In our attempt to develop effective EGFR-TKIs, two series of 1H-pyrazolo[3,4-d]pyrimidine derivatives were designed and synthesized. All the newly synthesized compounds were evaluated in vitro for their inhibitory activities against EGFR^WT. Compounds 15_b, 15_j, and 18_d potently inhibited EGFR^WT at sub-micro molar IC₅₀ values comparable to that of erlotinib. Moreover, thirteen compounds that showed promising IC₅₀ values against EGFR^WT were tested in vitro for their inhibitory activities against mutant EGFR^T790M. Compounds 17_d and 17_f exhibited potent inhibitory activities towards EGFR^T790M comparable to osimertinib. Compounds that showed promising IC₅₀ values against EGFR^WT were further tested for their anti-proliferative activities against three cancer cell lines bearing EGFR^WT (MCF-7, HepG2, A549), and two cancer cell lines bearing EGFR^T790M (H1975 and HCC827). Compounds 15_g, 15_j, 15_n, 18_d and 18_e were the most potent anticancer agents against the EGFR^WT containing cells, while compounds 15_e, 17_d and 17_f showed promising anti-proliferative activities against EGFR^T790M containing cells. Furthermore, the most active compound 18_d was selected for further studies regarding to its effects on cell cycle progression and induction of apoptosis in the HepG2 cell line. The results indicated that this compound is good apoptotic agent and arrests G₀/G₁and G₂/M phases of cell cycle. Finally, molecular docking studies were performed to investigate binding pattern of the synthesized compounds with the prospective targets, EGFR^WT (PDB: 4HJO) and EGFR^T790M (PDB: 3W2O).

在我们尝试开发有效的EGFR-TKIs的过程中，设计并合成了两个系列的1 H-吡唑并[3,4- d ]嘧啶衍生物。所有新合成的化合物在体外对EGFR ^WT的抑制作用进行了评价。化合物15 _b，15 _Ĵ，和18 _d有效地抑制EGFR ^WT在亚微摩尔的IC ₅₀值相媲美的埃罗替尼。此外，在体外测试了13种显示出有希望的针对EGFR ^WT的IC ₅₀值的化合物具有抑制突变型EGFR ^{T790M的作用}。化合物17 _d和17 _˚F表现出朝向EGFR强效抑制活性^T790M媲美osimertinib。进一步测试了针对EGFR ^WT表现出有希望的IC ₅₀值的化合物对三种带有EGFR ^WT的癌细胞系（MCF-7，HepG2，A549）和两种带有EGFR ^T790M的癌细胞系（H1975和HCC827）的抗增殖活性。。化合物15 _g，15 _j，15 _n，18 _d和18 _e它们是针对含有EGFR ^WT的细胞最有效的抗癌药，而化合物15 _e，17 _d和17 _f对含有EGFR ^T790M的细胞显示出有希望的抗增殖活性。此外，选择最具活性的化合物18 _d对其在HepG2细胞系中对细胞周期进程和细胞凋亡诱导的影响进行进一步研究。结果表明该化合物是良好的凋亡因子，阻滞G ₀ / G ₁和G _2。/ M阶段的细胞周期。最后，进行分子对接研究以研究合成化合物与预期靶标EGFR ^WT（PDB：4HJO）和EGFR ^T790M（PDB：3W2O）的结合模式。