Design, Synthesis, and In Vitro, and In Silico Evaluation of N2‐ and/or C5‐Substituted Pyrazolo[4′,3′:5,6]Pyrano[2,3‐ <i>d</i> ]Pyrimidine Derivatives as Promising Anticancer Agents Targeting Leukemia and Breast Cancer

Abstract

Abstract A novel series of N2‐ and/or C5‐substituted pyrazolo[4″,3″:5,6]pyrano[2,3‐ d ]pyrimidine derivatives was achieved via direct N 2‐alkylation and C5‐chlorine substitution using various alkylating agents and primary amines. Structural characterization was performed by NMR ( 1 H, 13 C, DEPT‐135), HRMS, FT‐IR, and X‐ray diffraction analysis of single crystals. Derivatives 2 , 3 , 7 , 9 , and 10 were evaluated for their in vitro anticancer activity against 60 human cancer cell lines from which 7 ( NSC: D‐851904/1 ) exhibited broad‐spectrum anticancer activity against several tumor cell lines, notably against leukemia and breast cancer cell lines. It achieved 100% growth inhibition (GI%) in HL‐60 (TB), MOLT‐4, and HOP‐62 cells, with associated lethality (LC%) values of 19.2%, 25.5%, and 15.3%, respectively. It also showed potent inhibition in RPMI‐8226 (93.6%) and MDA‐MB‐468 (98.4%) cell lines, and moderate activity against SNB‐75 (67.27%) and MCF7 (61.1%) cell lines. Molecular docking studies revealed strong binding affinities for 7 to three key cancer targets: EGFR kinase domain (3POZ, ‐10.2 kcal/mol), 6O0K (−9.2 kcal/mol), and 3ERT (−8.9 kcal/mol). MM‐GBSA binding free energy calculations further confirmed its affinity, with 3ERT‐ 7 displaying the most favorable value (−81.48 kcal/mol). Molecular dynamics simulations demonstrated the stability of the docked complexes, particularly for 3ERT‐ 7 , which maintained stable interactions with key residues, suggesting selective modulation of breast cancer‐related targets. ADME‐T in silico predictions are also reported. These results provide valuable insights for the development of anticancer agents.