Design and Synthesis of New CDK2 Inhibitors Containing Thiazolone and Thiazolthione Scafold with Apoptotic Activity

Abstract

Cyclin dependent kinase 2 (CDK2) inhibition is a well-established strategy for treating cancer. Different series of novel thiazolone (1, 7–9) together with fused thiazolthione (2–6, and 10) derivatives were designed, then synthesized and evaluated for their biological inhibitory activity against CDK2. Additionally, the cytotoxicity of the new compounds was explored against breast and colon cancer cell lines. The novel thiazolone and the fused thiazolthione derivatives exhibited potent CDK2/cyclin A2 inhibitory effect of an IC₅₀ values ranging 105.39–742.78 nM. Amongst them compounds 4 and 6 revealed highest IC₅₀ of 105.39 and 139.27 nM, respectively. Most compounds showed significant inhibition on both breast cancer and colon cancer cell lines with IC₅₀ range 0.54–5.26 and 0.83–278 µM, respectively. Further investigations involved flow cytometry analysis on MCF-7 cancer cell line for compounds 5 and 7 which resulted in arrest cell-cycle at two phases Pre G1/G2-M and re-enforced apoptosis via activation of caspase-7. Molecular modeling simulation of the designed compounds revealed that they were well fitted into CDK2 active site and their complexes were stabilized through the essential hydrogen bonding. Three dimensional quantitative structure activity relationship (3D QSAR) pharmacophore, and absorption, distribution, metabolism, excretion, and toxicity (ADMET) studies were also carried out showing proper pharmacokinetic and drug-likeness which aided in the prediction of the structure requirements responsible for the observed antitumor activity.

Introduction

The cyclin dependent kinases (CDK) are responsible for all cell cycle initiation and succession. Activity of members of CDK family is corrupted in many tumor cells where they are essential for phosphorylation of key components for cell proliferation.^1,2) CDK2 has important catalytic role in the complex of cyclin-dependent protein kinase, its activity is essential for cell cycle progress.^3,4) The cyclin protein family modulate 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.^3,4) 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.⁵⁾ During G1 phase cell cycle, complexes of both CDK4 and CDK6 with D-type cyclins are formed, which in turn activates CDK2 through cyclin E association. While in S phase, cyclin A binds CDK2. However, in the G2/M phase, CDK1 is complexed with cyclin B.^3,4) The activation of CDK2 through cyclin E is claimed to be an essential step through the progression from G1 to S phase and this makes CDK2 be principal target for most inhibitory drugs.^1–4) Heterocyclic compounds such as 2-thioxothiazolidin-4-ones, pyrimidines and pyrazolines have been widely studied for their importance in medicinal chemistry regarding their wide biological inhibitory activities of CDK2/CyclinA. These compounds displayed wide pharmacological activities, including antibacterial,^6–8) antiviral,⁹⁾ antidiabetic,¹⁰⁾ anticancer,¹¹⁾ anti-inflammatory,¹²⁾ antioxidant,¹³⁾ anti-tubercular,⁸⁾ anticonvulsant,¹⁴⁾ and cytotoxic activities.^15–24)

Derivatives of pyridine scaffold also were widely used as both fungicides and anticancer drugs.²⁵⁾ Based on these activities, the aim of this research was to design and synthesize new pyridine 2, pyrazoline 3, 4, pyrimidine 5, 6, thiazolone 7, 8a, 8b, 9 and triazepine 10 derivatives from thiophen-2-yl methylene thioxthiazolidine containing chalcone and evaluate their antimicrobial and anticancer activities maintaining the pharmacophoric essential features. Three dimensional quantitative structure activity relationship (3D QSAR) study was also performed to validate the observed pharmacological properties in order to investigate the most important parameter controlling their antitumor properties.

Rational Design

Designing the targeted compounds was based on Bioisosteric modifications and structure optimization of Roscovitine (the main lead compound), AT7519, and NU6102 (Fig. 1) as lead compounds, which includes the studies of SAR of reported lead compounds and molecular modeling.^26,27) The pyrazole moiety of the lead AT7519 occupying the adenine part in the ATP binding pocket was isosterically replaced by thiazole ring in compounds 7, 8a, b and 9. Also, the amide linkage at position 3 was replaced by thiosemicarbazide in compound 7, morphiline or piperidine in compounds 8a and b and 2-thiopyranose moiety in compound 9 to maintain the same interaction pattern as that of the reference compound. The two halogen atoms of in AT7519 occupying the hydrophobic pockets were replaced by the lipophilic moiety thienomethylene at position 5 of dihydrothiazole ring in order to occupy the hydrophobic pocket for compounds 7, 8a, b and 9 (Fig. 2).

Fig. 1. Structure of CDK2 Inhibitors

Fig. 2. Features’ Similarities for the AT7519 and the Designed Compounds 7, 8a, b and 9

While the purine scaffold of the reference compound Roscovitine was bioisosterically replaced by different ring system as thiazolopyridine in compound 2, thiazolopyrazole in compounds 3, 4 thiazolopyrimidine in compounds 5, 6 and thiazolotriazepine in 10 to locate it in the adenine region of ATP binding site as in Roscovitine. The phosphate binding region occupied by 6-substituted amino moiety in Roscovitine was replaced by thiophene moiety in all the proposed compounds. The amino derivative at 4-position of Roscovitine was either maintained as in compound 2 or substituted by mercapto or hydroxyl groups as in compounds 5 and 6, respectively. Also, phenyl group in compound 4 was added to occupy the hydrophobic region not occupied by ATP. Also, the introduction of thione group in thiazole ring of compounds 2–6 was to form an additional hydrogen bond interaction in the active site (Fig. 3).

Fig. 3. Features’ Similarities for the Roscovitine and the Designed Compounds 2–6

The design of the target compounds revealed using Discovery Studio 4.0 Software that two hydrogen bonds with Leu83 (1 Hydrogen Bond Acceptor (HBA), 1 Hydrogen Bond Donor (HBD)) together with hydrophobic interaction with Phe82, Ile10, Leu134 and Ala31 are essential for activity. The two Leu83 hydrogen bonding were retained by thione and NH of thiazole ring in compounds 4 and 6 (Fig. 4) together with an additional HBA with Lys89 as in compound 6, While in compound 5 NH of thiazole and N of pyrimidine developed the mentioned Leu83 hydrogen bonding. In compound 2 NH and CN of the pyridine moiety was involved. While in compound 3 different binding mode was observed with Lys33 with S of thiophene and Phe82 with thione of thiazole. Also, in compound 10 different binding mode was observed as 3HB one of them with Lys33 with thione of triazepine and two HB with Lys89 with thione and NH of thiazole.

Fig. 4. Binding Modes of the Roscovitine (PDB ID: 2A4L) and (a) Compound 4, (b) Compound 6 Which Revealed the Two Essential H-Bond with LEU83 Using Discovery Studio 4.0 Software

While the design of compounds of 4 oxothiazole with methylene linker to thiophene core 1, 7, 8a, 8b and 9 was studied. Where in compound 1 Leu83 hydrogen bonding was retained by NH and 4 oxo of thiazole ring. While in compound 7, S and NH of thiourea with Leu83 hydrogen bonding was observed. In compound 8a different binding mode was observed where only 1HB with Lys33 via 4 oxothiazole. Compound 9 only 1HBA LEU83 with S of thiophene and 1HBA with Lys89 with oxo of thaizole.

