ABSTRACT
Using a high-throughput screening strategy, a series of 1-aryl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-ones was identified that inhibit the cyclin-dependent kinase (CDK) 4/cyclin D1 complex-mediated phosphorylation of a protein substrate with IC(50)s in the low micromolar range. On the basis of preliminary structure-activity relationships (SAR), a model was proposed in which these inhibitors occupy the ATP-binding site of the enzyme, forming critical hydrogen bonds to the same residue (Val96) to which the amino group in ATP is presumed to bind. X-ray diffraction studies on a later derivative bound to CDK2 support this binding mode. Iterative cycles of synthesis and screening lead to a novel series of potent, CDK2-selective 6-(arylmethyl)pyrazolopyrimidinones. Placement of a hydrogen-bond donor in the meta-position on the 6-arylmethyl group resulted in approximately 100-fold increases in CDK4 affinity, giving ligands that were equipotent inhibitors of CDK4 and CDK2. These compounds exhibit antiproliferative effects in the NCI HCT116 and other cell lines. The potency of these antiproliferative effects is enhanced in anilide derivatives and translates into tumor growth inhibition in a mouse xenograft model.
Subject(s)
Cyclin-Dependent Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Pyrazoles/chemical synthesis , Pyrimidines/chemical synthesis , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Binding Sites , Cell Line, Tumor , Cells, Cultured , Crystallography, X-Ray , Cyclin D1/antagonists & inhibitors , Cyclin D1/metabolism , Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinases/metabolism , Drug Screening Assays, Antitumor , Humans , Mice , Models, Molecular , Molecular Structure , Phosphorylation , Proto-Oncogene Proteins/metabolism , Pyrazoles/chemistry , Pyrazoles/pharmacology , Pyrimidines/chemistry , Pyrimidines/pharmacology , Structure-Activity Relationship , Transplantation, HeterologousABSTRACT
The identification of indeno[1,2-c]pyrazol-4-ones as inhibitors of cyclin-dependent kinases (CDKs) has led to the discovery of a series of novel and potent compounds. Herein, we report the effects of substitutions at C3 of the indeno[1,2-c]pyrazol-4-one core with alkyls, heterocycles, and substituted phenyls. Substitutions at the para position of the phenyl ring at C3 were generally well-tolerated; however, larger groups were generally inactive. For alkyls directly attached to C3, longer chain substituents were not tolerated; however, shorter alkyl groups and cyclic alkyls were acceptable. In general, the heterocycles at C3 gave the most potent analogues. One such heterocycle, 24j, was examined in detail and was determined to have a biological profile consistent with CDK inhibition. An X-ray crystal structure of one of the alkyl compounds, 13q, complexed with CDK2 was determined and showed the inhibitor residing in the adenosine 5'-triphosphate pocket of the enzyme.
