Discovery of New Pyrazolopyridine, Furopyridine, and Pyridine Derivatives as CDK2 Inhibitors: Design, Synthesis, Docking Studies, and Anti-Proliferative Activity

New pyridine, pyrazoloyridine, and furopyridine derivatives substituted with naphthyl and thienyl moieties were designed and synthesized starting from 6-(naphthalen-2-yl)-2-oxo-4-(thiophen-2-yl)-1,2-dihydropyridine-3-carbonitrile (1). The chloro, methoxy, cholroacetoxy, imidazolyl, azide, and arylamino derivatives were prepared to obtain the pyridine-−C2 functionalized derivatives. The derived pyrazolpyridine-N-glycosides were synthesized via heterocyclization of the C2-thioxopyridine derivative followed by glycosylation using glucose and galactose. The furopyridine derivative 14 and the tricyclic pyrido[3′,2′:4,5]furo[3,2-d]pyrimidine 15 were prepared via heterocyclization of the ester derivative followed by a reaction with formamide. The newly synthesized compounds were evaluated for their ability to in vitro inhibit the CDK2 enzyme. In addition, the cytotoxicity of the compounds was tested against four different human cancer cell lines (HCT-116, MCF-7, HepG2, and A549). The CDK2/cyclin A2 enzyme inhibitory results revealed that pyridone 1, 2-chloro-6-(naphthalen-2-yl)-4-(thiophen-2-yl)nicotinonitrile (4), 6-(naphthalen-2-yl)-4-(thiophen-2-yl)-1H-pyrazolo[3,4-b]pyridin-3-amine (8), S-(3-cyano-6-(naphthaen-2-yl)-4-(thiophen-2-yl)pyridin-2-yl) 2-chloroethanethioate (11), and ethyl 3-amino-6-(naphthalen-2-yl)-4-(thiophen-2-yl)furo[2,3-b]pyridine-2-carboxylate (14) are among the most active inhibitors with IC50 values of 0.57, 0.24, 0.65, 0.50, and 0.93 µM, respectively, compared to roscovitine (IC50 0.394 μM). Most compounds showed significant inhibition on different human cancer cell lines (HCT-116, MCF-7, HepG2, and A549) with IC50 ranges of 31.3–49.0, 19.3–55.5, 22.7–44.8, and 36.8–70.7 μM, respectively compared to doxorubicin (IC50 40.0, 64.8, 24.7 and 58.1 µM, respectively). Furthermore, a molecular docking study suggests that most of the target compounds have a similar binding mode as a reference compound in the active site of the CDK2 enzyme. The structural requirements controlling the CDK2 inhibitory activity were determined through the generation of a statistically significant 2D-QSAR model.


Introduction
The cyclin-dependent kinases (CDK) (a serine/threonine kinase family) [1] are responsible for the initiation and the succession of each cell cycle phase; CDK2 activity is required for progression through G1 to the S phase [2]. The activity of members of the CDK family is corrupted in many tumor cells where they are essential for the phosphorylation of key components for cell proliferation [3,4]. CDK2 has an important catalytic role in the complex of cyclin-dependent protein kinase; its activity is essential for cell cycle progress [5]. The cyclin protein family modulates CDK activities throughout the cell cycle where specific activating phosphorylation for CDK apoenzymes is required through CDK-activating kinase, which is a CDK complex, and also, complex formation with cyclins is needed for optimum kinase activity [5,6]. CDK2 is associated mainly with the regulatory subunits including either cyclin A or E with overexpression in human cancer as in ovarian, breast, endometrial, lung and thyroid carcinomas, osteosarcoma, and melanoma [7]. The activation of the CDK2 through cyclin A is claimed to be an essential step through the progression from the S phase, and this makes CDK2 the principal target for most inhibitory drugs [3][4][5][6].
Heterocyclic ring systems have emerged as powerful scaffolds for many biological evaluations [8] and play an important role in the design and discovery of new physiological/pharmacologically active molecules [9]. The incorporation of more than one heterocyclic core in one hybrid structure, known as molecular hybridization, gained considerable interest in the design and synthesis of bioactive compounds [10][11][12]. The pyridine core is found in a variety of biologically active compounds possessing a broad spectrum of bioactivities [13,14]. The pyrazolo [3,4-b]pyridine framework is a promising heterocyclic scaffold that has gained renewed interest displaying a broad spectrum of biological activities such as antitumor inhibiting CDK2 [15][16][17][18][19] and glycogen synthase kinase-3 (GSK-3) inhibitors [20]. In addition, pyrazolo [3,4-b]pyridine derivatives represent important building blocks in both natural and synthetic bioactive compounds [21]. Moreover, fused heterocyclic-containing pyrazolopyridine systems have been described as associated with several biological and medicinal activities [22][23][24][25]. Oxygen heterocycles exhibit diverse biological and pharmacological activities due to the similarities with many natural and synthetic molecules [26]. Figure 1 illustrates some important pyrazolopyridine-based compounds with potent activities for CDK2 enzyme [27,28] and its bicyclic congener Roscovitine, which is a CDK2 inhibitor undergoing clinical trials to treat cancers [29].

