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
We disclose a novel series of indenopyrazole-based cyclin-dependent kinase (CDK) inhibitors. Kinetic experiments confirmed our initial molecular modeling studies that the compounds are competitive with respect to adenosine 5'-triphosphate (ATP) and bind in the kinase ATP pocket. A unique combination of active pharmacophores led us to a series of semicarbazide-based inhibitors that are highly potent against CDK2 and CDK4 while maintaining selectivity against other relevant serine/threonine kinases. These compounds were active against a transformed human colon cancer cell line (HCT116) while maintaining an acceptable margin of activity against a normal fibroblast cell line. The compounds were found to be highly protein bound in our cell-based assay with the exception of 11k, which maintained a reasonable level of activity in the presence of human plasma proteins.
