ABSTRACT
A series of N-6 substituted 9-hydroxy-4-phenylpyrrolo[3,4-c]carbazole-1,3(2H,6H)-diones were prepared from N-substituted (5-methoxyphenyl)ethenylindoles. The target compounds were tested for their ability to inhibit the G2/M cell cycle checkpoint kinases, Wee1 and Chk1. Analogues with neutral or cationic N-6 side chains were potent dual inhibitors. Acidic side chains provided potent (average IC(50) 0.057 microM) and selective (average ratio 223-fold) Wee1 inhibition. Co-crystal structures of inhibitors bound to Wee1 show that the pyrrolo[3,4-c]carbazole scaffold binds in the ATP-binding site, with N-6 substituents involved in H-bonding to conserved water molecules. HT-29 cells treated with doxorubicin and then target compounds demonstrate an active Cdc2/cyclin B complex, inhibition of the doxorubicin-induced phosphorylation of tyrosine 15 of Cdc2 and abrogation of the G2 checkpoint.
Subject(s)
Carbazoles/chemical synthesis , Carbazoles/pharmacology , Cell Cycle Proteins/antagonists & inhibitors , Nuclear Proteins/antagonists & inhibitors , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/antagonists & inhibitors , Carbazoles/chemistry , HT29 Cells , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Protein Kinase Inhibitors/chemistry , Structure-Activity RelationshipABSTRACT
High-throughput screening has identified a novel class of inhibitors of the checkpoint kinase Wee1, which have potential for use in cancer chemotherapy. These inhibitors are based on a 4-phenylpyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione template and have been shown by X-ray crystallography to bind at the ATP site of the enzyme. An extensive study of the effects of substitution around this template has been carried out, which has identified substituents which lead to improvements in potency and selectivity for Wee1. While retention of the maleimide ring and pendant 4-phenyl group is necessary for potency, replacement of the carbazole nitrogen by oxygen is well tolerated and results in improved Wee1 selectivity against the related checkpoint kinase Chk1. Wee1 potency and selectivity are also enhanced by the incorporation of lipophilic functionality at the 2'-position of the 4-phenyl ring, and Wee1 selectivity against Chk1 is favored by C3-C5 alkyl substitution of the carbazole nitrogen. These studies provide a basis for the design of active analogues of the pyrrolocarbazole lead with improved physical properties.
Subject(s)
Benzene Derivatives/chemical synthesis , Carbazoles/chemical synthesis , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/chemistry , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/chemistry , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/chemistry , Pyrroles/chemical synthesis , Benzene Derivatives/chemistry , Carbazoles/chemistry , Checkpoint Kinase 1 , Crystallography, X-Ray , Humans , Models, Molecular , Protein Binding , Protein Kinases/chemistry , Pyrroles/chemistry , Structure-Activity RelationshipABSTRACT
The inhibition of cyclin-dependent kinase 4 (Cdk4) causes cell cycle arrest and restores a checkpoint that is absent in the majority of tumor cells. Compounds that inhibit Cdk4 selectively are targeted for treating cancer. Appropriate substitution of 2-aminoquinazolines is demonstrated to produce high levels of selectivity for Cdk4 versus closely related serine-threonine kinases.
Subject(s)
Antineoplastic Agents/chemical synthesis , Cyclin-Dependent Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Quinazolines/chemical synthesis , Antineoplastic Agents/pharmacology , Cyclin-Dependent Kinase 4 , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Humans , Inhibitory Concentration 50 , Quinazolines/pharmacology , Structure-Activity RelationshipABSTRACT
Inhibition of the cell cycle kinase, cyclin-dependent kinase-4 (Cdk4), is expected to provide an effective method for the treatment of proliferative diseases such as cancer. The pyrido[2,3-d]pyrimidin-7-one template has been identified previously as a privileged structure for the inhibition of ATP-dependent kinases, and good potency against Cdks has been reported for representative examples. Obtaining selectivity for individual Cdk enzymes, particularly Cdk4, has been challenging. Here, we report that the introduction of a methyl substituent at the C-5 position of the pyrido[2,3-d]pyrimidin-7-one template is sufficient to confer excellent selectivity for Cdk4 vs other Cdks and representative tyrosine kinases. Further optimization led to the identification of highly potent and selective inhibitors of Cdk4 that exhibit potent antiproliferative activity against human tumor cells in vitro. The most selective Cdk4 inhibitors were evaluated for antitumor activity against MDA-MB-435 human breast carcinoma xenografts in mice.
Subject(s)
Antineoplastic Agents/chemical synthesis , Cyclin-Dependent Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Pyridines/chemical synthesis , Pyrimidines/chemical synthesis , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cyclin-Dependent Kinase 4 , Cyclin-Dependent Kinases/chemistry , Drug Screening Assays, Antitumor , Humans , Mice , Mice, Nude , Models, Molecular , Proto-Oncogene Proteins/chemistry , Pyridines/chemistry , Pyridines/pharmacology , Pyrimidines/chemistry , Pyrimidines/pharmacology , Stereoisomerism , Transplantation, HeterologousABSTRACT
A series of 2-anilino-6-phenylpyrido[2,3-d]pyrimidin-7(8H)-ones were synthesized and evaluated for their inhibitory properties against the non-receptor kinase c-Src and the G2/M checkpoint kinase Wee1. Overall, the compounds were 10-100-fold more potent inhibitors of c-Src than Wee1, and variation of substituents on the 6-phenyl ring did not markedly alter this preference. Solubilizing substituents off the 2-anilino ring in many cases increased Wee1 activity, thus lowering this preference to about 10-fold. 5-Alkyl substituted analogs were generally Wee1 selective, but at the expense of absolute potency.