Chemistry
The ease synthesis and high biological activity of the starting 6-(naphthalen-2-yl)-2oxo-4-(thiophen-2-yl)-1,2-dihydropyridine-3-carbonitrile (1) prompted us to prepare some of its derivatives starting from available and inexpensive compounds. The reaction of compound 1 with chloroacetyl choloride yielded 3-cyano-6-(naphthalen-2-yl)-4-(thiophen-2-yl)pyridin-2-yl-2-chloroacetate (2) via substitution reaction in 84% yield. The structure of compound 2 was elucidated based on elemental and spectral analyses. The IR spectrum of 2 revealed strong absorption bands at ν 2216 and 1697 cm −1 for CN and CO groups, respectively. The 1 H-NMR of the same compound showed signals at δ 4.40 ppm referring to CH 2 Cl in addition to the signals of the aromatic protons. The formation of imidazolthione 3 was obtained through the heterocyclization of compound 2 by reaction with thiourea in DMF. Its IR spectrum showed the disappearance of the absorption band at ν 1697 cm −1 of the carbonyl group and the appearance of the characteristic bands at ν 3424 and 1260 cm −1 due to NH and C=S groups, respectively. The 1 H-NMR spectrum of compound 3 exhibited very distinct singlet signals resonating at δ 4.13 (imidazole-H) and δ 12.98 (exchangeable NH) confirmed the successful formation of 3. 13 C-NMR spectrum of compound 3 showed signals at δ 49.6, 168.1, and 188.4 ppm for imidazoles-C and C=S groups, respectively beside the aromatic carbons at their specific regions (see supplementary materials).
On the other hand, pyridine-2(1H) thione derivative 7 [45] was reacted with hydrazine hydrate in ethanol under reflux conditions to afford 3-amino-1H-pyrazolo [3,4-b]pyridine derivatives (8) in a good yield. The IR spectrum of 8 revealed bands at ν 3375-3228 cm −1 referred to NH 2 and NH groups. The 1 H-NMR exhibited a singlet signal at δ 6.45 ppm referring to the exchangeable NH 2 protons and also a singlet signal at δ 11.65 ppm corresponding to the exchangeable NH proton. 13 C-NMR spectra agreed with such investigation and confirmed their structures, which showed that C-pyrazoles have been revealed at δ 90.2, 151.3, and 152.2 ppm.
Compound 8 was further reacted with the monosaccharides such as d-glucose or d-galactose as six-carbon sugars in ethanol, and a few drops of glacial acetic acid afforded compounds 9 and 10, respectively, which have been shown to incorporate sugar moieties in the cyclic form. Their IR spectra showed strong and broad absorption bands characterizing the polyhydroxy chain and NH groups in the range ν 3520-3390 cm −1 . The 1 H-NMR spectra of the same compounds showed signals for NH (D 2 O exchangeable) and OH groups at their specific regions (see supplementary materials). On the other hand, pyridine-2(1H) thione derivative 7 [45] was reacted with hydrazine hydrate in ethanol under reflux conditions to afford 3-amino-1H-pyrazolo [3,4-b]pyridine derivatives (8) in a good yield. The IR spectrum of 8 revealed bands at ν 3375-3228 cm −1 referred to NH2 and NH groups. The 1 H NMR exhibited a singlet signal at δ 6.45 ppm referring to the exchangeable NH2 protons and also a singlet signal at δ 11.65 ppm corresponding to the exchangeable NH proton. 13 C NMR spectra agreed with such investigation and confirmed their structures, which showed that C-pyrazoles have been revealed at δ 90.2, 151.3, and 152.2 ppm.
Compound 8 was further reacted with the monosaccharides such as d-glucose or dgalactose as six-carbon sugars in ethanol, and a few drops of glacial acetic acid afforded compounds 9 and 10, respectively, which have been shown to incorporate sugar moieties Scheme 1. Synthesis of substituted pyridine derivatives (2,(4)(5)(6) and (imidazolyl)oxynicotinonitrile derivatives 3.
The reaction proceeds according to the proposed mechanism [43]. In addition, compound 11 was synthesized by stirring compound 7 with chloroacetyl chloride in acetone and anhydrous potassium carbonate at room temperature. The IR spectrum of 11 showed the appearance of CH aliphatic, C=O and CN, bands at ν 2921, 1698, and 1590 cm −1 , respectively. The 1 H-NMR spectrum of 11 showed a sharp singlet signal at δ 4.30 ppm belonging to the CH 2 Cl protons beside the singlet signal at δ 8.19 ppm for pyridine proton, thus confirming the formation of compound 11. The azido compound 12 was produced when compound 11 was stirred at room temperature with sodium azide in N, N-dimethyl formamide. The IR spectrum of compound 12 revealed strong absorption bands assigning the N 3 at ν 2366 cm −1 . The 1 H-NMR spectra of that compound showed a characteristic singlet signal for CH 2 N 3 at δ 2.42 ppm in addition to aromatic protons. 13 C-NMR spectra of compound 12 revealed signals at δ 65.1 and 191.0 ppm attributed to CN 3 and CO groups respectively beside the aromatic carbons at their specific region (Scheme 2).  The ester 13 was refluxed with sodium ethoxide to give ethyl 3-amino-6-(naphthalen-2-yl)-4-(thiophen-2-yl)furo[2,3-b]pyridine-2-carboxylate (14). This compound might be subjected to further structural modification. The formation of the furan ring with two functionalities (the amino and the carboethoxy groups) resulted, whose further modifications could provide the opportunity for new cyclization processes. The IR spectrum of 14 showed absorptions bands at ν 3437 cm −1 referring to the NH 2 group and a strong absorption band at ν 1739 cm −1 referring to the ester carbonyl group. The 1 H-NMR spectrum exhibited a singlet signal of the amino group at δ 5.61 ppm, in addition to triplet and quartet signals at δ 1.2 and 4.3 ppm for CH 3 and CH 2 groups. Compound 14 was condensed with electrophiles namely, formamide, to afford the pyridofuropyrimidine 15. The chemical structure of 15 was elucidated by its correct elemental analysis and spectral data. Its IR spectrum showed absorption bands at ν 3419 and 1662 cm −1 for the imino (NH) and the amidic carbonyl group (C=O). The 1 H-NMR spectrum revealed singlet signals at δ 8.26 and 8.34 ppm for pyridine and pyrimidine protons, multiplet signals in the range of δ 7.24-8.11 ppm for aryl protons, and a single signal at δ 11.18 corresponding to the imine proton NH (D 2 O exchangeable) (Scheme 3).

