N-Substituted colchicine analogues incorporating ferrocenyl and ruthenocenyl moieties: potent antiproliferative activity and substituent-dependent MDR profiles.

Colchicine-site-binding antimicrotubule agents remain promising antimitotic compounds, particularly when structural modification enables retention of tubulin-directed activity while modulating multidrug resistance (MDR) phenotypes. Building on the N-benzyl colchicine scaffold, we synthesised and evaluated a focused series of N-substituted analogues in which the native N-acetyl group was replaced by adamantylmethyl, ferrocenylmethyl or ruthenocenylmethyl, or isomeric o-, m-, and p-ferrocenylbenzyl substituents. The derivatives displayed low-nanomolar antiproliferative activity in drug-sensitive colorectal cancer cell lines, with selected ferrocenylbenzyl analogues and the ruthenocenylmethyl derivative showing particularly high potency. The N-substituent strongly influenced MDR performance: directly attached metallocenylmethyl analogues exhibited pronounced cross-resistance in the ABCG2-overexpressing model, whereas the N-benzyl analogue and selected ferrocenylbenzyl derivatives retained substantial activity and markedly reduced resistance relative to colchicine, especially in the ABCB1-overexpressing model. Cell-free tubulin polymerisation assays confirmed concentration-dependent inhibition of microtubule assembly across the series, and cell-cycle analysis revealed substituent-dependent redistribution consistent with an antimitotic mechanism, with several newly synthesised analogues producing stronger G2/M accumulation than colchicine under the selected assay conditions.