Results and Discussion

Chemistry

The 2-thioxothiazolidin-4-one undergoes condensation reaction together with thiophen-2-carbaldehyde to obtain the chalcone 1.²⁸⁾

Interaction of Chalcone 1 with malononitrile and ammonium acetate in ethanol was performed to produce the pyridine derivatives 5-amino-6-isocyano-7-(thiophen-2-yl)thiazolo [4,5-b]pyridine-2(3H)-thione 2. In compound 2 stretching vibration was found in IR analysis of the NH₂ and NH groups at ν3298.5–3357.6 and 3205.6 cm⁻¹, respectively, as well as the presence of CN group at ν2216.6 cm⁻¹. This IR pattern appears to be consistent with the assigned structure.

New pyrazoline derivatives were prepared by reacting chalcone 1 with hydrazine hydrate and phenyl hydrazine in ethanol in the presence of hydrochloric acid forming the new corresponding derivatives3-(thiophen-2-yl)-2H-pyrazolo[3,4-d]thiazole-5(6H)-thione 3 and 2-phenyl-3-(thiophen-2-yl)-2H-pyrazolo[3,4-d]thiazole-5(6H)-thione 4. In compound 3 IR spectrum displayed absorption of two bands of the amine (NH) at ν3191.2 and 3104.5 cm⁻¹. The ¹H-NMR spectrum of compound 3 exhibited two NH singlet signals (D₂O exchangeable) at δ = 11.9 and 12.1 ppm respectively. The ¹³C-NMR indicate the presence of C=N and C=S groups resonate around 151.68; 187.91 ppm. While IR spectrum of compound 4 confirmed the characteristic amine group absorption band at ν3149.1 cm⁻¹. The ¹H-NMR spectrum of 4 showed signals of the amine proton at δ = 8.7 ppm. The ¹³C-NMR showed that C–N; C=N and C=S groups are resonated around 137.2, 150.86, 185.41 ppm. On the other hand new pyrimidine derivatives were prepared by reacting chalcone 1 with thiourea and urea, producing 5-mercapto-7-(thiophen-2-yl)thiazolo[4,5-d]pyrimidine-2(3H)-thione 5 and 5-hydroxy-7-(thiophen-2-yl)thiazolo[4,5-d]pyrimidine-2(3H)-thione 6, respectively. IR spectrum of derivative 5 showed strong absorption bands of NH and SH groups at ν3138.6 and 2550 cm⁻¹, respectively. The ¹H-NMR spectrum of 5 revealed the presence of NH as a singlet at δ = 8.01 ppm and the SH signal at δ = 12.52 ppm both NH and SH disappeared by D₂O. The ¹³C-NMR of C–N; C=N and C=S groups results showed that carbon atoms resonate around 147.69, 148.99, 171.57 ppm. IR spectrum of 6 displayed strong absorption bands of OH and NH groups at ν3447.4 and 3349.5 cm⁻¹, respectively. The ¹H-NMR spectrum of compound 6 confirmed the presence of OH as a singlet at δ = 8.72 ppm and the disappearance of NH signal at δ = 5.44 ppm and they diaper by D₂O. While, carbon atoms present in C=N, C–OH and C=S groups resonate around 144.86, 153.87, 182.21 ppm in ¹³C-NMR.

A mixture of chalcone 1 and appropriate thiosemicarbazide in ethanol were used for the preparation of open compound 2-(4-oxo-5-(thiophen-2-ylmethylene)-4,5-dihydrothiazol-2-yl)hydrazine carbothioamide derivative 7.²⁹⁾ The characteristic absorption bands for the NH₂ group at ν3444.43–3449.57 cm⁻¹ was revealed by the IR spectrum where strong absorption bands of two NH groups in the region ν3147.72–3082.10 cm⁻¹were displayed. Furthermore, ¹H-NMR spectrum presented two NH singlets (D₂O exchangeable) at δ = 2.491, 2.504 ppm and a singlet (1H) at δ = 8.073 ppm assignable to NH₂ proton (D₂O exchangeable) which confirmed their proposed structures. Carbon atoms present in C=O and C=S groups resonate around 162.26 and 182.5 ppm in ¹³C-NMR. Piperdine and morpholine derivatives were synthesized via the reaction between chalcone (1) and different cyclic amines to produce the piperidine derivative 2-(piperidin-1-yl)-5-(thiophen-2-ylmethylene)thiazol-4(5H)-one 8a, or the morpholine derivative2-morpholino-5-(thiophen-2-ylmethylene) thiazol-4(5H)-one 8b.³⁰⁾

While reacting compound 1 with 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide, acetylated thioglycoside derivatives of 2-(acetoxymethyl)-6-(4-oxo-5-(thiophen-2-ylmethylene)-4,5-dihydrothiazol-2-yl)thio) tetrahydro-2H-pyran-3,4,5-triyl triacetate 9 was formed.³¹⁾ Four singlet signals (12H) at δ = 2.48–2.51 ppm were shown in ¹H-NMR spectra, their appearance was assignable to four acetyl methyl groups beside the signals of the sugar protons in the region δ = 3.20–4.86 ppm. The ¹³C-NMR showed resonance of carbon atoms in C=N and C=O groups around 158.8; 167.2 ppm. A mixture of chalcone 1 with thiosemicarbazide was dissolved in dioxane then glacial acetic acid 5-(thiophen-2-yl)-5,5a,8,8a-tetrahydro-3H-thiazolo[5,4-f][1,2,4]triazepine-3,7(4H)-dithione 10 was produced closed ring,²⁹⁾ IR spectrum of 10 showed absorption band of NH at ν3416.49 cm⁻¹ and absence of the C=O band. The ¹H-NMR spectrum of 10 revealed two singlet at δ = 3.498, 5.122 ppm to 2NH proton (D₂O exchangeable), as shown in experimental (Chart 1).

Chart 1. Synthesis of Chalcone Derivatives (1–10)

R1=2,3,4,6 Tetra-O-acetyl-alpya-D-glucopyranosyl bromide

Biological Evaluation

CDK2/Cyclin A2 Activity

The in vitro CDK2/cyclin A2 assays of the synthesized compounds 1, 2, 4–7, 8b and 9 were carried out using Promega Kinase-Glo Plus luminescence kinase kit. This assay measures ADP formed by a kinase reaction, where ADP is turned to ATP, which is further converted into light.³²⁾ The luminescent signal is correlated to ATP amount while it is inversely correlated to the kinase activity. The IC₅₀ result of the target compounds was presented in Table 1. All the new compounds revealed good inhibitory effect toward CDK2/cyclin A2 protein Kinase with an IC₅₀ values ranging 105.39 ± 7.18–742.78 ± 22.4 nM compared to imatinib reference compound of IC₅₀ 167.71 ± 5.36 nM. Amongst them compounds 4 and 6 revealed highest IC₅₀ of 105.39 ± 7.18 and 139.27 ± 5.92 nM, respectively. While compound 2 showed the least inhibitory activities (IC₅₀ of 742.78 ± 22.4 nM). Compounds 3, 8a and 10 were not identified due to their difficult solubility.