CDK2/Cyclin A2 Activity
The in vitro CDK2/cyclin A2 assays of the newly synthesized compounds 1-5, 7, 8, 11, 13, and 14 were carried out using Promega Kinase-Glo Plus luminescence kinase kit. It measures kinase activity by quantitating the amount of ATP remaining in solution following a kinase reaction. The luminescent signal from the assay is correlated with the amount of ATP present and is inversely correlated with the amount of kinase activity. The percent inhibition of the tested compounds against CDK2/cyclin A2 protein kinase was compared to reference kinase inhibitor roscovitine at a single concentration of 10 µM. The computer software Graphpad Prism was used for analyzing the luminescence data. The difference between luminescence intensities in the absence of CDK2/cyclin A2 protein kinase (Lut) and in the presence of kinase (Luc) was defined as 100% activity (Lut − Luc). Using luminescence signal (Lu) in the presence of the compound, the percentage of activity was calculated as: % activity = {(Lut − Lu)/(Lut − Luc)} × 100%, where Lu = the luminescence intensity in the presence of the compound. The percentage of inhibition was calculated as: % inhibition = 100 (%) − % activities [46].
The IC50 results of the target compounds are presented in Table 1. All the new compounds revealed a potent inhibitory effect against CDK2/cyclin A2 protein kinase with an IC50 values ranging from 3.52 to 0.24 µM compared to roscovitine reference compound of IC50 0.39µM. Amongst them, compound 4 revealed the highest IC50 of 0.24 µM more potent than roscovitine, while compounds 1, 8, 11, and 14 showed good inhibitory activities comparable to the reference compound with IC50 0.57, 0.65, 0.50, and 0.93 µM, respectively. Moreover compounds 2, 3, 5, 7, and 13 revealed promising activity with IC50 values ranging 1.01-4.45 µM.

