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In this work, six new mixed ligand Schiff base metal complexes of [M2(L1)(X)2Cl4] type, (where, M = CoII, NiII, CuII, L1 = Schiff base ligand derived from 1‐pyrenecarboxaldehyde and 1,4‐bis[3‐aminopropyl]piperazine and X = pyrazine‐2‐carboxylic acid or 2,2′‐biimidazole) were synthesized and studied their biological activity and cytotoxicity. The synthesized Schiff base ligands, their metal complexes were characterized by Infrared, UV‐Visible spectroscopy, and elemental analysis. Density functional theory calculations were performed to examine the molecular geometry and frontier molecular orbital properties of complexes (1–6). The DNA binding ability of these complexes (1–6) was evaluated by in vitro spectroscopic (absorption and fluorescence) titrations, viscosity measurements, and in silica by molecular docking measurements. The results showed a good binding propensity with the binding constant from 2.65 × 104 to 3.38 × 105 M−1 in the order 6 > 5 > 4 > 3 > 2 > 1, respectively. All complexes exhibited a good binding affinity to BSA proteins with relatively higher binding constant values similar to the trend of DNA binding studies and where Cu (II) complexes have greater efficiency than Co (II) and Ni (II) complexes. The in vitro cytotoxic study of all the complexes was investigated in the cervical cancer (HeLa) cell line, which displayed IC50 values of 1.36–24.2 μM, signifying the potential of complexes for an operative anticancer drug. Metal complexes were also screened for antimicrobial properties, which showed virtuous inhibition than the free ligands.

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