Table 1. The IC₅₀ Result of the Target Compounds against CDK2 Enzyme

Name	Results IC50 (nM)
1	191.14 ± 6.39
2	742.78 ± 22.4
4	105.39 ± 7.18
5	366.56 ± 13.7
6	139.27 ± 5.92
7	263.67 ± 8.24
8b	352.67 ± 13.5
9	665.04 ± 26.1
Imatinib	167.71 ± 5.36

In Vitro Cytotoxicity Activity

The cytotoxicity of the newly synthesized compounds 1, 2, 4–7, 8b and 9 against two different human cancer cell lines named MCF-7 (Breast) and HCT-116 (Colon) was evaluated.³³⁾ The tested compounds 1, 4–7, 8b and 9 showed high potent activity against MCF-7 with IC₅₀ range (0.54–5.26 µM) compared to control drug Staurosporine (IC₅₀ = 7.25 µM). The obtained results are presented in Table 2. The cytotoxicity of the screened compounds compared to Stauroporine revealed that compounds 9, 5 and 7 were the most potent with IC₅₀ equal to 0.54, 0.77 and 1.34 µM, respectively while compound 2 showed the least activity (IC₅₀ = 15.20 µM). However, compounds 2, 4, 7 and 9 showed high inhibitory activity more than Stauroporine (IC₅₀ = 6.94 µM) against HCT-116. Where compound 4 was the most active (IC₅₀ = 0.83 µM) and compounds 1, 5, 6 and 8b showed moderate inhibitory activity with IC₅₀ of 14.62, 9.52, 21.01 and 10.54, respectively.

Table 2. Cytotoxic Activities of Synthesized Compounds 1, 2, 4–7, 8b and 9

Entry	Name	IC50µM
		MCF-7	HCT-116
1	1	5.26 ± 0.16	14.62 ± 0.48
2	2	15.20 ± 0.82	2.78 ± 0.06
3	4	2.14 ± 0.07	0.83 ± 0.02
4	5	0.77 ± 0.02	9.52 ± 0.3
5	6	1.87 ± 0.05	21.01 ± 1.2
6	7	1.34 ± 0.04	1.40 ± 0.04
7	8b	1.83 ± 0.04	10.54 ± 0.44
8	9	0.54 ± 0.01	1.68 ± 0.03
9	Staurosporine	7.25 ± 0.1	6.94 ± 0.1

Flow Cytometry Cell Cycle Analysis

Among the highly potent screened compounds against breast cancer cell line (MCF-7) compared to control drug Staurosporine, compounds 5 and 7 were selected for cell cycle analysis to determine the mechanism and understand the mode of action of the new compounds.³⁴⁾ The assay results showed Pre-G1 and cell growth arrest at G2-M stage, where checkpoint arrest of G2 phase was initiated in response to DNA damage, the CDC25-dependent activation of either cyclin A/CDK2 or cyclin B1/CDC2 is blocked. Ectopic expression of CDC25B, and not CDC25C, in G2 phase arrested cells activated both cyclin A/CDK2 and cyclin B1/CDC2.³⁵⁾ Results were compared to normal control cells, in Dip Pre-G1 with an increase from 1.72 to 27.88 and 21.59% and an increase in Dip G2-M phase from 3.5 to 47.89 and 39.31% for compounds 5 and 7, respectively (Figs. 5, 6).

Fig. 5. Cell Cycle Analysis for Compounds 5 and 7 on MCF-7

Fig. 6. Cell Cycle Analysis for Compounds 5 and 7 on MCF-7 Evaluated by Flow Cytometry

Caspase-7 Study

The sensitive colorimetric assay using Caspase-7 was performed to examine the potentiality of compounds 5 and 7 in apoptosis induction against the breast cancer cell line.

Figure 7 showed that compounds 5 and 7 exerted nine fold increases, in absorbance on MCF-7 cell line compared to the control. Which indicated their capability to stimulate caspase-7 and enforcing a cancer cells death program.

Fig. 7. Caspase-7 Absorbance Data Obtained for Compounds 5 and 7 on MCF-7 Cell Line Compared with Control

In Silico Studies

Molecular Docking

Molecular docking study was carried out using C-Docker protocol in Discovery Studio 4.0 Software. CHARMm forcefield was applied for calculation with MMFF94 partial charge. Where the prepared target compounds were docked into the determined binding site of CDK2 enzyme. The binding modes analysis of the designed compounds was studied to interpret their biological results and to obtain further explanation of the binding orientations and the activity of the new compounds. The X-ray crystallographic enzyme (CDK2) substrate being complexed with Roscovitine (PDB ID: 2A4L) showed the existence of the two essential hydrogen bonds with LEU83. The pose selected out of ten that showed proximal similarity to the binding mode of the ligand is considered the best pose. Re-docking of the lead compound (Roscovitine) in the active site of CDK2 kinase was used to confirm validation of C-Docker protocol which showed good coincidence with RMSD value = 0.5 A°. This ensured the docking protocol validity. The presented docking study insured comparable binding modes between the lead compound and the docked molecules. The binding mode and the C-Docker interaction energy of the biologically active synthesized compounds are summarized in Table 3.

Table 3. The CDOKER Interaction Energy of the Newly Synthesized Compounds

Where the molecular docking of the new compounds using C-Docker protocol revealed that Compounds 1, 2, 4–7 retained the two essential H-bonds with LEU83 when compared with the reference compound Roscovitine also having the highest inhibition activity against CDK2/A2 for compounds 4 and 6. While compounds 5, 7, 8b, 6, 4 and 1 showed potent antitumor activity against MCF-7 and compounds 4, 7, 9, and 2 showed higher activity against HCT-116 which indicate that the antitumor effect is due to CDK2 enzyme inhibition .Whereas compound 9 forming one H-bond only with LEU83 with different binding mode than that of the lead compound but with high MCF-7 and HCT-116 antitumor activity.

The results explain the superior activity of compound 4 regarding IC₅₀ values towards both MCF-7 and HCT-116 as 2.14 ± 0.07 and 0.83 ± 0.02 µM, respectively in addition to the best CDK2 inhibitory activity as IC₅₀ 105.39 ± 7.18 nM compared to the reference. Where the phenyl substitution of pyrazole ring is essential as a bulky group for ATP binding site occupation. Also thiazolpyrazole presence in compound 4 or the thiazolopyrimidine occurrence in compounds 5 and 6 are responsible for better CDK2 activity (compound 6 CDK2 IC₅₀ 139.27 nM). However, The thione presence in either thiazolpyrazole containing compounds as in compound 4 or thiazolopyrimidine containing compounds in compound 6 is essential for antitumor activities and CDK2 activity, while its substitution with thiosemicarbazide group in compound 7 or thiopyranose moiety in compound 9 retains antitumor activity but with lower CDK2 inhibitory effect. The 4-oxo group in thiazole of compounds 7 and 9 or the SH and OH groups of pyrimidine in compounds 5 and 6, respectively explain the antitumor activity but with lower CDK2 activity than that of compound 4 except for compound 6 where hydroxyl pyrimidine is present rather than mercapto substitution in compound 5. Compound 2 is the only thiazolopyridine derivative, it showed the least CDK2 activity compared to reference with IC₅₀ of 742.78 nM. However, it showed HCT-116 cell line cytotoxicity.

