Recent advances in understanding the role of JAK2 V617F mutation in Bcr-Abl negative myeloproliferative (MPD) diseases pathogenesis opened up a possibility to develop highly targeted therapies against these debilitating ailments. We used a Ba/F3 cell line expressing the V617F mutant of JAK2 to screen a focused small molecule library for potential inhibitors of JAK2 V617F-dependent proliferation. Further extensive SAR of initial hits resulted in identification of R723, a potent and selective JAK2 inhibitor. This molecule is strongly antiproliferative (IC50 130–200 nM) against mouse BaF3 cells used for initial screening as well as against human UKE1 and SET2 cell lines harboring the same mutation. On the other hand, R723 has only weak activity in IL2-dependent (i.e. JAK1/JAK3-dependent) proliferation assays performed with human primary T (IC50 1300 nM) and mouse T-cell leukaemia CTLL2 cells (IC50 600 nM). A 10 to 20 fold cell-based selectivity of R723 was further confirmed by measuring inhibition of constitutive STAT5 phosphorylation in SET2 and BaF3 cells versus inhibition of IL-2 inducible STAT5 phosphorylation in human primary T and mouse CTLL2 cells using FACS-based approach. Compound R723 has low nonspecific antiproliferative activity against JAK2-independent MOLT4, A549 and H1299 cell lines with an IC50 ranging from 4 to 6 uM. The molecule has been also proven to be potent (IC50 of 2 nM against JAK2 in biochemical assay) and highly selective (window of more than 500 fold over JAK1 and 10 fold over JAK3) inhibitor of JAK2 kinase in vitro. Moreover, when tested in biochemical assay against a panel of more than 200 kinases at a concentration of 20 nM (IC90 for JAK2), R723 inhibited none of them. The selectivity of R723 was further confirmed using a variety of cell-based assays probing T-, B- and mast cell activation. Compound R723 was further evaluated in a stress-induced erythropoiesis mouse model, where kinetics of EPO-dependent hematocrit recovery from phenylhydrazine-induced anemia was assessed. Significant delay in recovery was observed at doses of 75 and 100 mg/kg bid indicating strong compound effect on EPOR signaling in vivo. The result could not be attributed to general toxicity effects as 14 day toxicology study did not identify any abnormalities at doses tested. As a result, R723 could become the basis for next generation of potent and selective compounds targeting JAK2-dependent myeloproliferative diseases.

Disclosures: Markovtsov:Rigel Pharmaceutical: Employment, Equity Ownership, Patents & Royalties. Tonkin:Rigel Pharmaceutical: Employment. Fang:Rigel Pharmaceutical: Employment. Liu:Rigel Pharmaceutical: Employment. Gelman:Rigel Pharmaceutical: Employment. Lang:Rigel Pharmaceutical: Employment. Romero:Rigel Pharmaceutical: Employment. McLaughlin:Rigel Pharmaceutical: Employment. Bhamidipati:Rigel Pharmaceutical: Employment. Clough:Rigel Pharmaceutical: Employment. Singh:Rigel Pharmaceutical: Employment. Low:Rigel Pharmaceutical: Employment. Reitsma:Rigel Pharmaceutical: Employment. Taylor:Rigel Pharmaceutical: Employment. Siu:Rigel Pharmaceutical: Employment. Park:Rigel Pharmaceutical: Employment. Sweeney:Rigel Pharmaceutical: Employment. Pine:Rigel Pharmaceutical: Employment. Torneros:Rigel Pharmaceutical: Employment. Clemens:Rigel Pharmaceutical: Employment. Duan:Rigel Pharmaceutical: Employment. Daniel:Rigel Pharmaceutical: Employment. Payan:Rigel Pharmaceutical: Employment, Equity Ownership, Patents & Royalties. Holland:Rigel Pharmaceutical: Employment. Hitoshi:Rigel Pharmaceutical: Employment.

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