Discovery of potent bisindole-based pyrazolopyridine derivatives as topoisomerase inhibitors: DNA damage induction and synergistic antileukemic activity

Introduction The development of novel anticancer agents targeting DNA replication and repair mechanisms remains a priority in leukemia therapy. In this study, newly synthesized derivatives incorporating bis-indole and pyrazolo3,4- b pyridine scaffolds were evaluated for their antiproliferative potential against leukemia cell lines. Methods The antiproliferative activity of the synthesized compounds was assessed in four cancer cell lines, including acute myeloid leukemia (MV4-11) and chronic myeloid leukemia (K562). Growth inhibition (GI 50 ) values were determined. DNA relaxation assays were performed to evaluate inhibition of topoisomerase I and IIα activities. Cell cycle distribution, apoptosis induction, and DNA damage response markers were analyzed using cellular and molecular assays. Combination studies were conducted using CHK1, ATR, and PARP-1 inhibitors. Results Compounds 7b , 7d , and 7e demonstrated the most potent antiproliferative activity, with GI 50 values below 2.5 μM in leukemic cell lines. Compound 7e exhibited notable cytotoxicity, with GI 50 values of 1.1 μM (MV4-11) and 2.7 μM (K562). Compounds 7b and 7e significantly inhibited topoisomerase I activity and effectively suppressed topoisomerase IIα-mediated DNA relaxation. Cellular studies revealed S-phase cell cycle arrest, activation of apoptotic pathways (caspase cleavage and PARP-1 degradation), and induction of DNA damage response markers (γH2AX, p-CHK1, p53). In MV4-11 cells, combination treatment with CHK1 or ATR inhibitors resulted in pronounced synergistic cytotoxicity, whereas co-treatment with a PARP-1 inhibitor produced minimal synergy. Discussion These findings identify bis-indole and pyrazolo3,4- b pyridine derivatives, particularly compound 7e , as potent dual topoisomerase inhibitors with significant antileukemic activity. Their ability to induce DNA damage and enhance cytotoxicity in combination with DNA damage response inhibitors highlights their potential therapeutic value, especially in combination strategies targeting replication stress pathways in leukemia.