3D QSAR Pharmacophore Based Study

The pharmacophore mapping protocol was applied using Discovery Studio 4.0 Software, to define the main features and declare the steric and electronic topographies of the newly synthesized molecules exhibiting the same biological activity by applying 3D Pharmacophore Generation from Create Pharmacophore Automatically. The training set for the cytotoxic study on the specified MCF-7 was composed of 6 compounds; 1, 5–7, 8b and 9. The pharmacophore model with best results regarding fit value and total cost compared to Null and Fixed cost was chosen. Three chemical common features were shown to the training set; two hydrogen bond acceptor (HBA) and one hydrophobic (H), displayed in Fig. 8A. The pharmacophore model for the cytotoxic study on colon cancer cells HCT-116 was generated using compounds 1, 2, 4, 5, 8b and 9 as training set. Three chemical common features were generated by the mapped pharmacophore; two HBAs and one Ring Aromatic (RA), displayed in Fig. 8B. The constraint distance and angles were determined and shown in Table 4 and Fig. 9. It is worth noting that the 3D QSAR pharmacophore-based study predicted and real activities were very close to be used for predication of more effective structures similar to that of the potent antitumor compound.

Fig. 8. Features Mapping for Compound 9 A) in Breast Cancer MCF-7 and B) in Colon Cancer HCT-116 Pharmacophore Model

Table 4. Constraint Distances (Å) and Angles (°) between Features of the Generated Pharmacophores

Cancer cell	Constraint distances (Å)	Constraint angles (°)
MCF-7 (breast)	(HBA-1)–(HBA-2), 4.13; (HBA-2)–(H), 6.89; (H)–(HBA-1), 3.13.	(HBA-1)–(HBA-2 vector)-(H), 13.76; (HBA-1)–(HBA-2 vector)-(H), 14.75; (HBA-2)–(HBA-1 vector)-(H), 45.25; (HBA-2)–(HBA-1 vector)-(HBA-1), 36.23
HCT-116 (colon)	(HBA-1)–(RA), 4.40; (HBA-1)-(HBA-2), 4.17; (HBA-2)–(RA), 3.20.	(RA vector)-(RA)-(HBA-1), 63.78; (HBA-1)-(HBA-2)-(RA), 71.99; (RA vector)-(RA)-(HBA-2), 123.53

Fig. 9. MCF-7 Pharmacophore Model A, B: A) Distance between Chemical Features, B) Angles between Chemical Features, HCT-116 Pharmacophore Model C, D: C) Distance between Chemical Features, D) Angles between Chemical Features

The estimated IC₅₀ values and the experimental ones were compared by their relative energies and fit values (Tables 5, 6).

Table 5. Results of 3D QSAR Pharmacophore Based of MCF-7 Training Set

No.	Name	Experimental value of IC50	Estimated value of IC50	Residual activity	Fit value	Log obtained activity	Log estimated activity	Relative energy
1	1	5.26	5.70	0.44	4.49	1.66	1.74	1.65
2	5	0.77	1.18	0.41	4.73	−0.26	0.16	0.01
3	6	1.87	1.80	0.07	4.54	0.62	0.56	0.01
4	7	1.34	1.48	0.14	4.63	0.29	0.39	18.85
5	8b	1.83	1.76	0.07	4.55	0.60	0.56	17.31
6	9	0.54	0.36	0.18	5.24	−0.61	−1.01	17.57

Table 6. Results of 3D QSAR Pharmacophore Based of HCT-116 Training Set

No.	Name	Experimental value of IC50	Estimated value of IC50	Residual activity	Fit value	Log obtained activity	Log estimated activity	Relative energy
1	1	14.62	17.65	3.03	3.62	2.68	2.87	1.65
2	2	2.78	3.79	1.01	4.29	1.02	1.33	0
3	4	0.83	1.12	0.29	4.82	−0.18	0.11	6.74
4	5	9.52	5.28	4.24	4.14	2.25	1.68	0.04
5	8b	10.54	8.74	1.8	3.93	2.25	2.16	5.00
6	9	1.68	1.91	0.23	4.59	0.51	0.64	9.49

Validation

Validation of the generated pharmacophore models was performed through internal validation through cost analysis and external validation utilizing two of the compounds; 2, 4 as external test set for MCF-7 pharmacophore model and 6, 7 as external test set for HCT-116 model.³⁶⁾ The test set exhibited potent and mild activity against the specified tumor cell. The experimental and the estimated activities by the generated models were shown in Table 7.

Table 7. Estimated and Observed Activities of MCF-7 and HCT-116 Test Sets

No.	Name	Experimental value of IC50	Estimated value of IC50	Residual activity	Fit value
MCF-7 3D Pharmacophore model
1	2	15.20	13.87	1.33	4.88
2	4	2.14	1.10	1.04	4.95
HCT-116 3D Pharmacophore model
1	6	21.01	19.61	1.40	3.58
2	7	1.40	1.90	0.50	4.58

In Vitro Predictive Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) Study

The ADMET study was concerned with the molecule chemical structure and involves several parameters calculation including using Discovery Studio 4.0 Software; Absorption level (Absorp LEV), Aqueous solubility level (AQ SOl LEV), Blood Brain Barrier value (BBB), Blood Brain Barrier Level (BBB LEV), Atom based Log P98 (A Log P98), 2D polar surface area (ADMET 2D PSA), CYP2D6, CYP2D6 Probability (CYP PROB), Hepatotoxicity (HEPATOX), Hepatotoxicity Probability (HEPATOX PROB) and Plasma protein binding logarithmic level (PPB LEV).

In ADMET plot, most of the compounds had BBB level of 1, hence they are able to pass the blood brain barrier, and consequently they can be used as promising candidates in the treatment of brain tumors. Ninety-nine percent of the compounds had absorption level = 0, thus estimated to have good human intestinal absorption, whilst only compound 9 showed low absorption. Most of the compounds showed ADME aqueous solubility level between 2 and 3 which indicates good aqueous solubility. The key property (PSA) was linked to drug bioavailability. Therefore molecules which are passively absorbed and PSA < 140 are thought to have lower bioavailability. Thus, all the synthesized compounds were predicted to present good passive oral absorption.

The obtained results are showed as a 2D ADMET plot, drawn using calculated PSA_2D and A log P98 properties (Fig. 10).

