Encouraged by this observation a systematic SAR study on

Encouraged by this observation, a systematic SAR study on the A ring -alkoxy was conducted as summarized in . The A ring methoxy-containing KRCA-0008 was proven an optimal substituent in terms of potency after all and its bigger size derivatives were detrimental in both biochemical and H3122 cellular activities (–). Since difluoromethoxy analog () is equipotent with KRCA-0008, the alkoxy size on the position proved critical on their ALK activity. It is noteworthy that the preparation of the hydroxyl analog (–OH) on the position failed presumably because of the instability of the 2-hydroxy-1,4-diaminobenzene moiety. Besides alkoxys on the A ring position, halide and amino replacements were also examined. Chlorine and fluorine derivatives (, ) possess similar biochemical and cellular potency to KRCA-0008, and amine analogs (, ) resulted in a significant loss in the biochemical ALK . activities. KRCA-0008 is potent to ALK . and mutants in biochemical assay (ALK IC=12nM, ALK L1196M IC=75nM, ALK C1156Y IC=4nM, ALK F1174L IC=17nM, ALK R1275Q IC=17nM). It has 0.08μM IC in H3122 (EML4-ALK driven tumor cell) cell proliferation and possesses 3.6μM cellular selectivity to H1993 (c-MET driven non small cell lung cancer). Immunoblotting of phospho-ALK and its downstream targets (phospho-Akt and phospho-Erk) was carried out in H3122 (EML4-ALK) and A549 (ALK ) PF-3845 and proved specific inhibition to EML4-ALK driven H3122 cell using and KRCA-0008as depicted in . Ambit assay (>100 kinase panel assay done by Millipore service) shows is very potent only to ALK (IC=25nM) and ACK1 (IC=18nM), and very selective over the rest other kinases including IGF-1R and IR (insulin receptor) at 1μM., KRCA-0008 retains good drug-like properties: good water-solubility (54μM in 5% DMSO–water, 150μM in 5% DMSO–PBS buffer) with moderate plasma protein binding (93% in rat) and low brain exposure (/=∼0.02). It has good liver microsomal stability (% remaining after 30min: 52% in mouse, 89% in rat, 72% in human) and little to no CYP inhibition (1A2, 2C9, 2D6, 3A4 @ 10μM). It does not appear to cause hERG blockade (patch clamp IC=30μM) and is negative on Ames test (1000μg/plate), chromosomal aberration assay and micronucleus assay. KRCA-0008 also shows promising pharmacokinetic parameters in both mice and rat (oral bioavailability=66–94.5%) as summarized in . Finally, KRCA-0008 shows a modest tumor growth inhibition activity in H3122 human lung cancer bearing mice model comparable to Crizotinib without significant body weight change (). It is important to mention the KRCA-0008 25 mpk and 50 mpk groups did not show dose-dependent tumor growth inhibition. In summary, the synthesis of bis--alkoxy-para-piperazinesubstituted-2,4-dianilinopyrimidines is described and their structure-activity-relationship study to an aplastic lymphoma kinase (ALK) is discussed. KRCA-0008 is selective and potent to ALK and Ack1, and has drug-like properties without hERG concerns. KRCA-0008 demonstrates efficacy comparable to Crizotinib in xenograft mice model. Currently KRCA-0008 analogs are under investigation and those will be reported in the future. Acknowledgment This work was supported by the Korea Research Institute of Chemical Technology (KK-1303), the Korea Chemical Bank (SI-1305) and Ministry of Science, ICT and Future Planning of Korea (KN-1235), and authors thank Sanghun Alex Lee for manuscript proofreading.