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A new series of steroidal pyrimidines (7–9) has been synthesized by reacting steroidal thiosemicarbazones (4–6) with ethyl cyanoacetate. The compounds were characterized by IR, 1 H NMR, 13 C NMR, MS and analytical data. The interaction studies of compounds 7–9 with DNA were carried out by UV–vis and luminescence spectroscopy. Compounds (7–9) bind to DNA preferentially through electrostatic and hydrophobic interactions, with K b values found to be 6.56× 10 3 M− 1, 1.54× 10 4 M− 1 and 9.34× 10 3 M− 1, respectively, indicating the higher binding affinity of compound 8 towards DNA. Gel electrophoresis pattern demonstrated that compound 8 shows strong interaction with DNA and that, during its cleavage activity with pBR322 DNA, it seems to follow the mechanistic pathway involving the generation of singlet oxygen and a superoxide anion, which are responsible for initiating DNA strand scission. The docking study suggested that the intercalation of compounds in between the nucleotide base pairs is due to the presence of a pyrimidine moiety in the steroid molecule. MTT assay was carried out to check the toxicity of new compounds 7–9 against the different human cancer as well as non-cancer cell lines A545, MCF-7, HeLa, HL-60, SW480, HepG2, HT-29, A549, 184B5, MCF10A, NL-20, HPC, and HPLF. Apoptotic degradation of DNA in the presence of steroidal pyrimidines 7–9 was analysed by agarose gel electrophoresis and visualized by ethidium bromide staining (comet assay).