Fms-like tyrosine kinase 3 (FLT3) mutation has been strongly associated with increased risk of relapse, and the irreversible covalent FLT3 inhibitors had the potential to overcome the drug-resistance. In this study, a series of simplified 4-(4-aminophenyl)-6-methylisoxazolo[3,4-b] pyridin-3-amine derivatives containing two types of Michael acceptors (vinyl sulfonamide, acrylamide) were conveniently synthesized to target FLT3 and its internal tandem duplications (ITD) mutants irreversibly. The kinase inhibitory activities showed that compound C14 displayed potent inhibition activities against FLT3 (IC₅₀ = 256 nM) and FLT3-ITD by 73 % and 25.34 % respectively, at the concentration of 1 μM. The antitumor activities indicated that C14 had strong inhibitory activity against the human acute myeloid leukemia (AML) cell lines MOLM-13 (IC₅₀ = 507 nM) harboring FLT3-ITD mutant, as well as MV4-11 (IC₅₀ = 325 nM) bearing FLT3-ITD mutation. The biochemical analyses showed that these effects were related to the ability of C14 to inhibit FLT3 signal pathways, and C14 could induce apoptosis in MV4-11 cell as demonstrated by flow cytometry. Fortunately, C14 showed very weak potency against FLT3-independent human cervical cancer cell line HL-60 (IC₅₀ > 10 μM), indicating that it might have no off-target toxic effects. In light of these data, compound C14 represents a novel covalent FLT3 kinase inhibitor for targeted therapy of AML.

Fms 样酪氨酸激酶 3 (FLT3) 突变与复发风险增加密切相关，不可逆的共价 FLT3 抑制剂有可能克服耐药性。在本研究中，我们方便地合成了一系列简化的 4-(4-氨基苯基)-6-甲基异恶唑并[3,4-b] 吡啶-3-胺衍生物，其中含有两种迈克尔受体（乙烯基磺酰胺、丙烯酰胺）以靶向 FLT3及其内部串联重复 (ITD) 突变体不可逆转。激酶抑制活性表明，化合物C14在 1 μM 浓度下对 FLT3 (IC ₅₀  = 256 nM) 和 FLT3-ITD 的抑制活性分别为 73 % 和 25.34 %。抗肿瘤活性表明C14_{对携带 FLT3-ITD 突变体的人急性髓性白血病 (AML) 细胞系 MOLM-13 (IC 50}  = 507 nM) 以及携带 FLT3-ITD 突变体的 MV4-11 (IC ₅₀  = 325 nM)具有强抑制活性。生化分析表明，这些作用与C14抑制FLT3信号通路的能力有关，流式细胞仪显示C14可诱导MV4-11细胞凋亡。幸运的是，C14对不依赖 FLT3 的人宫颈癌细胞系 HL-60（IC ₅₀  > 10 μM）显示出非常弱的效力，表明它可能没有脱靶毒性作用。根据这些数据，化合物C14代表一种用于 AML 靶向治疗的新型共价 FLT3 激酶抑制剂。