Fig. 10. The ADMET Plot Using Calculated PSA_2D and A Log P98 Properties

Molecules with PSA < 140 are passively absorbed and show low bioavailability. The Values of PSA for the tested compounds ranged from 30.11–142.47, that’s why they are predicted to show high passive oral absorption (Table 8).

Table 8. ADMET Predictions of the Newly Synthesized Compounds

Name	BBB LEVEL	ADMET absorption level	ADMET A log P	ADMET PSA 2D	CYP2D6	Hepatotoxicity	ADMET aq solubility level
1	1	0	2.567	30.111	6.51911	3.09562	3
2	2	0	3.101	73.546	5.3172	3.35945	2
3	1	0	3.316	39.126	4.65827	2.5217	2
4	1	0	4.111	35.332	4.00186	3.29541	2
5	1	0	3.509	56.147	4.50843	2.26559	2
6	3	0	1.786	80.784	8.28892	1.37338	3
7	1	0	3.051	31.976	3.54952	0.789047	2
8a	2	0	1.822	40.906	6.05482	2.2512	3
8b	2	0	1.798	42.902	6.21511	2.1954	3
9	4	2	2.495	142.477	8.35658	2.29416	3
10	1	0	3.896	48.266	4.99787	1.35158	2

Structure–Activity Relationship

3D QSAR pharmacophore revealed that the controlling features for breast cancer MCF-7 activity contain three essential chemical features that are responsible for providing relative alignment for the molecule controlling the mode of binding towards the proposed site of the receptor. The chemical features were mainly two hydrogen bond acceptor HBA and one hydrophobic as shown in Fig. 8A. While chemical features of the active molecules within the colon cancer model HCT-116 were mainly three, two of them were hydrogen bond acceptor HBA and an additional ring aromatic RA as shown in Fig. 8B.

Conclusion

New chalcone derivatives of 5-(thiophen-2-ylmethylene)-2-thioxothiazolidin-4-one (1) were designed and synthesized. All compounds exhibited potent CDK2/cyclin A2 inhibitory effect of an IC₅₀ values ranging 105.39 ± 7.18–742.78 ± 22.4 nM. Amongst them compounds 4 and 6 revealed highest IC₅₀ of 105.39 ± 7.18 and 139.27 ± 5.92 nM, respectively. Most of the new compounds exhibited potent antitumor activity against MCF-7 where IC₅₀ range was (0.54–5.26 µM) and potent to moderate effect against colon cancer cell lines with IC₅₀ range of (0.83–14.62 µM) comparable to Staurosporine (IC₅₀ 7.25 and 6.94 against MCF-7 and HCT-116, respectively). Compounds 5 and 7 exhibited arrest of cell-cycle in the Pre G1and G2-M phases on MCF-7 cancer cells and enforced apoptosis via activation of caspase-7. The results revealed agreement between both the experimental and estimated data through docking and 3D QSAR pharmacophore modelling providing an effective technique for predicting structure requirements for pharmacological properties. ADMET study results showed a reasonable BBB penetration of most of the compounds to be used for brain tumor treatment with good passive oral absorption.

Experimental

Chemistry

General

All chemicals were used without further purification unless mentioned in the procedure and were purchased from Sigma-Aldrich. The starting material [compound 1] was prepared adopting the reported method.²⁸⁾ TLC plates covered with 60 F254 silica gel was used to check the purity of the new compounds. CDK2/CyclinA2 Kinase Enzyme system used from Promega Cat. Number V2971. And Human CASP7 (Caspase 7) enzyme-linked immunosorbent assay (ELISA) Kit with Catalog No: MBS2505226 was used.

Melting points of all the new compounds are listed uncorrected and were measured by a Reichert Thermovar apparatus. Yields Registered are of the new compounds. The IR spectra were determined using Perkin-Elmer spectrometer (KBr disc), model 1720 FTIR. ¹H-NMR, and ¹³C-NMR spectra were done using a Bruker AC-300 or DPX-300 spectrometers. Chemical shifts were reported in δ scale (ppm) using TMS as a reference standard and the coupling constants J values are given in Hz. The progress of the reactions was determined using TLC aluminum silica gel plates 60 F245. IR, the analysis (¹H-NMR, ¹³C-NMR and Elemental analyses) were proceeded at the, Cairo University Micro analytical Centre, Egypt.

Synthesis of (Z)-5-(Thiophen-2-ylmethylene)-2-thioxothiazolidin-4-one (1).

A flask contains a solution of 5 g of sodium hydroxide in 20 mL of water and 10 mL ethanol was immersed in a bath of crushed ice, and then (1.65 g, 10 mmole) of freshly distilled 2-thioxothiazolidin-4-one was added. The reaction mixture was stirred with addition of (1.12 g, 10 mmole) of pure thiophen-2-carbaldehyde drop wise. The temperature of the mixture was kept at about 0 °C with stirring for 4h until the mixture became so thick. Then the reaction mixture was left overnight in an ice chest. The formed precipitate was filtered off and was washed with cold water until the washings are neutral to litmus paper and then with 20 mL of ice cold rectified spirit, the precipitate was crystallized from ethanol to get compound (1),²⁸⁾ mp 223 °C, yellow powder.

Synthetic Procedure for Compound (2)

5-Amino-6-isocyano-7-(thiophen-2-yl)thiazolo[4,5-b]pyridine-2(3H)-thione (2)

Refluxing mixture of chalcone (1) (2.3 g, 10 mmol), malononitrile (0.66 g, 10 mmol) and ammonium acetate, (0.5 g) in 30 mL ethanol for 4 h. Afterwards, evaporation of the solvent. Then the precipitate was filtered after the residue been poured on ice-water and crystallized from ethanol to obtain compound 2.

Yields 77%, mp 267–270 °C, Brown powder; IR (KBr) cm⁻¹: 3298.5, 3357.6.5, 3205.6; 3088, 2216.6 1591.9, 1296.7. ¹H-NMR (300 MHz, dimethyl sulfoxide (DMSO)-d₆) δ: 4.95 (s, 1H, NH, D₂O exchangeable), 6.96 (s, 2H, NH₂, D₂O exchangeable), 7.45–7.92 (m, 3H, thiophene). ¹³C-NMR (300 MHz, DMSO-d₆) δ: 82.23 (C), 112.3 (CN); 125.53 (CH); 126 (CH); 127.925 (CH); 122.63 (C–S); 137.8, 140.07 (2C–C), 149.86 (C=N); 158.87 (C–NH₂); 182.21 (C=S), MS m/z: 290 (M), Anal. Calcd for C₁₁H₆N₄S₃ (290.39): C, 45.50; H, 2.08; N, 19.29; S, 33.13. Found: C, 45.42; H, 2.01; N, 19.08; S, 33.05.

General Synthetic Procedure for Compounds (3, 4)

Mixing chalcone (1) (2.3 g, 10 mmol), with nucleophilic reagents: hydrazine hydrate or phenyl hydrazine (10 mmol) and the mixture were dissolved in ethanol (40 mL) as solvent with the addition of drops of HCl. Refluxing mixture for 4 h. The solid was collected after poured into ice water, filtered off, dried and recrystallized from ethanol to produce compounds 3, 4 respectively.