CDK2/Cyclin A2 Activity
The in vitro CDK2/cyclin A2 assays of the newly synthesized compounds 1-5, 7, 8, 11, 13, and 14 were carried out using Promega Kinase-Glo Plus luminescence kinase kit. It measures kinase activity by quantitating the amount of ATP remaining in solution following a kinase reaction. The luminescent signal from the assay is correlated with the amount of ATP present and is inversely correlated with the amount of kinase activity. The percent inhibition of the tested compounds against CDK2/cyclin A2 protein kinase was compared to reference kinase inhibitor roscovitine at a single concentration of 10 µM. The computer software Graphpad Prism was used for analyzing the luminescence data. The difference between luminescence intensities in the absence of CDK2/cyclin A2 protein kinase (Lut) and in the presence of kinase (Luc) was defined as 100% activity (Lut − Luc). Using luminescence signal (Lu) in the presence of the compound, the percentage of activity was calculated as: % activity = {(Lut − Lu)/(Lut − Luc)} × 100%, where Lu = the luminescence intensity in the presence of the compound. The percentage of inhibition was calculated as: % inhibition = 100 (%) − % activities [46].
The IC 50 results of the target compounds are presented in Table 1. All the new compounds revealed a potent inhibitory effect against CDK2/cyclin A2 protein kinase with an IC 50 values ranging from 3.52 to 0.24 µM compared to roscovitine reference compound of IC 50 0.39µM. Amongst them, compound 4 revealed the highest IC 50 of 0.24 µM more potent than roscovitine, while compounds 1, 8, 11, and 14 showed good inhibitory activities comparable to the reference compound with IC 50 0.57, 0.65, 0.50, and 0.93 µM, respectively. Moreover compounds 2, 3, 5, 7, and 13 revealed promising activity with IC 50 values ranging 1.01-4.45 µM.

In Vitro Cytotoxicity Activity
In this study, ten compounds were examined in vitro for their activities against HCT-116, MCF-7, HepG2, and A549 human cancer cells using the MTT assay. The percentages of intact cells were calculated and compared to those of the control. The activities of these compounds against the four cell lines were compared to the activity of doxorubicin as well. All compounds suppressed the four human cells in a dose-dependent manner . In order to study the efficacy of the synthesized compounds, a comparison of the cytotoxic effect of each compound has been related to the cytotoxicity of the reference drug as follows.

In Vitro Cytotoxicity Activity
In this study, ten compounds were examined in vitro for their activities against HCT-116, MCF-7, HepG2, and A549 human cancer cells using the MTT assay. The percentages of intact cells were calculated and compared to those of the control. The activities of these compounds against the four cell lines were compared to the activity of doxorubicin as well. All compounds suppressed the four human cells in a dose-dependent manner . In order to study the efficacy of the synthesized compounds, a comparison of the cytotoxic effect of each compound has been related to the cytotoxicity of the reference drug as follows.
Meanwhile, only compound 7 has more potent anticancer activity against HepG2 with IC 50 22.7 µM, compared to that of the reference compound ( Figure 4 and Table 2).
For A549 human lung cancer cells, seven compounds (7, 8, 13, 5, 14, 11, and 3, respectively) have better anticancer activities; the rest of the compounds have insignificantly less anticancer activity compared to that of doxorubicin ( Figure 5 and Table 2).

