br Conflict of interest br Financial support br Acknowledgme

Conflict of interest
Financial support
Acknowledgments This work was supported by the Japanese Millennium project. We thank all members of the Center for Genomic Medicine of RIKEN and Dainippon-Sumitomo Pharma Co., Ltd. for supporting this study. We are also grateful to members of the Hiroshima Liver Research Group and Toranomon Hospital, Japan, for their support of this study.
Anaplastic lymphoma kinase (ALK) belongs to the insulin receptor superfamily of tyrosine kinase. ALK-fused Reparixin such as NPM-ALK, EML4-ALK, KIF5-ALK are putative oncogenes and responsible for many cancers including anaplastic large-cell lymphomas (ALCL), inflammatory myofibroblastic tumors (IMT), diffuse large B cell lymphoma (DLBCL) and a variety of solid tumor types. Crizotinib (Xalkori®, Pfizer) is the first EML4-ALK inhibitor in clinical use for non-small-cell-lung cancer (NSCLC) treatment. However, despite dramatic responses to Crizotinib, its efficacy is ultimately limited by the development of acquired drug resistance that occurred within a year including EML4-ALK L1196M, F1174L, R1275Q and G1269S and grade 3 or 4 adverse event of ALT and AST elevation (∼10%). To overcome these limitations, other novel ALK inhibitors such as CH5424802, AP26113, NVP-TAE684, and LDK378 have been studied and are currently under development (). The pyrimidine ALK inhibitors, NVP-TAE684 and AP26114, reportedly retain potency over Crizotinib-resistant ALK mutants. They possess either a sulfone or phosphine oxide at the aniline position of the C4 pyrimidine to make a selective hydrogen interaction to ALK Lys1150. Of many ALK pyrimidine analogs, Cephalon recently reported a methoxyl instead of the carbonyl and achieved moderate potency but excellent selectivity over homologous insulin receptor (IR) which is likely responsible for compound-dependent glucose fluctuation in preclinical animal study. Inspired by this report, we decided to explore the alkoxy structure–activity-relationship (SAR) and improve its potency by replacing the morpholines with more polar piperazine derivatives which in general allows lower plasma–protein-binding (PPB) for improved efficacy. A general synthetic chemistry is outlined in . 5-Fluoro-2-nitrophenol was alkylated with various alkyl halide in the presence of potassium carbonate in ,-dimethylformamide. Thus alkylated arylfluoride was then reacted with -acetylpiperazine to provide 1-(4-(3-alkoxy-4-nitrophenyl)piperazin-1-yl)ethanone . Subsequent hydrogenation afforded the key building block 1-(4-(4-amino-3-alkoxyphenyl)piperazin-1-yl)ethanone as a dark solid. The aniline was then condensed with 2,4,5-trichloropyrimidine in ,-dimethylformamide at 80°C to make a regioselective adduct . The adduct was heated with 1-(4-(4-amino-3-methoxyphenyl)piperazin-1-yl)ethanone in the presence of catalytic amounts of hydrogen chloride in ethoxyethanol at 115°C to provide an ALK inhibitor . First of all, we compared the -acetylpiperazine substitution on the A ring (KRCA-0008) to the morpholine analogue on ALK activity (). To our surprise, was unexpectedly less potent and the -acetylpiperazine analogue (KRCA-0008) was far superior to in both biochemical and cellular assay. Although we do not fully understand the role of -acetylpiperazine on the A ring at this time, we next explored the -methoxy effect on both A & C rings as shown in . When compared to (no -OMe on A & C rings), (-OMe on the C ring) displays slightly improved biochemical ALK activity and similar H3122 cell (EML4-ALK driven) proliferation potency with slightly improved H1993 cell (c-MET driven) selectivity. When the -OMe is placed on the A ring (), ALK potency in both biochemical and H3122 cell improved noticeably, although the H1993 selectivity decreased from that of . Combined with these results, KRCA-0008, a bis--OMe analog attached on both the A and C rings, gratifyingly improved the biochemical activity and H3122 cellular potency significantly while retained good H1993 selectivity. This observation was in general consistent with other close analogs (data not shown) and we could draw a conclusion regarding the -OMe effect on the pyrimidine ALK inhibitors: The A ring -Me b rings ALK activity in both biochemical & cellular assay while keeping IR selectivity, and the C ring -Me adds ALK activity and selectivity over other kinases.