3-(Thiophen-2-yl)-2H-pyrazolo[3,4-d]thiazole-5(6H)-thione (3)

Yields 76%, mp 197–200 °C, brown powder, IR (KBr) cm⁻¹: 3191.2, 3104.5; 1624, 1563.4, 1118.9. ¹H-NMR (300 MHz, DMSO-d₆) δ: 7.52–7.89 (m, 3H, thiophene), 11.95, 12.12 (s, 2H, NH, D₂O exchangeable); ¹³C-NMR (300 MHz, DMSO-d₆) δ: 90 (C–S); 125.53 (CH); 126 (CH); 127.25 (CH); 128.75 (C-NH); 138.57 (C–C), 151.68 (C=N); 187.91 (C=S). MS m/z: 240 (M⁺¹), 219, 141, 111, Anal. Calcd for C₈H₅N₃S₃ (239.34): C, 40.15; H, 2.11; N, 17.56; S, 40.19. Found: C, 39.82; H, 2.06; N, 17.24; S, 39.95.

2-Phenyl-3-(thiophen-2-yl)-2H-pyrazolo[3,4-d]thiazole-5(6H)-thione (4)

Yields 69%, mp 235 °C, red powder, (ethanol); IR (KBr) cm⁻¹: 3149.1; 3069.12, 1589.4, 1229.54. ¹H-NMR (300 MHz, DMSO-d₆) δ: 7.17–7.41 (m, 3H, thiophene), 7.75 (t, 2H, J = 8.13, Ar–H), 7.83 (t, 1H, Ar–H), 7.92 (d, 2H, J = .8.04, Ar–H), 8.76 (s, 1H, NH, D₂O exchangeable), ¹³C-NMR (300 MHz, DMSO-d₆) δ: 92 (C–S); 120.53 (CH); 124 (2CH); 126.25 (CH); 127.53 (CH); 128 (CH); 128.25 (2CH); 120 (C=C); 137.2 (C–N); 138.09 (C–C); 150.86 (C=N); 185.41 (C=S). MS m/z: 316 (M⁺¹), 304, 140, 65, Anal. Calcd for C₁₄H₉N₃S₃ (315.44): C, 53.31; H, 2.88; N, 13.32; S, 30.50. Found: C, 52.94; H, 2.98; N, 13.24; S, 30.47.

General Synthetic Procedure for Compounds (5, 6)

Chalcone (1) (2.3 g, 10 mmol) was mixed with thiourea and urea (10 mmol) respectively, then dissolved in ethanolic sodium hydroxide refluxing the mixture for 3 h. Then left to cool, and poured over crushed ice. Then, neutralized using diluted HCl and kept for 24 h in refrigerator. The obtained precipitate was filtered and washed then recrystallized from ethanol. Affording 5, 6 respectively.

5-Mercapto-7-(thiophen-2-yl)Thiazolo[4,5-d]pyrimidine-2(3H)-thione (5)

Yields 86%, mp 216–218 °C, yellow powder; IR (KBr) cm⁻¹: 3138.6; 2550, 1683.9, 1583.8, 1226.3. ¹H-NMR (300 MHz, DMSO-d₆) δ: 7.27–7.99 (m, 3H, thiophen)_, 8.01 (s, 1H, NH, D₂O exchangeable), 12.52 ppm (s, 1H, SH, D₂O exchangeable), ¹³C-NMR (300 MHz, DMSO-d₆) δ: 126.53 (CH); 127 (CH); 127.85 (CH); 128.85 (C–S); 137.02 (C–C); 147.69 (C–N); 148.99 (C=N); 171.57 (C–SH); 189.21 (C=S). MS m/z: 283 (M) 87, 59, Anal. Calcd for C₉H₅N₃S₄ (283.42): C, 38.14; H, 1.78; N, 14.83; S, 45.26, Found: C, 37.95; H, 1.83; N, 14.44; S, 45.37.

5-Hydroxy-7-(thiophen-2-yl)thiazolo[4,5-d]pyrimidine-2(3H)-thione (6)

Yields 75%, mp 190 °C, yellow powder; IR (KBr) cm⁻¹: 3447.4, 3349.5; 1623. ¹H-NMR (DMSO-d₆, 300 MHz): δ(ppm): 5.44 (s, 1H, NH, D₂O exchangeable), 7.01–7.93 (m, 3H, thiophen); 8.72 (s, 1H, OH, D₂O exchangeable), ¹³C-NMR (DMSO-d₆) δ: 113.8 (C–S); 126.43 (CH); 127 (CH); 127.28 (CH); 136.49 (C–C); 144.86 (C=N); 113.8 (C–S), 150.8 (C–N); 153.87 (C–OH); 182.21 (C=S). MS m/z: 269 (M⁺²) 260, 157, 108, Anal. Calcd for C₉H₅N₃OS₃ (267.35): C, 40.43; H, 1.89; N, 15.72; S, 35.98. Found: C, 40.19; H, 1.81; N, 15.43; S, 36.03.

Synthetic Procedure for Compound (7)

2-(4-Oxo-5-(thiophen-2-ylmethylene)-4,5-dihydrothiazol-2-yl)hydrazinecarbothioamide (7)

Vigorous stirring for mixture of Chalcone (1) (2.3 g, 10 mmol), thiosemicarbazide (0.91g, 10 mmol) in 35 mL ethanol and potassium carbonate (0.5 g), furthermore, refluxing the product for 4 h was performed. After that the mixture was poured over crushed ice then left it 24 h at room temperature. Then, filtered off, washed with water, dried and recrystallized from ethanol.

Yields 82%, mp 215 °C, yellow powder; IR (KBr) cm⁻¹: 3444.43, 3449.57, 3147.72, 3082.10, 1683.93 1583.81, 1226. ¹H-NMR (300 MHz, DMSO-d₆) δ: 2.49, 2.50 (s, 2H, NH, D₂O exchangeable), 7.23 (s, 1H, CH), 7.56–7.92 (m, 3H, thiophen), 8.07(s, 2H, NH₂, D₂O exchangeable); ¹³C-NMR (300 MHz, DMSO-d₆) δ: 127.83 (CH); 129 (CH); 130 (CH); 137 (C-S); 136.2 (C–C); 147.63 (C=C); 158. 3 (C=N); 162.26 (C=O); 182.5 (C=S). MS m/z: 284 (M), 261, 167, 226, Anal. Calcd for C₉H₈N₄OS₃ (284.38): C, 38.01; H, 2.84; N, 19.70; S, 33.83. Found: C, 38.19; H, 2.81; N, 19.13; S, 34.03.

