An intact C=C bond in the vinyl side chain is required for the pro-apoptotic effect of nitrostyrene and its derivatives

We have recently shown that nitrostyrene and some its derivatives are potent pro-apoptotic agents in tumour cells. In a previous structure activity analysis we could outline the importance of the nitro group in the side chain. Here, we will demonstrate that also an intact conjugation between the nitro group and the phenyl ring is necessary for apoptosis induction. The importance of this C=C bond became apparent when we tested Michael adducts of nitrostyrene, which by adduct formation lack the C=C bond. While the triamino-pyrimidine adduct (TAP-NS) between 10 and 25 µM displayed a distinct pro-apoptotic potency and like nitrostyrene induced DNA-strand breaks in comet assays, the diamino-pyridine adduct (DAP-NS) did neither damage DNA nor was cytotoxic even at 100 µM. Nitrostyrene can reactwith thiol groups via the C=C group which results in the loss of an absorption peak at 320 nm (Park & Pei, 2004). When UV-vis absorption of the adducts was measured, both initially displayed no peak at 320 nm. While the active TAP-NS adduct time-dependently developed such a peak, the spectrum of the non-functional DAP-NS adduct did not change with time. Apparently the functional adduct liberates nitrostyrene with its characteristic C=C bond explaining the observed effects, whereas the DAP-NS adduct did not. This conclusion was further substantiated with various nitrostyrene analogues lacking the C=C bond, thus displaying no peak at 320 nm, which were without cytotoxic effects. On the other hand, adducts that were significantly less potent than TAP-NS in former tests revealed a less intensive increase in adsorption at 320 nm than TAP-NS. Thus, our data suggest that an intact C=C is required for the pro-apoptotic effect of nitrostyrene and its adducts. Therefore, adducts with a pro-apoptotic effect can be regarded as prodrug forms of nitrostyrene with different potencies.