Molecular Docking Study
The molecular simulation study was carried out through docking of the target compounds in the binding pocket of the CDK2 enzyme using C-Docker protocol in Discovery Studio 4.0 Software. In this work, molecular docking study and analysis of the binding modes for the designed compounds were achieved to interpret the biological results and to gain further insight into orientations and interactions with the key amino acids. It has been found that both the X-ray crystallographic enzyme (CDK2) substrate complex with roscovitine (PDB code 2A4L) [47] revealed the formation of the essential two H-bonds with Leu83.
Validation of the C-Docker protocol in this investigation was performed by re-docking of the co-crystallized lead compound in the CDK2 kinase active site. The calculated RMSD value (RMSD = 0.46 A • ) indicated the ability of the used docking protocol to retrieve valid docking poses. The docking of the target compounds under study, into the active site of the CDK2 enzyme, revealed comparable binding modes to the lead compound. The binding interactions of the docked compounds together with their binding energies and the key amino acids involved are presented in Table 3 and Figures 6-11.
By exploring the C-Docker interaction energy and the binding mode of the highly biologically active compounds 1, 4, 8, 11, and 14, it was found that compounds 1, 4, 11, and 14 conserved the formation of the two H bonds with Leu83 as the reference compound (roscovitine), while compound 8 showed one H bond with Leu83 and another additional two H bonds with Asp86 (Figures 7-11). These compounds (1, 4, 8, 11, and 14) exhibited high antitumor activity against cancer cell lines (HCT-116, MCF-7, HepG2, A549) with high inhibitory activities against the CDK2 enzyme. Whereas compounds 2, 3, 5, 7, and 13 showed low binding energy compared to that of the lead compound with the formation of only one H bond with Leu83 and compounds 2, 5, and 7 formed an additional H bond with Lys89. Additional interaction was found between most of the docked compounds and Lys89, Lys33, Phe82, and Ile10. In addition, an alignment study was performed for compound 11, which showed good alignment with approximately the same pharmacophoric elements of the reference compound (roscovitine) (Figure 12). The alignment study revealed that (i) the purine ring system of the compound roscovitine was perfectly aligned with the purine 3-cyano pyridine moiety of compound 11, (ii) a thiocarboxamide side chain superimposed with N-benzyl substituent of roscovitine, and (iii) additionally, thiophene moiety and naphtyl group were aligned with N-isoprpyl and N(1-hydroxy-but-2-yl) of roscovitine, respectively.
Correlation between the binding energy and the inhibitory activity of the investigated molecules showed that the most potent pyridine derivatives 4, 11, and 1 (IC 50 values of 0.24 ± 0.01, 0.50 ± 0.03, and 0.57 ± 0.03 µM, respectively) showed the highest binding energy ( Figure 13). Whereas compounds with low IC 50 values (high potent) revealed high binding energy with -ve (high docking score, more stable in binding site), while compounds with a high IC 50 (less potent) revealed low binding energy (less docking score). This consistent relation resulted in the successful designing of promising analogs with potent CDK2 inhibitory activity.      igure 7. Two-dimensional (2D) and 3D docking of compound 1 into the CDK2 active site. Amino acids with green color epresent the Van der Waals interaction; the purple amino acids represent the electrostatic interaction, while the blue mino acid represents the hydrophobic interaction.
igure 8. Docking of compound 4 into the CDK2 active site. Purple color represent carbon atoms, gray color represent ydrogen atom, blue color represent nitrogen atom, orange color represent sulfur atom and green color represent chlorine tom.

