SAR and in vivo evaluation of 4-aryl-2-aminoalkylpyrimidines as potent and selective Janus kinase 2 (JAK2) inhibitors.

We report the discovery of a series of 4-aryl-2-aminoalkylpyrimidine derivatives as potent and selective JAK2 inhibitors. High throughput screening of our in-house compound library led to the identification of hit 1, from which optimization resulted in the discovery of highly potent and selective JAK2 inhibitors. Advanced lead 10d demonstrated a significant dose-dependent pharmacodynamic and antitumor effect in a mouse xenograft model. Based upon the desirable profile of 10d (XL019) it was advanced into clinical trials.

EXEL-7647 inhibits mutant forms of ErbB2 associated with lapatinib resistance and neoplastic transformation.

PURPOSE: Mutations associated with resistance to kinase inhibition are an important mechanism of intrinsic or acquired loss of clinical efficacy for kinase-targeted therapeutics. We report the prospective discovery of ErbB2 mutations that confer resistance to the small-molecule inhibitor lapatinib. EXPERIMENTAL DESIGN: We did in vitro screening using a randomly mutagenized ErbB2 expression library in Ba/F3 cells, which were dependent on ErbB2 activity for survival and growth. RESULTS: Lapatinib resistance screens identified mutations at 16 different ErbB2 amino acid residues, with 12 mutated amino acids mapping to the kinase domain. Mutations conferring the greatest lapatinib resistance cluster in the NH2-terminal kinase lobe and hinge region. Structural computer modeling studies suggest that lapatinib resistance is caused by multiple mechanisms; including direct steric interference and restriction of conformational flexibility (the inactive state required for lapatinib binding is energetically unfavorable). ErbB2 T798I imparts the strongest lapatinib resistance effect and is analogous to the epidermal growth factor receptor T790M, ABL T315I, and cKIT T670I gatekeeper mutations that are associated with clinical drug resistance. ErbB2 mutants associated with lapatinib resistance transformed NIH-3T3 cells, including L755S and T733I mutations known to occur in human breast and gastric carcinomas, supporting a direct mechanism for lapatinib resistance in ErbB2-driven human cancers. The epidermal growth factor receptor/ErbB2/vascular endothelial growth factor receptor inhibitor EXEL-7647 was found to inhibit almost all lapatinib resistance-associated mutations. Furthermore, no ErbB2 mutations were found to be associated with EXEL-7647 resistance and lapatinib sensitivity. CONCLUSIONS: Taken together, these data suggest potential target-based mechanisms of resistance to lapatinib and suggest that EXEL-7647 may be able to circumvent these effects.

Inhibition of the T790M gatekeeper mutant of the epidermal growth factor receptor by EXEL-7647.

PURPOSE: Agents inhibiting the epidermal growth factor receptor (EGFR) have shown clinical benefit in a subset of non-small cell lung cancer patients expressing amplified or mutationally activated EGFR. However, responsive patients can relapse as a result of selection for EGFR gene mutations that confer resistance to ATP competitive EGFR inhibitors, such as erlotinib and gefitinib. We describe here the activity of EXEL-7647 (XL647), a novel spectrum-selective kinase inhibitor with potent activity against the EGF and vascular endothelial growth factor receptor tyrosine kinase families, against both wild-type (WT) and mutant EGFR in vitro and in vivo. EXPERIMENTAL DESIGN: The activity of EGFR inhibitors against WT and mutant EGFRs and their effect on downstream signal transduction was examined in cellular assays and in vivo using A431 and MDA-MB-231 (WT EGFR) and H1975 (L858R and T790M mutant EGFR) xenograft tumors. RESULTS: EXEL-7647 shows potent and long-lived inhibition of the WT EGFR in vivo. In addition, EXEL-7647 inhibits cellular proliferation and EGFR pathway activation in the erlotinib-resistant H1975 cell line that harbors a double mutation (L858R and T790M) in the EGFR gene. In vivo efficacy studies show that EXEL-7647 substantially inhibited the growth of H1975 xenograft tumors and reduced both tumor EGFR signaling and tumor vessel density. Additionally, EXEL-7647, in contrast to erlotinib, substantially inhibited the growth and vascularization of MDA-MB-231 xenografts, a model which is more reliant on signaling through vascular endothelial growth factor receptors. CONCLUSIONS: These studies provide a preclinical basis for clinical trials of XL647 in solid tumors and in patients bearing tumors that are resistant to existing EGFR-targeted therapies.

Essential embryonic roles of the CKI-1 cyclin-dependent kinase inhibitor in cell-cycle exit and morphogenesis in C elegans.

Following a phase of rapid proliferation, cells in developing embryos must decide when to cease division and then whether to survive and differentiate or instead undergo programmed death. In screens for genes that regulate embryonic patterning of the endoderm in Caenorhabditis elegans, we identified overlapping chromosomal deletions that define a gene required for these decisions. These deletions result in embryonic hyperplasia in multiple somatic tissues, excessive numbers of cell corpses, and profound defects in morphogenesis and differentiation. However, cell-cycle arrest of the germline is unaffected. Cell lineage analysis of these mutants revealed that cells that normally stop dividing earlier than their close relatives instead undergo an extra round of division. These deletions define a genomic region that includes cki-1 and cki-2, adjacent genes encoding members of the Cip/Kip family of cyclin-dependent kinase inhibitors. cki-1 alone can rescue the cell proliferation, programmed cell death, and differentiation and morphogenesis defects observed in these mutants. In contrast, cki-2 is not capable of significantly rescuing these phenotypes. RNA interference of cki-1 leads to embryonic lethality with phenotypes similar to, or more severe than, the deletion mutants. cki-1 and -2 gene reporters show distinct expression patterns; while both are expressed at around the time that embryonic cells exit the cell cycle, cki-2 also shows marked expression starting early in embryogenesis, when rapid cell division occurs. Our findings demonstrate that cki-1 activity plays an essential role in embryonic cell cycle arrest, differentiation and morphogenesis, and suggest that it may be required to suppress programmed cell death or engulfment of cell corpses.