Novel 1,3,4-thiadiazolo[2,3-c]-1,2,4-triazin-4-one derivatives 3a-e, 4a-f and 5a-f were designed as Type I c-Met kinase inhibitors based on scaffold hopping of our previous Type II c-Met kinase lead. Target compounds were then synthesized under the guidance of molecular docking analysis to identify the potential inhibitors that fit the binding pocket of c-Met kinase in the characteristic manner as the reported Type I c-Met kinase inhibitors. All synthesized derivatives were evaluated for their c-Met kinase inhibitory activity at 10 µM concentration, where 3d, 5d and 5f displayed >80% inhibition. Further IC₅₀ investigation of these compounds identified 5d as the most potent c-Met kinase inhibitor with IC₅₀ value of 1.95 µM. Moreover, 5d showed selective antitumor activity against c-Met over-expressing colon HCT-116 and lung A549 adenocarcinoma cells with IC₅₀ values of 6.18 and 10.6 µg/ml, respectively. More significantly, 5d effectively inhibited c-Met phosphorylation in the Western blot experiment. Also, 5d induced cellular apoptosis in HCT-116 cancer cells as well as cell cycle arrest with accumulation of cells in G2/M phase. Finally, kinase selectivity profiling of 5d against nine oncogenic kinases revealed its selectivity to only Tyro3 kinase (% inhibition = 80%, IC₅₀ = 3 µM). All these experimental findings clearly demonstrate that 5d is a potential dual acting inhibitor against c-Met and Tyro3 kinases, standing out as a viable lead that deserves further investigation and development to new generation of antitumor agents.

新型 1,3,4-噻二唑并[2,3- c ]-1,2,4-triazin-4-one 衍生物3a-e、4a-f和5a-f被设计为基于 I 型 c-Met 激酶抑制剂在我们之前的 II 型 c-Met 激酶引线的支架跳跃上。然后在分子对接分析的指导下合成目标化合物，以确定以特征方式适合 c-Met 激酶结合口袋的潜在抑制剂作为报道的 I 型 c-Met 激酶抑制剂。对所有合成衍生物在 10 µM 浓度下的 c-Met 激酶抑制活性进行了评估，其中3d、5d和5f显示出 >80% 的抑制。进一步的 IC ₅₀对这些化合物的研究发现5d是最有效的 c-Met 激酶抑制剂，IC ₅₀值为 1.95 µM。此外，5d显示出针对 c-Met 过表达结肠 HCT-116 和肺 A549 腺癌细胞的选择性抗肿瘤活性，IC ₅₀值分别为 6.18 和 10.6 µg/ml。更重要的是，在蛋白质印迹实验中，5d有效地抑制了 c-Met 磷酸化。此外，5d诱导 HCT-116 癌细胞的细胞凋亡以及细胞周期停滞，细胞在 G2/M 期积累。最后，5d 的激酶选择性分析针对 9 种致癌激酶显示其仅对 Tyro3 激酶的选择性（抑制百分比 = 80%，IC ₅₀  = 3 µM）。所有这些实验结果清楚地表明，5d是一种潜在的针对 c-Met 和 Tyro3 激酶的双重作用抑制剂，作为一种可行的先导物脱颖而出，值得进一步研究和开发新一代抗肿瘤药物。