Synthesis, Structural Characterisation and Biological Evaluation of Novel Pyrimido[1,6-a]pyrimidine Derivatives Bearing Phenoxy, Phenylamino and Pyrazolyl Moieties as Potent Antimicrobial and Cytotoxic Agents

Abstract

A new series of pyrimido[1,6-a]pyrimidine-based heterocycles (HP1-HP10) was synthesised and structurally characterised using FTIR, ¹H/¹³C NMR, HRMS and elemental analysis. The key intermediate, 6-((4-chlorophenyl)amino)-8-hydroxy-2-(methylthio)-4-oxo-4H-pyrimido[1,6-a]pyrimidine-3-carbonitrile (HP1), was synthesised via base-catalysed cyclisation of ethyl 2-cyano-3,3-bis(methylthio)-acrylate with 6-amino-2-((4-chlorophenyl)amino)pyrimidin-4-ol. Subsequent nucleophilic substitutions of HP1 afforded phenoxy (HP2-4), phenylamino (HP5-7) and pyrazolo-fused (HP8-10) derivatives. The biological evaluations revealed that the compounds HP3 and HP6 exhibited significant antibacterial activity, particularly against Staphylococcus aureus and Escherichia coli, outperforming the standard drug streptomycin. HP3 also demonstrated the strongest antifungal efficacy against Candida albicans (26 mm), exceeding fluconazole. Cytotoxicity assessed via brine shrimp lethality bioassay showed that HP1 and HP5 possessed the lowest LD₅₀ values (35 µM), suggesting promising bioactivity. Structure-activity relationship (SAR) analysis highlighted the importance of electron-donating and halogenated substituents in modulating antimicrobial potency. These findings suggest that the HP1-HP10 scaffold offers a viable platform for further medicinal chemistry development. Molecular docking with AutoDock Vina revealed strong interactions of the tested ligands with the C. albicans protein target. Fluconazole, used as the reference, showed a binding affinity of -7.3 kcal/mol via hydrogen bonds with Thr411 and Glu70, while HP9 demonstrated the highest affinity (-9.3 kcal/mol) through hydrophobic contacts with Phe52, Trp54 and Glu70, followed by HP8 (-9.2 kcal/mol) and HP4 (-9.0 kcal/mol). Several ligands, including HP5, HP10 and HP2, formed multiple hydrogen bonds with active-site residues, while HP6 and HP7 exhibited strong nonpolar interactions. Low RMSD values (0.678 Å) confirmed reliable binding poses.