| Lung cancer is the most common cause of cancer death with non-small cell lung cancer(NSCLC)accounting for about 80%of cases.In recent years,EML4-ALK translocation genes and ROS1 translocation genes have been identified as driver genes in NSCLC,and combined as a joint target for the development of new treatments for NSCLC.In this paper,the author reviewed recent progresses of ALK and ROS1 as ’druggable’ targets for patients with NSCLC,especially focusing on the discovery of drugs in this area,including first-generation agent(Crizotinib),second-generation agents(Ceritinib,Alectinib,Brigatinib)and others at different clinical stages.Despite notable and typically durable responses,the vast majority of patients treated with ALK inhibitors will be doomed to develop disease progression.Over one-half of Ceritinibresistant patients developed ALK resistance mutations,and the most common ALK mutation was ALKG1202R.Based upon the co-crystal structure of Ceritinib with ALKWT(PDB 4MKC)as well as the binding model of Ceritinib with ALKG1202,84 4-arylaminopyrimidine derivatives were designed and synthesized to explore ALK/ROS1 dual inhibitors to overcome resistant mutants,including 2,4-diarylaminopyrimidine analogues bearing thiazole moieties(YA-1～YA17),1,2,3-triazole moieties(YB-1～YB-13),sulfone moieties(YC-1～YC-17)or crotonamides moieties(YD-1～YD-5),and 4-arylaminopyrimidine derivatives possessing hydrazone moieties(WA-1～WA-32).All target compounds were confirmed by MS and1H NMR,and some were also identified by 13C NMR and 2D NOESY.In order to evaluate ALK and ROS1 inhibition,84 compounds were directly evaluated in a cellular context by measuring the anti-proliferation of ALK-positive cell lines KARPAS299(human anaplastic large-cell lymphomas cells)and H2228(human NSCLC cells)as well as ROS1-positive cell line HCC78(human lung adenocarcinoma cells).Meanwhile,tumor cell lines A549(human NSCLC cells),H1975(EGFR-positive human NSCLC cells),H460(large cell lung cancer cells),and HT-29(human colon cancer cells)whose growth was not dependent on ALK and/or ROS1,were used to test the potential off-target effects.As a general trend,most compounds exhibited moderate to excellent anti-proliferative potencies in ALK-addicted cell lines and ROS1-positive cell line with IC50 values below 500 nM,and showed little potency against A549,H1975,H460 and HT-29 cells,confirming that these compounds are selective for ALK and ROS1.Some promising compounds,such as YA-7,YB-9,YB-10,YD-4,WA-6,WA-12,WA-24 and WA-26 displayed excellent anti-tumor activities against KARPAS299 and HCC78 in the double-digit nanomolar range,approximately as potent as Ceritinib and superior than Crizotinib.Based on the anti-proliferation activities,20 representative compounds were selected for further in vitro kinase inhibitory assays,including ALK,ALKL1196M,ALKG1202R,ROS1,c-Met and EGFR.These compounds displayed preferable ALK and ROS1 kinases inhibition with IC50 values ranging from 1.1～19 nM,while most shown little inhibition on c-Met and EGFR.In particular,YB-9 and YD-4 were much more potent against ALKL1196M and ALKG1202R than Ceritinib and Crizotinib with IC50 values below 10 nM.In addition,YB-9 exhibited potent activities similar to Ceritinib on against a panel of secondary ALK mutants regularly found in resistant patients with IC50 values below 5 nM,including ALKG1269A,ALKC1156Y,ALKT1151ins and ALKs1206Y.Furthermore,Acridine Orange/Ethidium Bromide(AO/EB)double staining assay and Western-Blot assay confirmed that YB-9 was capable of inducing cell apoptosis by strongly inhibiting cellular ALK and ROS1 activity.Finally,the binding models of YB-9 with ALKwT,ALKL1196M and ALKG1202R provided structural bases for SARs observations.According to the results of antitumor activities,the structure-activity relationships were summarized,and the preliminary mechanism was explored.These studies will lead to further develop novel ALK/ROS1 dual inhibitors to overcome resistance mutations. |
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