Discovery of Potent Bisindole-based Pyrazolopyridine Derivatives as Topoisomerase Inhibitors: DNA Damage Induction and Synergistic Antileukemic Activity

Wagdy Mohamed Eldehna^1*Haytham O. Tawfik^2*Denisa Veselá³Miroslav Peřina³Ahmed Thabet Negmeldin^4*Zainab M. Elsayed¹Taghreed A Majrashi⁵Veronika Vojáčková³Mostafa M. Elbadawi¹Moataz Ahmed Shaldam¹Vladimír Kryštof³Hatem A. Abdel-Aziz⁶

• ¹Kafrelsheikh University Faculty of Pharmacy, Kafr El-Shaikh, Egypt
• ²Faculty of Pharmacy, Tanta University, Tanta, Egypt
• ³Univerzita Palackeho v Olomouci, Olomouc, Czechia
• ⁴Gulf Medical University, Ajman, United Arab Emirates
• ⁵King Khalid University, Abha, Saudi Arabia
• ⁶Applied Organic Chemistry Department, National Research Center, Dokki, Cairo, Egypt

The anti-proliferative properties of newly synthesized derivatives containing bis-indole and pyrazolo[3,4-b]pyridine scaffolds were evaluated in four cancer cell lines, including acute myeloid leukemia (MV4-11) and chronic myeloid leukemia (K562). Among them, compounds 7b, 7d, and 7e emerged as the most potent candidates, with GI₅₀ values below 2.5 μM in leukemic cell lines. In particular, compound 7e showed notable cytotoxic activity with GI₅₀ values of 1.1 μM and 2.7 μM in MV4-11 and K562, respectively. DNA relaxation assays revealed that 7b and 7e significantly inhibited topoisomerase I activity and also effectively suppressed topoisomerase IIα mediated DNA relaxation. Further cellular studies showed that these compounds triggered S-phase cell cycle arrest, activated apoptotic pathways, as evidenced by caspase cleavage and PARP-1 degradation, and induced DNA damage response markers (γH2AX, p-CHK1, p53). Furthermore, 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 only minimal synergy. These findings highlight the potential of these new derivatives as potent topoisomerase inhibitors with prospective therapeutic utility for leukemia, particularly when used in combination with agents targeting the DNA damage response pathways.