Academic Press All rights reserved Synthesis and cytotoxic evaluation of pyrido-dipyrimidine derivatives: Mechanism of action, DNA and HSA binding studies Milović, Lazar Stanojković, Tatjana Jeremić, Svetlana Petrović, Maja Marković, Milica Rilak Simović, Ana Janković, Nenad Grozdić, Nađa Đorović Jovanović, Jelena https://phaidrabg.bg.ac.rs/o:38559 doi:10.1016/j.bioorg.2026.109740 ISSN: 0045-2068 2026 Pyrido-dipyrimidines; Cytotoxicity; DNA binding; HSA interaction; Molecular docking application/pdf 8722927 bytes info:eu-repo/semantics/article Abstract: This study presents the synthesis of a novel series of pyrido[2,3-d:6,5-d’]dipyrimidine derivatives (4a-v) via an ‘on-water’ multicomponent reaction, utilizing thiobarbituric acid, diverse aldehydes, and amines at room temperature. The compounds were evaluated for cytotoxic activity against human cancer cell lines (HeLa, K562, LS174, A549) and normal fibroblasts (MRC-5) using MTT assays. Notable potency was observed against HeLa and K562 cells, with IC50 values ranging from 9.82 to 192.47 μM. Compound 4k exhibited the strongest activity against HeLa (IC50 = 9.82 ± 1.07 μM) with a selectivity index (SI) >12, while 4u was most effective against K562 (IC50 = 17.36 ± 1.39 μM, SI >9). LS174 and A549 showed limited sensitivity. Structure-activity relationship (SAR) analysis revealed that para- hydroxyphenyl substituents at position 5 and thiocarbonyl groups enhanced cytotoxicity, particularly against leukemia cells, via improved hydrogen bonding and target affinity. Mechanistic studies on 4k and 4u focused on DNA and human serum albumin (HSA) interactions. UV–Vis and fluorescence spectroscopy, including ethidium bromide displacement and viscosity measurements, indicated minor groove binding to CT- DNA (Kb ~ 104 M–1; KSV ~ 104 M–1) with partial intercalation. HSA binding occurred via static quenching at sites I and II Državni univerzitet u Novom Pazaru (KSV ~ 105 M–1; Kb ~105 M–1), confirmed by competitive assays with Eosin Y and ibuprofen. Cell cycle analysis and AO/EB staining demonstrated apoptosis induction, with increased subG1 populations. Caspase assays showed 4k activated both intrinsic (caspase-9) and extrinsic (caspase-8) pathways in HeLa cells, leading to caspase-3 execution; 4u induced caspase-independent apoptosis in K562. Molecular docking and dynamics simulations supported minor groove DNA preference and HSA site I/II binding, with stable complexes (ΔGbind ≤ –8 kcal/mol). RMSD, RMSF, and Rg analyses affirmed structural integrity. These derivatives emerge as promising selective anticancer agents targeting DNA and apoptosis, warranting further optimization and in vivo studies eng Bioorganic Chemistry startpage: 109740 endpage: 109740