2'-Benzoylpyridine thiosemicarbazones (BpT) are effective iron chelators and display potent anti-proliferative activity against tumour cells. In order to gain greater insight into the structure-activity relationships of the BpT chelators, ten new analogues containing phenyl substituents at the N4-position of the BpT structure were synthesised. Importantly, aromatic substitution at the latter position of the BpT scaffold has not been previously explored and these studies represent the first attempt to investigate their structure-activity relationships. These compounds demonstrated significantly enhanced anti-proliferative activity compared to the clinically used iron chelator, desferrioxamine (DFO). Furthermore, the compounds showed appreciable therapeutic indices against cancer cells over normal cells in vitro. Structure-activity analysis revealed that electron-donating substituents such as -CH3 and -OCH3 resulted in greater anti-proliferative activity than electron-withdrawing groups such as -Br and -Cl. These findings help to elucidate the effect of a variety of 4-phenyl substituents on the biological activity of BpT series of chelators and facilitate the future development of thiosemicarbazones with improved anti-tumour activity.