General Synthetic Procedure for Compounds (8a, b)

Refluxing a mixture containing chalcones (1) (10 mmol) and different cyclic amines (piperdine or morpholine) (10 mmole) in 25 mL of ethanol as solvent for 6 h, cooled to room temperature. Then the precipitated was filtered off and then crystallized from ethanol.³⁰⁾

(Z)-2-(Piperidin-1-yl)-5-(thiophen-2-ylmethylene)thiazol-4(5H)-one (8a)

Yields 73%, mp 184 °C, brown semi solid (ethanol); IR (KBr) cm⁻¹: 2941.88, 2806.88; 1671.02_, 1562.06. ¹H-NMR (300 MHz, DMSO-d₆): 1.51–1.66 (m, 6H, piperidine), 3.88 (m, N-(CH₂)₂, piperidine); 7.225(s, 1H, CH), 7.566- 7.892 (m, 3H, thiophen), MS m/z: 278 (M), Anal. Calcd for C₁₃H₁₄N₂OS₂ (278.39): C, 56.09; H, 5.07; N, 10.06; S, 23.04. Found: C, 56.19; H, 4.81; N, 9.93; S, 23.03.

(Z)-2-Morpholino-5-(thiophen-2-ylmethylene)thiazol-4(5H)-one (8b)

Yields 70%, mp 177 °C, brown semi solid (ethanol); IR (KBr) cm⁻¹: 275.04, 2922.91; 1678.33, 1595.06. ¹H-NMR (300 MHz, DMSO-d₆) δ: 3.64 (m, N-(CH₂)₂, morpholine), 3.89 (m, O-(CH₂)₂, morpholine); 7.252 (s, 1H, CH), 7.580–7.903 (m, 3H, thiophen), MS m/z: 280 (M), Anal. Calcd for C₁₂H₁₂N₂O₂S₂ (280.37): C, 51.41; H, 4.31; N, 9.99; S, 22.87. Found: C, 51.37; H, 4.41; N, 9.93; S, 23.03.

Synthetic Procedure for Compound (9)

2-(Acetoxymethyl)-6-((4-oxo-5-(thiophen-2-ylmethylene)-4,5-dihydrothiazol-2-yl)thio)tetrahydro-2H-pyran-3,4,5-triyl Triacetate (9)

Chalcone (1) (2.3 g, 10 mmol) was added to a stirred solution of 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide (2.11 g, 5 mmol) in acetone (20 mL) for 30 min, and then aqueous potassium hydroxide solution (0.28 g, 5 mmol, 2 mL) was added. The mixture was stirred for 8 h at room temperature. After evaporation of solvent, filtered off, washed using distilled water in order to remove any potassium bromide salt, dried and then recrystallized from ethanol providing 9.

Yields 86%, mp 180–182 °C, orange powder; IR (KBr) cm⁻¹: 2928.64, 1630.81; 1579.80, ¹H-NMR (300 MHz, DMSO-d₆) δ: 2.48–2.51 (4s, 12H, 4CH₃CO); 3.20–3.28 (d, 2H, methylene), 3.40–4.86 (m, 5H, CH, tetrahydropyran), 7.15–7.18 (s, 1H, CH), 7.36–8.52 (m, 3H, thiophene), ¹³C-NMR (300 MHz, DMSO-d₆) δ: 20 (4 CH₃); 59 (CH₂); 67.6 (5 C-O); 79.4 (2 C); 127.53 (CH); 128 (CH); 129.01 (CH); 132.63 (C-S); 149.7 (C=C); 158.8 (C=N); 165.2; 170 (5 C=O). MS m/z: 557 (M), Anal. Calcd for C₂₂H₂₃NO₁₀S₃ (557.61): C, 47.39; H, 4.16; N, 2.51; S, 17.25. Found: C, 47.26; H, 3.98; N, 2.38; S, 16.55.

Synthetic Procedure for Compound (10)

5-(Thiophen-2-yl)-5,5a,8,8a-tetrahydro-3H-thiazolo[5,4-f][1,2,4]triazepine-3,7(4H)-dithione (10)

Refluxing for 14 h a solution of Chalcone (1) (2.3 g, 10 mmol) with thiosemicarbazide (0.91g, 10 mmol) was dissolved in dioxane (30 mL), then adding 1 mL glacial acetic acid, after that, The reaction contents were poured into crushed ice. The mixture was kept at room temperature overnight. Then the resulted solid was filtered off, washed with water and dried, afterwards recrystallized from methanol was performed.

Yields 72%, mp 109 °C, brown crystal; IR (KBr) cm⁻¹: 3416.49; 1628.47, 1413.17, 1383.65. ¹H-NMR (300 MHz, DMSO-d₆) δ: 3.27 (s, 1H, CH), 3.49, 5.12 (s, 2H, NH, D₂O exchangeable); 7.28–8.05 (m, 3H, thiophen), ¹³C-NMR (300 MHz, DMSO-d₆) δ: 79.3 (C–N–); 97.35 (C–S); 126.53 (CH); 127 (CH); 128.3 (CH); 133 (C–NH); 135.63 (C–C); 180.86 (C=S); 199.2 (C=S). MS m/z: 298 (M), 143, 165, 138, Anal. Calcd for C₉H₆N₄S₄ (298.43): C, 36.22; H, 2.03; N, 18.77; S, 42.98. Found: C, 36.15; H, 2.06; N, 18.73; S, 42.99.

Biological Assays

CDK2/Cyclin A2 Assay

The In vitro assay of CDK2/cyclin A2 protein kinase was carried out on the synthesized compounds 1, 2, 4–7, 8b and 9 in Egypt using Kinase-Glo Plus luminescence kinase Assay kit (Promega).³²⁾ Protocol steps were followed by diluting the enzyme and the substrate. Also, ATP and inhibitors dilution in Kinase Buffer was performed before the addition of 1 µL of inhibitor or 5% DMSO, 2 µL of enzyme and 2 µL of substrate/ATP mix. After 10 min room temperature incubation, 5 µL of ADP-Glo™ Reagent was added then incubated for 40 min. Afterwards, an amount of Kinase Detection Reagent (10 µL) was added and incubated for 30 min at room temperature. Luminescence was then detected (Integration time range 0.5–1 s). The luminescent signal was directly correlated with the quantity of ATP present but inversely correlated with the kinase activity.

Cell Viability 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay

The in vitro cytotoxicity of synthesized compounds 1, 2, 4–7, 8b and 9 against cancer cell lines of breast cancer (MCF-7) and colon cancer (HCT-116) was performed in Egypt with the MTT assay procedure.³⁷⁾ Cell Line cells source was from American Type Culture Collection. Plate cells used (cells density 1.2–1.8 × 10000 cells/well) were of 100 µL complete growth medium +100 µL of each tested compound in each well of a 96-well plate for 24 h prior to MTT assay. The MTT method is appropriate for usage with multiwell plates. Final cell count should not be more than 10⁶ cells/cm². A blank should be included for each test containing the medium without the cells. Absorbance was measured spectrophotometrically at 570 nm wavelength. The background absorbance was measured of multiwell plates at 690 nm and then subtracted from the 450 nm absorbance. Tests performed in multiwell plates can be determined using the suitable plate reader type or by using appropriate size cuvettes for spectrophotometric measurement.

