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Title: Design and synthesis of purine isosteres as inhibitors of Nek2 and CDK2
Author: Turner, David Michael
Awarding Body: University of Newcastle Upon Tyne
Current Institution: University of Newcastle upon Tyne
Date of Award: 2013
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Nek2 and CDK2 are serine/threonine protein kinases that are implicated in cell cycle control and cancer. The Nek family of enzymes contains 11 known serine/threonine protein kinases (Nek1-11) and are involved in mitotic cell cycle control. There is evidence that of these 11 kinases, Nek2, Nek6, Nek7 and Nek9 play an important role in the regulation of mitotic events through microtubule control. Particular interest has been placed upon Nek2, which has been shown to have a critical role in mitosis, assisting centrosome disjunction. 6-Ethnyl purine 39, was identified as a submicromolar irreversible inhibitor of Nek2 (IC50 = 150 nM), exhibiting good selectivity over other members of the Nek family. It was believed that this compound formed a triplet of H-bonds with the amino acids of the Nek2 ATP-binding site hinge region via the 2-amino N-H, the purine N3 and the imidazole N9H, allowing an initial noncovalent binding interaction, which facilitates subsequent covalent modification. To validate this binding motif and to act as control compounds, N-methylated purines 52-54 were synthesised, along with the 2-phenoxypurine 55 and the 2-benzylpurine 56. These compounds were all essentially inactive in the Nek2 inhibition assay, which demonstrated that the purine 2-amino N-H group and imidazole N9H were essential for non-covalent binding interactions. 5 To investigate the influence of the purine heterocycle upon Nek2 inhibitory activity and ethynyl group reactivity, heterocyclic derivatives of 39 were synthesised. Deazapurines 87 and 88 were weakly active in the Nek2 inhibition assay (IC50 = 30 μM and 24 μM, respectively), whilst pyrazolopyrimidine 89 and triazolopyrimidine 90 had sub-micromolar activity comparable with the parent purine 39. The 5- formylpyrimidine 101 and triazine 91 were modest inhibitors of Nek2 (IC50 = > 10 μM and 17 μM, respectively). By contrast, pyrimidine 95 (10% inhibition at 100 μM) demonstrated weak Nek2 inhibitory activity. Quantitative 1H NMR (q1H-NMR) kinetic studies were performed to model the reaction of ethynyl-functionalised heterocycles with Cys-22 at the Nek2 ATP-binding site. Compounds 54, 56, and 88-89 were reacted with N-acetylcysteine methyl ester in DMSO-d6 at 24 oC under pseudo first order reaction conditions. The most reactive compounds were triazolopyrimidine 90 and pyrazolopyrimidine 89. The 2- benzylpurine 56 and N-methylanilinopurine 54 had moderate reactivity, comparable with the parent purine 39. The least reactive compound was pyrrolopyrimidine 88, which was approximately 150-fold less reactive than purine 39. 6 CDK2 is a member of the cyclin-dependent kinase (CDK) family of enzymes and a mediator of cell cycle progression. Of the 11 known human CDKs, four (CDK1, CDK2 CDK4 and CDK6) have been directly implicated in cell cycle regulation. Mutation of genes coding for CDKs are common in a variety of cancers, making CDKs attractive chemotherapeutic targets. Purines 249 and 37 were identified as moderate (IC50 = 17 μM) and potent (IC50 = 5 nM) ATP-competitive inhibitors of CDK2, respectively. The focus of this research was to determine whether subtle changes to the core heterocycle would allow retention of CDK-inhibitory activity. CDK2 inhibitors based on the pyrazolopyrimidine and triazolopyrimidine heterocycles (256, 252, 257 and 253) were of comparable potency with the corresponding purines (249 and 37). The most potent compound was triazolopyrimidine 253 with an IC50 value of 3 nM against CDK2. Interestingly, the pyrrolopyrimidine 255 proved only weakly active (IC50 > 100 μM), whereas the functionalised pyrrolopyrimidine 251 had potent CDK2 inhibitory activity (IC50 = 26 nM). Finally, imidazopyridine 254 exhibited activity comparable with purine 249; however, imidazopyridine 250 was found to be approximately 30-fold less potent (IC50 = 140 nM) than the parent 37, for reasons that remain unclear. N.B. diagrams not included in this abstract
Supervisor: Not available Sponsor: Engineering and Physical Sciences Research Council (EPSRC) ; Cancer Research UK
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available