of the glycyl moiety.

In silico predictive ADMET study
The design and development of novel drug molecules need early investigation of pharmacokinetic properties including absorption, distribution, metabolism, and excretion (ADME), at a stage where access to the physical samples is limited 41 . It was observed that the limited pharmacokinetic or toxicity profile of new drug-like molecules had a great influence on the low percentage of the drugs that could reach to clinical development stage to be ready to reach the market 42 .
Several ways for in silico predictive ADMET studies have been developed. Including web-based platforms, such as ADMETlab, Pro Tox-II, and SwissADME to predict ADMET properties 42 .  Here in this research ADMET study was performed mainly using Discovery Studio 4.0 Software for all the synthesised compounds.
Also, the ADMETlab web tool was used to confirm results on the highly potent molecules with promising molecular docking results only to assure the early investigation of their pharmacokinetic properties and compare to Roscovitine as a control drug 43 .
The ADMET study using Discovery Studio 4.0 Software is mainly concerned with the chemical structure of the molecule, it includes several parameters calculation; Blood Brain Barrier Level, Absorption level, Atom-based Log P98 (A LogP 98), 2D polar surface area (ADMET 2D PSA), Cytochrome P450 2D6 (CYP2D6), Hepatotoxicity Probability, Aqueous solubility level, and Plasma protein binding logarithmic level (PPB Level).
Results shown in Table 6 reveals that all the compounds had BBB level of 3 and 4. So, they are not able to pass the blood-brain barrier. Most of the compounds had absorption levels ¼ 0 or 1, thus estimated to have good to moderate human intestinal absorption, whilst only compounds 4, 5, 14, and 15 showed low absorption. All the compounds showed ADME aqueous solubility levels between 2 and 3 which indicates good aqueous solubility except compounds 7, 8, and 9 which showed low aqueous solubility. The key property (PSA) was linked to drug bioavailability. Therefore, molecules that are passively absorbed and PSA < 140 are thought to have lower bioavailability. Most of the synthesised compounds were predicted to present good passive oral absorption except compounds 4, 5, 14, and 15 which showed good bioavailability results with a PSA range of 168.67-199.705. Compound 4 showed no hepatotoxicity. Also, all the compounds are considered non-inhibitors to Cytochrome P450 2D6 (CYP2D6) and are . The IC 50 of tested compounds against MCF-7, HepG-2, and HCT-116 cancer cell lines. Data expressed as mean ± SD from three independent repeats (n ¼ 3). Ã Significant from sorafenib at p < 0.05. 0.061 ± 0.003 Ã Ã Significant from sorafenib at p < 0.05. # Significant from dinaciclib at p < 0.05. Figure 5. Inhibitory activity of most potent compounds on CDK/Cyclin A2. Ã Significant from sorafenib at P < 0.05, # Significant from dinaciclib at P < 0.05. poorly bounded to plasma proteins except compounds 2 and 3. (Table 6). Also, the ADMETlab web tool was used to predict the pharmacokinetics of the most promising synthesised compounds. The results were compared to the control drug (Roscovitine) 43 .
Also, Toxicity profiles, such as H-HT (Human Hepatotoxicity) and CYP2D6 were predicted using ADMETlab.
Lipinski's rule of five is an essential tool for rational drug design. The low permeability or poor absorption for tested compounds was related to a disturbance in one of Lipinski's rules of five 42 . Molecular weight, LogP, and the number of hydrogen bond acceptors (NHBAs) should lie within the values of <500, 3, and 10,   respectively. Hereby, all the compounds except compound 8 had NHBAs of 10 or more. Compounds 11, 14, and 15 showed low aqueous solubility and poor absorption. Where it was observed from log S results that all the tested drugs showed non-aqueous solubility values in the range of À5.401 to À4.731 compared to the control drug (optimal: À4-0.5 log mol/L). While, the logP results showed optimal lipophilicity values (Optimal: 0 < LogP < 3) ( Table 7).
It is found that the values of Caco-2-Permeability were in the range from À4.647 to À5.49 cm/s, suggesting to have optimal permeability except for compounds 14 and 15 which showed a À5.49 cm/s result. (optimal permeability more than À5.15 cm/s) The predicted HIA data of the tested compounds are represented categorically as 1, where Category 1: HIAþ (HIA ! 30%); Category 0: HIAÀ (HIA < 30%). The output value indicated a high HIA probability for the tested compounds except for compounds 14, 15, and the control drug. Most of the compounds showed good bioavailability, Compounds 14 and 15 showed 20-30% bioavailability.

