The results of western blot analysis reveal that 10g dose-dependently attenuates phosphorylation of ALK downstream signalling molecules (STAT3, ERK and PLC-gamma) as well as ALK autophosphorylation in ALK wt-TEL Ba/F3, ALK L1196M-TEL Ba/F3 and H2228 cell lines. 10c) resulted in little to no activity Rabbit polyclonal to OSBPL6 against ALK-wt. In contrast, the 4-methoxy containing derivative 10d has an Divalproex sodium enhanced activity against ALK-wt (IC50?=?69?nM) and it possesses a high activity (IC50?=?19?nM) against ALK-L1196M gatekeeper mutation, a value that is 50-fold higher than that (IC50?=?980?nM) of crizotinib. Moreover, replacement of the 4-methoxy group by a 4-dimethylamino group led to 10e, which was found to exhibit picomolar activity against ALK-L1196M. It is worthwhile to note that 10e is more potent against ALK-L1196M (IC50?=?0.7?nM) than against ALK-wt (IC50?=?7.3?nM). Picomolar inhibitory activity against ALK-wt was achieved with the 4-morpholino derivative 10f, which is also extremely active against ALK-L1196M (IC50?=?1.4?nM). The SAR study led us to Divalproex sodium identify 10g containing a 4-methylpiperazin-1-yl group as the most potent inhibitor against both ALK-wt (IC50?0.5?nM) and ALK-1196M (IC50?0.5?nM). Table 1. Kinase-inhibitory activities of 1H-pyrazolo[3,4-b]pyridine derivatives against ALK-wt and ALK-L1196M. Open in a separate window aRadiometric kinase assay. bInactive means that kinase activity is inhibited by less than 50% even at 10?M concentration of compound. cActivity value from the reference15. Antiproliferative activities of selected pyrazolo[3,4-b]pyridines Based on the results arising from studies of the kinase-inhibitory activities of the pyrazolo[3, 4-b]pyridine derivatives against ALK-wt and ALK-L1196M gatekeeper mutant, we Divalproex sodium selected the most potent inhibitors and measured their antiproliferative activities on Ba/F3 cells transformed with ALK-wt/ALK-L1196M and on H2228 non-small cell lung cancer cells harbouring EML4-ALK. Ba/F3 cell lines transformed with ALK-wt and ALK-L1196M mutant were employed to assess the ALK inhibition capability of the derivatives in a cellular context and parental Ba/F3 cells were utilised as controls to determine differential cytotoxicities. The antiproliferative activities Divalproex sodium of the selected pyrazolo[3,4-b]pyridines were further elucidated using the H2228 NSCLC cell line, which is an EML4-ALK positive cancer cell line. As the data in Table 2 show, the overall cellular activities of the selected pyrazolo[3,4-b]pyridines are relatively moderate compared with their enzymatic activities. In particular, it is difficult to understand why 10d is potent against ALK enzyme but inactive on H2228 and ALK-driven Ba/F3 cells. Among the compounds tested, 10g most strongly suppressed proliferation of both H2228 (GI50?=?0.219?M) and ALK-driven Ba/F3 cells Divalproex sodium (GI50?0.205?M). Table 2. Antiproliferative activities of 1H-pyrazolo[3,4-b]pyridine derivatives against Ba/F3 transformed with ALK and H2228 NSCLC cancer cell.
crizotinib0.249??0.061.654??0.130.141??0.080.726??0.2110dInactiveInactiveInactiveInactive10e8.538??0.78Inactive1.767??0.694.549??0.7210f1.693??0.40Inactive0.916??0.502.527??1.5010g0.219??0.053.495??1.130.205??0.060.129??0.0210hInactive15.18??0.523.352??0.242.276??0.59124.033??1.81Inactive3.869??1.541.980??0.2813c9.215??1.92inactive4.708??3.025.625??2.34 Open in a separate window aGI50 represents the concentration at which a compound causes half-maximal growth inhibition. GI50 value for parental, Ba/F3 transformed with ALK and H2228 cell lines were shown as the means??standard deviation (SD) of three independent experiments. bInactive means that the proliferation was suppressed by less than 10% even at 50?M concentration of compound. In order to understand the discrepancy between enzymatic and cellular activities, we first assessed the cell permeability of 10g using the human colon carcinoma cell line Caco-2. It was found that 10g has moderate permeability and is not a substrate of P-glycoprotein (P-gp) as evidenced by the fact that the efflux ratio of 10g is 1.85 (Table 3). This finding indicates that the cell permeability of 10g is not the reason for the discrepancy. We next measured the kinase-inhibitory activities of 10g against ALK-wt at three different ATP concentrations because the IC50 value derived from biochemical kinase assay depends on both Ki and Km, which are defined by ATP concentration43,44. As described in Table.