CYP1-bioactivated 2,4-diaryl-substituted pyridine analogues with remarkable activity in a breast cancer in vitro model.

A series of 2,4-diaylpyridine analogues 8(1-9) was synthesised and evaluated for antiproliferative activity against a panel of human tumour and non-tumour breast cell lines that was characterised for CYP1 A1, A2 and B1 isoforms expression. Diarylpyridine 8 (6) was the most potent analogue with high cytotoxicity towards MDA-MB-468 cells (IC = 0.08 μM) and no toxicity towards MCF-10 A cells (IC = 100 μM). In vitro enzyme inhibition studies revealed that CYP1 isozymes were responsible for the metabolism and consequent bioactivation of 8 (6). CYP1-catalysed metabolism experiments using 8 (6) revealed the formation of four main metabolites (M1-4) that were characterised by LC-MS analysis. It was found that the primary metabolisation route for 8 (6) consisted in the dealkylation of its 3,4-methylenedioxy A-ring functionality to generate the toxic catechol metabolite M2. The latter was synthesised (9), co-eluted with samples spiked with original CYP1-generated metabolites and evaluated for antiproliferative activity. Our studies confirmed that 9 was the CYP1-generated metabolite (M2) exhibiting cytotoxic activities at low micromolar level against all cell lines in the panel regardless of their expression of CYP1 enzymes. In summary, we demonstrated the pro-drug mode of action of the tumour-selective 8 (6), which upon CYP1-mediated conversion to toxic metabolites, was capable of exerting antiproliferative activity in breast cancer cells.