br Results and discussion br

Results and discussion
Conclusion The tyrosine kinase ALK represents one of the most successful molecular targets for the development of precise medicine to treat stratified subgroups of cancer patients. Three generations of ALK inhibitors have been awarded the FDA's approval or are being extensively investigated in the clinic. The second-generation ALK inhibitors were designed to suppress most of the ALK resistant mutations caused by treatment of the first-generation ALK inhibitor, especially the gatekeeper L1196M mutation, however, these inhibitors generally failed to interact with the most refractory mutation G1202R. Two third-generation ALK inhibitors have been reported recently capable of inhibiting nearly all of the resistant ALK mutants, including G1202R, but the clinical outcome has not been available yet. Since the G1202R mutation is located at the solvent front of the ALK kinase domain close to the inhibitor's binding pocket, in this report, we designed a series of new diarylaminopyrimidine analogues by incorporating a resorcinol moiety to interact with ALK kinase domain where the G1202R is located. These compounds showed high potency against most of ALK resistant mutants, especially the challenging G1202R. Compound 12d turns out as the most potent with IC50 values of 1.7 nM, 3.5 nM, and 1.8 nM against ALK wild type, the gatekeeper mutant L1196M, and the G1202R mutant, respectively. It exerted significant antiproliferative effects in a set of ALK-addicted cancer cells, including H3122, SU-DHL-1, and Karpas 299 with IC50 values of 4.8–10.4 nM, and dose-dependently decreased the phosphorylation of ALK as well as its downstream STAT3/AKT/ERK and SHP-2/ERK/AKT signaling pathways in ALK-dependent cells. More importantly, compound 12d has excellent inhibitory effects against the proliferation of BaF3 AT7867 mg specifically expressing ALK wild type, gatekeeper L1196M, and the most challenging mutant G1202R, with IC50 values all less than 1.5 nM. On the basis of the high potency profile of 12d, especially the remarkable antiproliferative effects against the cancer cells respectively expressing the gatekeeper mutation L1196M and the most refractory mutation G1202R, this compound is worthy of further investigation as a new more potent third-generation ALK inhibitor to circumvent drug resistance of both the first-generation and the second-generation inhibitors.
Experimental section
Overview of lung cancers
Role of ALK and ALK-fusion proteins in the pathogenesis of malignancies
Structure of ALK-drug complexes Crizotinib is a first generation antagonist that is approved for the first-line treatment of ALK+-NSCLC (Table 3). The X-ray crystal structure shows that the amino group of the aminopyrimidine scaffold of crizotinib (Fig. 5A) forms a hydrogen bond with the carbonyl group of the first hinge residue (E1197), the N1 nitrogen of the pyrimidine moiety forms another hydrogen bond with the NH group of the third hinge residue (M1199) (Fig. 6A). The drug occupies only the front cleft and does not extend past the gatekeeper residue. Additionally, crizotinib makes hydrophobic contact with L1122 within the glycine-rich loop, V1130 (CS7) after the loop, A1148 (CS8), the gatekeeper L1196 (Sh2), the 1198LMA1200 triad of the hinge, N1254 within the catalytic loop, and L1256 (CS6) in the large lobe. L1122 and V1130 occur above the plane of the pyrazopyridine scaffold and L1256 occurs below it. The 3-fluorophenyl group of the drug makes van der Waals contact with R1253 of the catalytic loop and with DFG-D1270. The piperidine ring, which is attached to the pyrazol-4-yl moiety, is directed away from the enzyme into the solvent. Crizotinib targets additional protein kinases including c-MET or the hepatocyte growth factor receptor (HGFR) protein-tyrosine kinase (its original drug target) [51] and ROS1 [52], another member of the insulin receptor superfamily of protein-tyrosine kinases [20]. The ALK-crizotinib complex has DFG-D pointed inward toward the active site, a linear R-spine, and an αC-helix “in” conformation and a disordered activation segment. However, the initial portion of the activation segment possesses an αAL helix indicating that the segment is in a closed or compact conformation and the enzyme is thus in a less active conformation. This corresponds to a type I½ inhibitor and not a type I inhibitor as previously classified [44]. Similarly, ceritinib is a type I½ inhibitor and not a type I inhibitor.