Distribution.
To study the distribution of drug candidates some parameters were considered as Plasma Protein Binding (PPB), Blood Brain Barrier (BBB) permeability, and Volume of distribution (VD) ( Table 7). The PPB was predicted to exceed 90% to bind significantly to blood proteins compared to the control drug. While BBB results showed that all the tested compounds have no BBB penetration effect except compounds 14 and 15.  The volume of distribution (VD) in L/Kg was predicted to be optimal when it lies in the range of: 0.04-20 L/kg. So, all compounds showed good VD with moderate clearance results (except compounds 11, 14, and 15 with low clearance results). The hydrophobic character of the compounds was contributed to good values of VD in tissues which confirmed absorption and distribution parameters. Also, all the tested compounds were non-inhibitors to CYP2D6 and showed no hepatotoxicity.
The most potent anti-proliferative compound 15, as compared to the control drug Roscovitine. Where related pharmacokinetic parameters showed the probability of the same physicochemical characteristics and related behaviour inside the human body with similar biological effects. Figure 10 showed related pharmacokinetic properties showing differences mainly in the number of nHD due to differences in their chemical structure.

Structure-activity relationship
The structure-activity relationship of the novel compounds was studied based on their biological evaluation in addition to the molecular studies results. Where in the pyrazolo [3,4-d]pyrimidine scaffold in compounds (4-6), it was observed that the N5 pyrazolopyrimidine D-Xylose substitution showed potent activity against HCT-116 cell lines and moderate activity against HepG-2 cell lines compared to D-Glucose derivative or the unsubstituted derivative that was inactive against the three cell lines (Figure 11).
While after the triazole ring fusion in pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin scaffold (7)(8)(9)(10)(11)(12)(13)(14)(15), the in vitro anti-proliferative activity results revealed that compounds 14 and 15 showed the best cytotoxic results that might be related to the thioglycosidic substitution on triazole ring. The thioalkyl substituents as the monoacetoxy alkyl group in compound 13 and diacetoxy alkyl in 11 showed the most potent cytotoxic results against HCT-116 cell lines with good activity against MCF-7 and moderate activity against HepG-2cell lines compared to other derivatives. While the dihydroxy alkyl derivative 10 showed only potent activity against MCF-7 cell lines. The acetonitrile derivative 8 showed potent activity against HepG-2cell lines, compared to the dimethoxy alkyl substitution in compound 9 with no activity against HepG-2cell lines. The monohydroxy alkyl derivative 12 was inactive against the three cell lines. While the thioxo derivative 7 showed only moderate activity against HepG-2cell line ( Figure 11). Figure 11 summarised the SAR investigation of the newly synthesised compounds after their in vitro and in silico evaluation. A molecular docking study revealed that all the potent antiproliferative tested compounds were of comparable binding mode to that of the ligand. In silico ADMET studies and drug-likeness showed good pharmacokinetic properties of the new potent compounds. This helped in the prediction of structure requirements necessary for the observed antitumor activity.

Chemistry
All melting points were measured using a Reichert Thermovar apparatus and are uncorrected. The IR spectra were recorded on a Perkin-Elmer model 1720 FTIR spectrometer for KBr disc. Routine NMR measurements were made on a JEOL ECA-500 II spectrometer with operating frequencies of 400 MHz for 1 H. Chemical shifts were reported in d scale (ppm) relative to TMS as a reference standard and the coupling constants J values are given in Hz. 13 C NMR were recorded at 125 M Hz. The progress of the reactions was monitored by TLC using aluminium silica gel plates 60 F245. Spectral measurements and Elemental analyses were performed at the Micro-analytical centre at the Faculty of Science, Mansoura University, Mansoura. Compound 1 was synthesised according to a reported procedure 44 .

