Potent anti-tumor activity of a macrocycle-quinoxalinone class pan-Cdk inhibitor in vitro and in vivo.

Deregulation of cell-cycle control is a hallmark of cancer. Thus, cyclin-dependent kinases (Cdks) are an attractive target for the development of anti-cancer drugs. Here, we report the biological characterization of a highly potent pan-Cdk inhibitor with a macrocycle-quinoxalinone structure. Compound M inhibited Cdk1, 2, 4, 5, 6, and 9 with equal potency in the nM range and was selective against kinases other than Cdks. This compound inhibited multiple events in the cell cycle in vitro, including retinoblastoma protein (pRb) phosphorylation, E2F-dependent transcription, DNA replication (determined by bromodeoxyuridine incorporation), and mitosis completion (assayed by flow cytometry) in the 10 nM range. Moreover, this compound induced cell death, as determined by induction of the subG1 fraction, activated caspase-3, and anexin V. In vivo, Compound M showed anti-tumor efficacy at a tolerated dose. In a nude rat xenograft tumor model, an 8-h constant infusion of Compound M inhibited pRb phosphorylation and induced apoptosis in tumor cells at ~ 30 nM, which led to the inhibition of tumor growth. Immunosuppression was the only liability observed at this dose, but immune function returned to normal after 10 days. Suppression of pRb phosphorylation in tumor cells was clearly correlated with tumor cell growth inhibition and cell death in vitro and in vivo. In vivo, Compound M inhibited pRb phosphorylation in both tumor and gut crypt cells. Rb phosphorylation may be a suitable pharmacodynamic biomarker in both tumors and normal tissues for monitoring target engagement and predicting the efficacy of Compound M.

Biological characterization of 2-aminothiazole-derived Cdk4/6 selective inhibitor in vitro and in vivo.

Abnormalities in the p16INK4a/ cyclin-dependent kinase (Cdk)4, 6/ Retinoblastoma (Rb) pathway frequently occur in various human cancers. Thus, Cdk4/6 is an attractive target for cancer therapy. Here we report the biological characterization of a 2-aminothiazole-derived Cdk4/6 selective inhibitor, named Compound A in vitro and in vivo. Compound A potently inhibits Cdk4 and Cdk6 with high selectivity (more than 57-fold) against other Cdks and 45 serine/threonine and tyrosine kinases. Compound A inhibits Rb protein (pRb) phosphorylation at Ser780, inhibits E2F-dependent transcription, and induces cell-cycle arrest at G1 in the T98G human glioma cell line. Among 82 human cells derived from various tissues, cell lines derived from hematological cancers (leukemia/lymphoma) tended to be more sensitive to Compound A in cell proliferation assay. Rb-negative cells tended to be insensitive to Compound A, as we had expected. In a nude rat xenograft model, Compound A inhibited pRb phosphorylation and bromodeoxyuridine (BrdU) incorporation in Eol-1 xenograft tumor at plasma concentration of 510 nM. Interestingly Compound A only moderately inhibited those pharmacodynamic and cell cycle parameters of normal crypt cells in small intestine even at 5 times higher plasma concentration. In F344 rats, Compound A did not cause immunosuppression even at 17 times higher plasma conc. These results suggest that Cdk4/6 selective inhibitors only moderately affects on the cell cycle of normal proliferating tissues and has a safer profile than pan-Cdk inhibitor in vivo.