D-QSAR Studies
Two-dimensional (2D)-QSAR studies were undertaken utilizing Discovery Studio 4.0 Software employing nine bio-active compounds (1-5, 7, 11, 13, and 14) as a training set, which exhibit variable inhibitory properties against the CDK2 enzyme. The "Create Multiple Linear Regression Model" protocol was used to generate the 2D-QSAR model predicting the controlled descriptors as statistically significant. The two descriptors controlling the QSAR model are presented in the following equation. Figure 14

Validation of QSAR
Internal and external validation of the determined QSAR equations was performed. The internal validation was presented in Table 4 including determination of r2 (the coefficient of determination), r2 adj (adjusted for the number of terms in the model), r2 pred (the prediction), q2 (from a leave-1-out cross-validation), Friedman L.O.F. (the Friedman lack-of-fit score), and S.O.R. p-value (the p-value for significance of regression). The external validation was carried out utilizing compound 8 and two reported compounds V, VI with similar scaffold (Figure 15) [6,48]. The estimated activity for the external test set was very close to its observed activity (Table 5), that is, support the established QSAR model  5, 7, 11, 13, and 14) as a training set, which exhibit variable inhibitory properties against the CDK2 enzyme. The "Create Multiple Linear Regression Model" protocol was used to generate the 2D-QSAR model predicting the controlled descriptors as statistically significant. The two descriptors controlling the QSAR model are presented in the following equation. Figure 14 shows

Validation of QSAR
Internal and external validation of the determined QSAR equations was performed. The internal validation was presented in Table 4 including determination of r2 (the coefficient of determination), r2 adj (adjusted for the number of terms in the model), r2 pred (the prediction), q2 (from a leave-1-out cross-validation), Friedman L.O.F. (the Friedman lack-of-fit score), and S.O.R. p-value (the p-value for significance of regression). The external validation was carried out utilizing compound 8 and two reported compounds V, VI with similar scaffold (Figure 15) [6,48]. The estimated activity for the external test set was very close to its observed activity (Table 5), that is, support the established QSAR model in the present work. In addition, QSAR model_ Applicability showed that all properties and OPS components are within expected ranges, with Applicability MD value 2.66667, and the applicability ranging from 0.236031 to 4.45003 for each component of the training set.       Structure-activity relationships among the newly synthesized pyridine derivatives have been closely investigated. The presence of the thione group in compound 7 with thiophene moiety increases antitumor activity against HepG2 and A549. Further investigations of the biological results concluded that the furopyridine derivative as in compound 14 and incorporating the ester functionality showed superiority in activity over the pyridine derivatives against a colon cancer cell line.

Chemistry
All melting points were measured using a Reichert Thermovar apparatus and are uncorrected. Yields listed are of isolated compounds. The IR spectra were recorded on a Perkin-Elmer model 1720 FTIR spectrometer for KBr disc. 1 H and 13 C-NMR spectra were recorded on a Bruker AC-300 or DPX-300 spectrometer. Chemical shifts were reported in δ scale (ppm) relative to TMS as a reference standard and the coupling constants J values are given in Hz. Mass spectra were recorded on a GC/MS SHIMADZU spectrophotometer. The progress of the reactions was monitored by TLC using aluminum silica gel plates 60 F245. IR, 1 H-NMR, 13 C-NMR, GC-MS, and elemental analyses were performed at the Micro analytical center at the Faculty of science, Cairo University, Cairo, Egypt. Compounds 1 and 13 were prepared according to a previous procedure [44].

