Synthesis and bioactivity investigation of polymyxin B analogues with Thr2, Dab3, and Thr10 modifications

To investigate the structure–activity (SAR) and structure–toxicity (STR) relationships of the three polar-face positions Thr2, Dab3, and Thr10 in polymyxin B (PMB) and identify substitutions retaining antibacterial potency while reducing HK-2 cytotoxicity. Twenty-two PMB analogues were synthesized and evaluated for antibacterial activity and HK-2 cytotoxicity. New compound 22 was further assessed in a mouse systemic infection model. Its lipopolysaccharide (LPS) binding was examined by biolayer interferometry (BLI). Isothermal titration calorimetry (ITC) characterized peptide–membrane interactions. Ser substitution at Thr2 or Thr10 retained antibacterial activity with modestly reduced HK-2 cytotoxicity. Replacing Dab3 with 2,3-diaminopropionic acid (Dap) or ornithine (Orn) preserved activity and markedly reduced HK-2 cytotoxicity. Compound 7 showed a selectivity index (SI) range of >2000.0 to >8000.0. Combining Dap3 with reduced hydrophobicity at Leu7 maintained or improved activity and increased selectivity. Compound 22 showed an SI range of >2000.0 to >16666.7, efficacy comparable to PMB in a preliminary mouse infection model, LPS binding, and a smaller ITC response than PMB. At positions 2 and 10, hydroxyl-bearing residues best preserved antibacterial activity, position 10 showed greater substitution tolerance. Position 3 required cationic character and offered the clearest opportunity to improve selectivity.