The success of imatinib, a BCR-ABL inhibitor for the treatment of chronic myelogenous leukemia, has created a great impetus for the development of additional kinase inhibitors as therapeutic agents. However, the complexity of cancer has led to recent interest in polypharmacological approaches for developing multi kinase inhibitors with low toxicity profiles. With this goal in mind, we analyzed more than 150 novel cyano pyridopyrimidine compounds and identified structure activity relationship trends that can be exploited in the design of potent kinase inhibitors. One compound, 8-Cyclopentyl-2-[4-(4-methyl-piperazin-1-yl)-phenylamino]-7-oxo-7,8-dihydro-pyrido[2,3-d]pyrimidine-6-carbonitrile (7x) was found to be the most active, inducing apoptosis of tumor cells at a concentration of approximately 30–100nM. In vitro kinase profiling revealed that 7x is a multi-kinase inhibitor with potent inhibitory activity against the CDK4/CYCLIN D1 and ARK5 kinases. Here, we report the synthesis, structure activity relationship, kinase inhibitory profile, in vitro cytotoxicity and in vivo tumor regression studies by this lead compound.

Highlights d Rigosertib does not bind directly to tubulin d Rigosertib dos not inhibit tubulin polymerization d Expression of TUBB L240F does not confer resistance to rigosertib d A small percentage of rigosertib-and BI2536-treated TUBB L240F cells undergo senescence

Rigosertib is a novel benzyl styryl sulfone that inhibits the growth of a wide variety of human tumor cells in vitro and in vivo and is currently in Phase III clinical trials. We recently provided structural and biochemical evidence to show that rigosertib acts as a RAS-mimetic by binding to Ras Binding Domains (RBDs) of the RAF and PI3K family proteins and disrupts their binding to RAS. In a recent study, Jost et al (2017) attributed the mechanism of action of rigosertib to microtubule-binding. In these studies, rigosertib was obtained from a commercial vendor. We have been unable to replicate the reported results with clinical grade rigosertib, and hence compared the purity of clinical grade and commercially sourced rigosertib. We find that the commercially sourced rigosertib contains a p p r o xi m a t e l y 5% ON01500, a potent inhibitor of tubulin polymerization. Clinical grade rigosertib, which is free of this impurity, does not exhibit tubulin binding activity. In vivo, cell lines that express mutant b-tubulin (TUBBL240F) were also reported to be resistant to the effects of rigosertib. However, our studies showed that both wild-type and TUBBL240F-expressing cells failed to proliferate in the presence of rigosertib at concentrations that are lethal to wild-type cells. Morphologically, we find that rigosertib, at lethal concentrations, induced a senescence-like phenotype in the small percentage of both wild-type and TUBBL240F-expressing cells that survive in the presence of rigosertib.Our results suggest that TUBBL240F expressing cells are more prone to undergo senescence in the presence of rigosertib as well as BI2536, an unrelated ATP-competitive pan-PLK inhibitor.The appearance of these senescent cells could be incorrectly scored as resistant cells in flow cytometric assays using short term cultures.