Flow Cytometry Cell Cycle Analysis

The protocol of cell cycle analysis is based on the quantitation of the content of DNA staining using propidium iodide. Cells were washed in phosphate buffered saline (PBS) then kept for 3 min at 4 °C by the addition of cold 70% ethanol drop wise using vortex, to ensure fixation and avoid any cell clumping. Then ribonuclease (50 µL from a stock of 100 µg/mL) was added to confirm staining of only DNA. Then, (200 µL from a stock concentration of 50 µg/mL) of propidium iodide was added.³⁴⁾

Caspase-7 Study

The assay protocol was followed where; caspase-7 buffer (100 µL) was added per well of the reaction plate. Afterwards, 100 µL of cell lysate was added. Immediate absorbance measurement of each sample at 405 nm is considered as the initial reading, while the final reading was recorded 30 min later. The resulted absorbance was compared to control normal cells. Where, any increase in absorbance was directly proportional to active caspase-7 amount per each culture sample.

Acknowledgments

Acknowledgement for Future University in Egypt (FUE) for performing 3D QSAR studying Computer Aided Drug Design labs.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

References

• 1) Chen J., Pang L., Wang W., Wang L., Zhang J. Z., Zhu T., J. Biomol. Struct. Dyn., 38, 985–996 (2020).
• 2) Chen J., Wang X., Zhang J. Z., Zhu T., ACS Omega, 3, 18052–18064 (2018).
• 3) Kontopidis G., McInnes C., Pandalaneni S. R., McNae I., Gibson D., Mezna M., Thomas M., Wood G., Wang S., Walkinshaw M. D., Fischer P. M., Chem. Biol., 13, 201–211 (2006).
• 4) Johnson L. N., De Moliner E., Brown N. R., Song H., Barford D., Endicott J. A., Noble M. E., Pharmacol. Ther., 93, 113–124 (2002).
• 5) Peyressatre M., Prével C., Pellerano M., Morris M. C., Cancers, 7, 179–237 (2015).
• 6) Bozdağ-Dündar O., Özgen Ö., Menteşe A., Altanlar N., Atlı O., Kendi E., Ertan R., Bioorg. Med. Chem., 15, 6012–6017 (2007).
• 7) Zervosen A., Lu W. P., Chen Z., White R. E., Demuth T. P. Jr., Frère J. M., Antimicrob. Agents Chemother., 48, 961–969 (2004).
• 8) Chawla A., Kaur H., Chawla P., Baghel U., J. Global Trends in Pharma. Sci., 5, 1641–1648 (2014).
• 9) Dayam R., Sanchez T., Clement O., Shoemaker R., Sei S., Neamati N., J. Med. Chem., 48, 111–120 (2005).
• 10) Terashima H., Hama K., Yamamoto R., Tsuboshima M., Kikkawa R., Hatanaka I., Shigeta Y., J. Pharmacol. Exp. Ther., 229, 226–230 (1984).
• 11) Ravi S., Chiruvella K. K., Rajesh K., Prabhu V., Raghavan S. C., Eur. J. Med. Chem., 45, 2748–2752 (2010).
• 12) da Rocha L. F. Junior., de Melo Rêgo M. J., Cavalcanti M. B., Pereira M. C., Pitta M. G., de Oliveira P. S., Gonçalves S. M., Duarte A. L., de Lima M. D., Pitta I. D., Pitta M. G., BioMed. research international., 2013, 926060 (2013).
• 13) Swathi N., Ramu Y., Subrahmanyam C., Satyanarayana K., Int. J. Pharm. Sci., 4, 561–566 (2012).
• 14) Mandal S. P., Garg A., Sahetya S. S., Nagendra S. R., Sripad H. S., Manjunath M. M., Soni M., Baig R. N., Kumar S. V., Kumar B. P., RSC Advances, 6, 58641–58653 (2016).
• 15) Alegaon S. G., Alagawadi K. R., Med. Chem. Res., 21, 816–824 (2012).
• 16) Mohamed S. F., Flefel E. M., Amr A. E., El-Shafy D. N., Eur. J. Med. Chem., 45, 1494–1501 (2010).
• 17) Barsoum F. F., Girgis A. S., Eur. J. Med. Chem., 44, 2172–2177 (2009).
• 18) Yusuf M., Jain P., Arab. J. Chem, 7, 553–596 (2014).
• 19) Siddiqui A. A., Rahman M. A., Shaharyar M., Mishra R., Chem. Sci. J., 8, 1–10 (2010).
• 20) Sharshira E. M., Hamada N. M., Molecules, 16, 7736–7745 (2011).
• 21) Hamadi N. B., Haouas A., Methamem M., Msaddek M., J. Chem. Res., 36, 563–565 (2012).
• 22) Havrylyuk D., Zimenkovsky B., Vasylenko O., Zaprutko L., Gzella A., Lesyk R., Eur. J. Med. Chem., 44, 1396–1404 (2009).
• 23) Sim M. M., Ng S. B., Buss A. D., Crasta S. C., Goh K. L., Lee S. K., Bioorg. Med. Chem. Lett., 12, 697–699 (2002).
• 24) Turan-Zitouni G., Chevallet P., Kilic F. S., Erol K., Eur. J. Med. Chem., 35, 635–641 (2000).
• 25) Bondock S., Naser T., Ammar Y. A., Eur. J. Med. Chem., 62, 270–279 (2013).
• 26) Ali G. M., Ibrahim D. A., Elmetwali A. M., Ismail N. S., Bioorg. Chem., 86, 1–14 (2019).
• 27) Thomas H. D., Wang L. Z., Roche C., Bentley J., Cheng Y., Hardcastle I. R., Golding B. T., Griffin R. J., Curtin N. J., Newell D. R., Eur. J. Cancer, 47, 2052–2059 (2011).
• 28) Joshi V. D., Kshirsagar M. D., Singhal S., Der Pharmacia Sinica, 3, 343–348 (2012).
• 29) Sharshira E. M., Hamada N. M., Am. J. Org. Chem., 2, 26–31 (2012).
• 30) Moharram H. H., Tammam G. H., Mansour S. A., ChemInform, 15 (1984).
• 31) Nassar I. F., Farargy El A. F., Abdelrazek F. M., Ismail N. S., Nucleosides Nucleotides Nucleic Acids, 36, 275–291 (2017).
• 32) Zegzouti H., Vidugiriene J., Goueli S., “ADP-Glo™ Kinase Assay Application Notes,” Springer, New York, 2016.
• 33) Girgis A. S., Stawinski J., Ismail N. S., Farag H., Eur. J. Med. Chem., 47, 312–322 (2012).
• 34) Pozarowski P., Darzynkiewicz Z., “Checkpoint Controls and Cancer,” Humana Press, NJ, 2004, pp. 301–311.
• 35) Goldstone S., Pavey S., Forrest A., Sinnamon J., Gabrielli B., Oncogene, 20, 921–932 (2001).
• 36) Nowroozi J., Mirzaii M., Norouzi M., Iran. J. Public Health, 1–7 (2004).
• 37) Fawzy I. M., Youssef K. M., Lasheen D. S., Ismail N. S., Abouzid K. A., Future Med. Chem., 10, 1421–1433 (2018).