Pim-1 kinase targeted recently has proved an essential goal of breast cancer therapy. We report the design, synthesis with full characterization analysis of pyrazolo[3,4-b]pyridine scaffold-based derivatives targeting Pim-1 kinase as anti-breast cancer agents. All the newly synthesized compounds were screened for their in vitro cytotoxic activity against two breast cancer cell lines MCF-7 and MDA-MB-231, and non-cancerous MCF-10A cells. Four derivatives notably, 17 and 19 exhibited a remarkable cytotoxic activity with IC₅₀ values 5.98 and 5.61 µM against MCF-7 (ERα-dependent) cells in a selective way, as they weren’t active against MDA-MB-231 (non-ERα-dependent) and safe against MCF-10A. The most active compounds through in vitro screening were subjected to PIM-1 kinase to elucidate the Pim-1 kinase inhibitory activity as the mechanistic mode of action. Among the tested derivatives, Compounds 17 and 19 showed the highest inhibitory activity with IC₅₀ values 43 and 26 nM, respectively, compared to the 5-FU with IC₅₀ value 17 nM. Moreover, apoptotic investigation through flow cytometry and gene expression analysis of the apoptosis-related genes for the most active compound 19 against MCF-7. It was found that compound 19 induced apoptotic MCF-7 cell death by cell cycle arrest at G2/M phase and by elevation the expression of pro-apoptotic genes and inhibition of anti-apoptotic genes expression. Finally, the PIM-1 inhibition activities for compounds 17 and 19 were in accordance with the molecular docking study that revealed good interaction with the Pim-1 kinase active site.

最近被靶向的Pim-1激酶已被证明是乳腺癌治疗的基本目标。我们报告设计，合成与针对Pim-1激酶作为抗乳腺癌药物的吡唑并[3,4- b ]吡啶支架基衍生物的全表征分析。筛选所有新合成的化合物对两种乳腺癌细胞系MCF-7和MDA-MB-231以及非癌MCF-10A细胞的体外细胞毒活性。值得注意的是，四种衍生物17和19对IC ₅₀表现出显着的细胞毒性活性对MCF-7（ERα依赖性）细胞有选择性的5.98和5.61 µM值，因为它们对MDA-MB-231（非ERα依赖性）没有活性，对MCF-10A安全。通过体外筛选，将最具活性的化合物置于PIM-1激酶下，以阐明Pim-1激酶的抑制活性是其作用机理。在测试的衍生物中，与IC ₅₀值为17 nM的5-FU相比，化合物17和19的抑制活性最高，IC ₅₀值为43和26 nM。此外，通过流式细胞术进行凋亡研究以及针对MCF-7活性最高的化合物19的凋亡相关基因的基因表达分析。发现化合物19通过在G2 / M期的细胞周期停滞和通过升高促凋亡基因的表达和抑制抗凋亡基因的表达来诱导凋亡的MCF-7细胞死亡。最后，化合物17和19的PIM-1抑制活性与分子对接研究一致，该研究显示与Pim-1激酶活性位点具有良好的相互作用。