الفهرس 
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Abstract
Cancer represents a pathological manifestation of uncontrolled cell division leading to uncontrolled proliferative growth and the spread of aberrant cells from their site of origin. There were many pieces of evidence to support that the inhibition of CDKs could play vital role in suppressing cancer. Human kinome is constituted by a total of 518 kinases out of which 388 are serine/threonine (Ser/Thr) kinases. 20 of these Ser/Thr kinases are further classified as Cyclin-dependent kinase (CDK). CDK2 is a member of protein kinase family. It plays an important role in regulating various events of eukaryotic cell division cycle. Accumulated evidences indicated that over expression of CDK2 should cause the abnormal regulation of cell-cycle. Therefore, CDK2 was regarded as a potentially therapeutic target for cancer therapy. Among the ten pharmacological inhibitors of CDK2 are currently in clinical trials, a potent CDK2 inhibitor with purine
scaffold has been synthesized, such as roscovitine (Seliciclib®) which undergoing phase 2
trials against non-small cell lung (NSCL) and nasopharyngeal cancers. The aim of this study was designed to generate more potent analogues of roscovitine.
In this work, a series of pyrazolo[1,5-a][1,3,5]triazine substituted with different amines was designed and synthesized in an attempt to improve inhibitory activity against CDK2/cyclinA. The design focused on exploration of the previous revealed SAR studies, bioisosteric modifications of the lead compound studies, and identification of the key interactions with the binding site. Synthesis of the designed compounds was then accomplished and their structures were confirmed by various spectral and microanalytical data.
This study involved the synthesis of the following unavailable reported intermediates:
1) 5-Amino-1H-pyrazole-4-carbonitrile (II)
2) Benzoyl chloride (IVa)
3) 4-Chlorobenzoyl chloride (IVb)
4) 4-Bromobenzoyl chloride (IVc)
5) Benzoyl isothiocyanate (Va)
6) 4-Chlorobenzoyl isothiocyanate (Vb)
7) 4-Bromobenzoyl isothiocyanate (Vc)
8) 1-Benzoyl-3-(4-cyano-1H-pyrazol-5-yl)thiourea (VIa)
Also, it comprises the following new intermediates:
1) 1-(4-Chlorobenzoyl)-3-(4-cyano-1H-pyrazol-5-yl)thiourea (VIb)
2) 1-(4-Bromobenzoyl)-3-(4-cyano-1H-pyrazol-5-yl)thiourea (VIc)
Moreover, these new target compounds were synthesized:
1) 2-(Ethylthio)-4-phenylpyrazolo[1,5-a][1,3,5]triazine-8-carbonitrile (VIIIa)
2) 4-(4-Chlorophenyl)-2-(ethylthio)pyrazolo[1,5-a][1,3,5]triazine-8-carbonitrile
(VIIIb)
3) 4-(4-Bromophenyl)-2-(ethylthio)pyrazolo[1,5-a][1,3,5]triazine-8-carbonitrile
(VIIIc)
4) 2-(Benzylamino)-4-phenylpyrazolo[1,5-a][1,3,5]triazine-8-carbonitrile (IXa)
5) 2-(Phenethylamino)-4-phenylpyrazolo[1,5-a][1,3,5]triazine-8carbonitrile (IXb)
6) 2-(3-Isopropoxypropylamino)-4-phenylpyrazolo[1,5-a][1,3,5]triazine-8- carbonitrile (IXc)
7) 2-(Butylamino)-4-phenylpyrazolo[1,5-a][1,3,5]triazine-8-carbonitrile (IXd)
8) 2-(Isobutylamino)-4-phenylpyrazolo[1,5-a][1,3,5]triazine-8-carbonitrile (IXe)
9) 2-(Ethylsulfonyl)-4-phenylpyrazolo[1,5-a][1,3,5]triazine-8-carbonitrile (IXf)
10) 2-(Benzylamino)-4-(4-chlorophenyl)pyrazolo[1,5-a][1,3,5]triazine-8-carbonitrile
(IXg)
11) 4-(4-Chlorophenyl)-2-(phenethylamino)pyrazolo[1,5-a][1,3,5]triazine-8 carbonitrile (IXh)
12) 4-(4-Chlorophenyl)-2-(3-isopropoxypropylamino)pyrazolo[1,5-a][1,3,5] triazine- 8-carbonitrile (IXi)
13) 2-(Butylamino)-4-(4-chlorophenyl)pyrazolo[1,5-a][1,3,5]triazine-8-carbonitrile
(IXj)
14) 4-(4-Chlorophenyl)-2-(isobutylamino)pyrazolo[1,5-a][1,3,5]triazine-8 carbonitrile (IXk)
15) 4-(4-Chlorophenyl)-2-(ethylsulfonyl)pyrazolo[1,5-a][1,3,5]triazine-8-carbonitrile
(IXl)
16) 4-(4-Chlorophenyl)-2-(cyclopropylamino)pyrazolo[1,5-a][1,3,5]triazine-8 carbonitrile (IXm)
17) 2-(Benzylamino)-4-(4-bromophenyl)pyrazolo[1,5-a][1,3,5]triazine-8-carbonitrile
(IXn)
18) 4-(4-Bromophenyl)-2-(phenethylamino)pyrazolo[1,5-a][1,3,5]triazine-8- carbonitrile (IXo)
19) 4-(4-Bromophenyl)-2-(3-isopropoxypropylamino)pyrazolo[1,5-a][1,3,5] triazine- 8-carbonitrile (IXp)
The biological activity of the compounds was evaluated at both molecular and cellular levels. All final compounds (19) were evaluated for their CDK2/cyclinA inhibition activity. The evaluation of CDK2/cyclinA inhibition at 10 µM was performed in BioScience Corporation. Compounds showing above 60% inhibition at 10 µM were further tested for determination of their IC50. Eight of the final compounds (IXc, IXf, IXg, IXh, IXj, IXk, IXl and IXm) were selected by the National Cancer Institute (NCI) for single dose screening program at 10 µM in the full NCI 60 cell panel to evaluated anticancer activity. Eleven of remaining compounds (VIIIa, VIIIb, VIIIc, IXa, IXb, IXd, IXe, IXi, IXn, IXo and IXp) were evaluated the cytotoxic activity against the renal cancer cell lines (UO-31) and the results were excellent when compared with doxorubicin as a reference drug.
Finally, a thorough molecular docking, using C-DOCKER protocol in Discovery Studio
2.5 Software was attempted to investigate the binding mode of the targeted compounds and interpret their variable inhibitory activity.