Inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) represents a promising strategy for the discovery of a new generation of anticancer chemotherapeutics. Our synthetic efforts, beginning from the lead compound 2, were directed at improving antiproliferative activity against cancer cells as well as various drug properties. These efforts led to the discovery of N-{3-[3-cyclopropyl-5-(2-fluoro-4-iodophenylamino)-6,8-dimethyl-2,4,7-trioxo-3,4,6,7-tetrahydro-2H-pyrido[4,3-d]pyrimidin-1-yl]phenyl}acetamide dimethylsulfoxide solvate (GSK1120212, JTP-74057 DMSO solvate; 1), a selective and highly potent MEK inhibitor with improved drug properties. We further confirmed that the antiproliferative activity correlates with cellular MEK inhibition and observed significant antitumor activity with daily oral dosing of 1 in a tumor xenograft model. These qualities led to the selection of 1 for clinical development.

The INK4 family members p16INK4a and p15 INK4b negatively regulate cell cycle progression by inhibition of cyclin-dependent kinase (CDK) 4/6. Loss of p16INK4a functional activity is frequently observed in tumor cells, and is thought to be one of the primary causes of carcinogenesis. In contrast, despite the biochemical similarity to p16 INK4a, the frequency of defects in p15INK4b was found to be lower than in p16 INK4a, suggesting that p15 INK4b-inductive agents may be useful for tumor suppression. Here we report the discovery of a novel pyrido-pyrimidine derivative, JTP-70902, which exhibits p15

A small molecule compound, JTP-74057/GSK1120212/trametinib, had been discovered as a very potent antiproliferative agent able to induce the accumulation of CDK inhibitor p15INK4b. To conduct its drug development rationally as an anticancer agent, molecular targets of this compound were identified as MEK1/2 using compound-affinity chromatography. It was shown that JTP-74057 directly bound to MEK1 and MEK2 and allosterically inhibited their kinase activities, and that its inhibitory characteristics were similar to those of the known and different chemotype of MEK inhibitors PD0325901 and U0126. It was further shown that JTP-74057 induced rapid and sustained dephosphorylation of phosphorylated MEK in HT-29 colon and other cancer cell lines, while this decrease in phosphorylated MEK was not observed in PD0325901-treated cancer cells. Physicochemical analyses revealed that JTP-74057 preferentially binds to unphosphorylated MEK (u-MEK) in unique characteristics of both high affinity based on extremely low dissociation rates and ability stabilizing u-MEK with high thermal shift, which were markedly different from PD0325901. These findings indicate that JTP-74057 is a novel MEK inhibitor able to sustain MEK to be an unphosphorylated form resulting in pronounced suppression of the downstream signaling pathways involved in cellular proliferation.

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Jun N-terminal kinase; LC3, microtubule associated protein 1 light chain 3; LC-MS/MS, liquid chromatography-tandem mass spectrometry; mTOR, mammalian target of rapamycin; PBS, phosphate-buffered saline; p70S6K, p70 S6 kinase; RFP, red fluorescent protein TagRFP; ROS, reactive oxygen species; qRT-PCR, quantitative reverse transcription polymerase chain reaction; RPS6, S6 ribosomal protein; siRNA, small interference RNA; Tm, melting temperature; TPP, thermal proteome profiling; TR, temperature range.