General procedure a.
A mixture of aminopyrazolopyrimidine compound 4 (3 g, 10 mmol), the aldose sugar namely; D-glucose or D-xylose [10 mmol suspended in water (1 ml)] and glacial acetic acid (0.5 ml) in ethanol (25 ml) was allowed to be refluxed for 6 h. Half of the amount of the solvent was evaporated under reduced pressure and the resulting solution was cooled to room temperature and then left to stand in a refrigerator at 5-8 C overnight. The resulting precipitated solid was filtered, dried, and recrystallized by methanol to afford the N-glycosyl compounds 5 or 6, respectively.  (25 ml) were heated under reflux with stirring for 10 h. The solvent was evaporated under reduced pressure and ice-cold water was added to the remaining residue with continuous stirring to afford a precipitate which was filtered, dried, and recrystallized from ethanol-water mixture 1:1 to afford the dimethoxy product 9 and 10, respectively.   (13) To a mixture of compound 12 (3.8 g, 10 mmol) and acetic anhydride (5 ml) in pyridine (15 ml To a suspension of potassium hydroxide (0.67 g, 10 mmol) in water (1 ml), a solution of pyrazolopyrimidine derivative 7 (3.4 g, 10 mmol) in acetone (20 ml) was added with continuous stirring for about 15 min, then a solution of the glycosyl bromide namely; 2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl bromide or 2,3,4,6-tetra-O-acetyl-a-D-galactopyranosyl bromide (12 mmol) in acetone (10 ml) was added portion-wise and the resulting mixture was stirred at room temperature for 5 h. The solvent was evaporated under reduced pressure and the residue was washed with cold water (15 ml) to remove any remained inorganic substance. Extraction of the product with ethyl acetate (50 ml) followed by removal of the solvent afforded a solid material which was treated with petroleum ether/diethyl ether mixture 3:1 (30 ml) with stirring for 15 min for further purification. The formed solid product was filtered and dried to give compounds 14 or 15, respectively.  37,38 . Briefly, 5000-10,000 cells per well were plated in a 96-well plate and allowed for growth for 24 h, then treated with the media of increased concentrations of tested compounds in 100 ml amount, complete growth medium was further mixed with 100 ml for each compound per well for 48 h before the assay. Then, the media were withdrawn, and 100 ml of MTT was added to each well, which was incubated for 4 h. The formed formazan crystals were solubilised by the addition of 100 ml of dimethyl sulfoxide (DMSO) solution. The cell viability was evaluated by measuring the optical density (OD) per well of the developed purple colour using spectrophotometric determination at 570 nm using an ELISA microplate reader (Epoc-2 C micro-plate reader, Bio Tek, VT, USA). The optical density of the produced colour was measured at 570 nm. The IC 50 values (the required concentration to inhibit cell viability by 50%) were calculated. The data was expressed as a percentage of control cells (100 percent of cell viability).

CDK2/cyclin A2 assay
The In vitro assay of CDK2/cyclin A2 protein kinase was carried out on all the synthesised compounds 4, 5, 6,7,8,9,10,11,12,13,14, and 15. The assay was proceeded in Egypt applying Kinase-Glo Plus luminescence kinase Assay kit (Promega) 39 . The Protocol steps were applied by enzyme and substrate dilution. Where, 1 ml of inhibitor, 5% DMSO, 2 ml of the enzyme, and 2 ml of substrate/ATP mix were added after ATP and inhibitors were diluted in Kinase Buffer. Incubation for 10 min at room temperature was allowed, then 5 ml of Reagent ADP-Glo TM was added and incubated for another 40 min. After that, an amount equivalent to 10 ml of Kinase Detection Reagent was added to be incubated at room temperature for another 30 min. Luminescence was (where Integration time range 0.5-1 s). The luminescent signal is in direct relation to the present quantity of ATP and in inverse relation to the activity of the kinase enzyme.

Flow cytometry cell cycle analysis
The cell cycle analysis protocol was performed on compound 14 on HCT-116 cells. The test is based on the content of DNA measurement via staining using propidium iodide. Cells were first washed in PBS before being kept at 4 C for 3 min through the dropwise using vortex addition of cold 70% ethanol, this is to avoid cell clumping and to ensure fixation as well. Then 50 ml from a stock of 100 mg/ml ribonuclease was added to selectively stain only DNA. Finally, an amount of 200 ml from a stock solution of 50 mg/ml of propidium iodide was added 40 .

Flow cytometric analysis of apoptosis
Annexin V-FITC-apoptosis detection kit (PN IM3546) was used to detect apoptosis in treated cells. Flow cytometric analysis was further performed according to manufacturer protocol. Where HCT-116 cells were allowed to grow in a 25 cm 3 flask until 70-80% confluence. Then HCT-116 cells were treated with compound 15 for 48 h followed by a wash in PBS and suspended in 1Â binding buffer. One mL of annexin V-FITC solution together with 5 mL of dissolved PI were added to 100 mL of cell suspensions, then incubated for 15 min in the dark. An aliquot of 400 mL of icecold 1Â binding buffer was gently added and mixed. The flow cytometric analysis method for the determination of the percentage of apoptotic cells was performed on a COULTER V R EPICS V R XL TM Flow Cytometer (USA) 41 .

Molecular modeling studies
A molecular docking study using CDOCKER protocol in Discovery Studio 4.0 Software was carried out. The targeted compounds were docked into the CDK2 active site. The X-ray crystallographic structure of CDk2 complexed with Roscovitine (PDB ID: 2A4L) was downloaded from PDB 9,12 . The binding mode of the designed compounds was studied to explain their biological results and to detect the essential hydrogen bonding with Leu83. Where the best pose out of ten for each compound was selected compared to the ligand binding mode. In silico ADMET studies using Discovery Studio 4.0 Software and ADMETlab web tool were carried out to predict the pharmacokinetic properties of the targeted compounds 43 , which helped in structure requirements prediction for the observed antitumor activity.

Data availability statement
Supporting data is supplied with the manuscript.