Development of New Pyrazolo [3,4-b]Pyridine Derivatives as Potent Anti-Leukemic Agents and Topoisomerase IIα Inhibitors with Broad-Spectrum Cytotoxicity

<b>Background/Objectives:</b> In the current medical era, Topoisomerase II is recognized as an essential enzyme that regulates DNA topology during critical biological processes such as DNA replication, transcription, and repair. This study aimed to design, synthesize, and biologically evaluate a new series of pyrazolo[3,4-<i>b</i>]pyridines (<b>8a</b>-<b>g</b>, <b>10a</b>-<b>g</b>, and <b>12</b>) as potential anticancer agents and Topoisomerase II inhibitors. <b>Methods:</b> The synthesized compounds were subjected to in vitro anticancer screening at the National Cancer Institute (NCI, USA). Active derivatives were further evaluated through a five-dose screening to determine their antiproliferative potency. Selected compounds were examined for their effects on leukemia cell lines (K562 and MV4-11), and mechanistic studies were performed to assess DNA damage, cell cycle distribution, and apoptosis-related protein modulation. Additionally, enzyme inhibition assays were conducted to determine Topoisomerase IIα (TOPIIα) inhibition. <b>Results:</b> Initial single-dose screening identified several active compounds, notably <b>8b</b>, <b>8c</b>, <b>8e</b>, <b>8f</b>, <b>10b</b>, <b>10c</b>, <b>10e</b>, and <b>10f</b>. Among these, compound <b>8c</b> exhibited potent and broad-spectrum antiproliferative activity across the NCI cancer cell line panel, with a GI₅₀ MG-MID value of 1.33 µM (range: 0.54-2.08 µM). The synthesized molecules showed moderate to good anti-leukemic efficacy against K562 and MV4-11 cells. Mechanistic investigations revealed that compound <b>8c</b> induced DNA damage and S-phase cell cycle arrest, leading to apoptosis as evidenced by the modulation of PARP-1, Bax, XIAP, and Caspases. Furthermore, target-based assays confirmed that compound <b>8c</b> significantly inhibited the DNA relaxation activity of TOPIIα in a dose-dependent manner, comparable to etoposide. <b>Conclusions:</b> The study highlights compound <b>8c</b> as a promising pyrazolo[3,4-<i>b</i>]pyridine derivative with potent antiproliferative activity and effective inhibition of Topoisomerase IIα. These findings suggest its potential as a lead scaffold for further optimization in anticancer drug development..