3-Cyano-6-(naphthalen-2-yl)-4-(thiophen-2-yl)pyridin-2-yl 2-chloroacetate (2)
To a solution of compound 1 (3.28 g, 10 mmol) and anhydrous potassium carbonate (1.38 g, 10 mmol) in dry acetone (30 mL), chloroacetyl chloride (0.79 mL, 10 mmol) was added drop wise. The reaction mixture was stirred at room temperature for 8 h. The mixture was poured onto cold water and extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated under vacuum to give compound 2. To a solution of compound 2 (4.06 g, 10 mmol) and anhydrous potassium carbonate (1.38 g, 10 mmol) in DMF (20 mL), thiourea (0.76 g, 10 mmol) was added, and the reaction mixture was heated under reflux for 8 h. After cooling, the reaction mixture was poured onto ice cold water. The formed precipitate was collected by filtration and crystallized from methanol to give compound 3.  (5) A solution of compound 4 (3.46 g, 10 mmol) in methanol (20 mL) and sodium methoxide (0.54 g, 10 mmol) was refluxed for 3 h. After cooling, the reaction mixture was poured onto water, and the solid obtained was crystallized from ethanol to give compound 5. To a solution of compound 8 (3.44 g, 10 mmol) in ethanol (30 mL) and glacial acetic acid (2 drops), d-glucose or d-galactose was added in water (1 mL). The reaction mixture was refluxed for 6 h (TLC; methanol/chloroform; 0.5/9.5) and then allowed to cool at room temperature. The excess ethanol was removed under reduced pressure, and the residue was treated with diethyl ether (15 mL). The solid that formed was filtered and crystallized from ethanol to form compounds 9, 10.  (11) To a solution of compound 7 (3.44 g, 10mmol) and anhydrous potassium carbonate (1.38 g, 10 mmol) in dry acetone (30 mL), chloroacetyl chloride (0.79 mL, 10 mmol) was added drop wise. The reaction mixture was stirred at room temperature for about 8 h. Then, the mixture was poured onto water and extracted with ethyl acetate, dried over anhydrous sodium sulfate, and concentrated under vacuum to give compound 11.  (12) A mixture of compound 11 (4.22 g, 10 mmol) and sodium azide (0.65 g, 10 mmol) in DMF (20 mL) was stirred at room temperature for 12 h. The reaction mixture was diluted with cold water (10 mL) and extracted with chloroform (3 × 10 mL). The combined organic layer was concentrated to afford compound 12.

Cell Culture and Maintenance
Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco, Paisley, UK. Dimethyl sulfoxide (DMSO) was of HPLC grade, and all other reagents and chemicals were of analytical reagent grade.
Cell cultures of HCT-116 (human colorectal carcinoma), MCF-7 (human breast adenocarcinoma), HepG2 (human hepatocellular carcinoma), and A549 (human lung carcinoma) cell lines were purchased from the American Type Culture Collection (Rockville, MD, USA) and maintained in DMEM medium that was supplemented with 10% heat-inactivated FBS (fetal bovine serum), 100 U/mL penicillin, and 100 U/mL streptomycin. The cells were grown at 37 • C in a humidified atmosphere of 5% CO 2 .

Cytotoxicity Measurement
The anticancer activity against HCT-116, HepG2, A549, and MCF-7 human cancer cell lines was estimated using the 3-[4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, which is based on the reduction of the tetrazolium salt by mitochondrial dehydrogenases in viable cells [49][50][51][52]. Cells were dispensed in a 96-well sterile microplate (5 × 10 4 cells/well) and incubated at 37 • C with a series of different concentrations, in DMSO, of each tested compound or doxorubicin (positive control) for 48 h in a serum-free medium prior to the MTT assay. After incubation, media were carefully removed, and 40 µL of MTT (2.5 mg/mL) were added to each well and then incubated for an additional 4 h. The purple formazan dye crystals were solubilized by the addition of 200 µL of DMSO. The absorbance was measured at 570 nm using a Spectra Max Paradigm Multi-Mode microplate reader. The relative cell viability was expressed as the mean percentage of viable cells compared to the untreated control cells. All experiments were conducted in triplicate and repeated on three different days. All the values were represented as mean ± SD. IC 50 s were determined by probit analysis by SPSS Incprobit analysis (IBM Corp., Armonk, NY, USA).

In Silico Studies Molecular Docking Study
Molecular modeling simulation study was performed through docking of the target compounds in the binding site of CDK2 enzyme using C-Docker protocol in Discovery Studio 4.0 Software. The X-ray crystal structure of Roscovitine in complex with CDK2 was downloaded from http://www.rscb.org/pdb (accessed on 9 March 2021) (PDB code 2A4L) in PDB format. Computational docking is an automated computer-based algorithm designed to estimate two main terms [53]. The first is to determine the suitable position and the orientation of a certain test set molecule's pose inside the binding site in comparison to that of the X-ray crystallographic enzyme-substrate complex. The second term is the calculation of the estimated protein ligand interaction energy, which is known as binding energy (docking scoring).

Data Availability Statement:
The data presented in this study are available in this article.