Nitrite-induced nitration pathways of retinoic acid, 5,6-epoxyretinoic acid, and their esters under mildly acidic conditions: toward a reappraisal of retinoids as scavengers of reactive nitrogen species.

All trans retinoic acid (1), a cancer chemopreventive agent and a pluripotent morphogen, was found to react efficiently with nitrite ions in a biphasic system consisting of CH(2)Cl(2)/0.1 M phosphate buffer (pH 3) 1:1 v/v to give a complex mixture of nitration products. Repeated TLC fractionation of the reaction mixtures after methylation allowed isolation of the main products, which could be identified as the 12-nitro derivatives 3a,b and the decarboxylated 12,14-dinitro and 5,6-epoxy-14-nitro derivatives 4 and 5a by spectral analysis. Use of (15)NO(2)(-) followed by extensive 2D NMR analysis, including (1)H,(15)N heteronuclear multiple bond correlation experiments, allowed identification of nitronitrate derivatives as additional constituents of the mixture. Under similar conditions, 1 methyl ester gave mainly 3a,b. 5,6-Epoxyretinoic acid (2) reacted smoothly with acidic nitrite to give mainly 5a and its isomer 5b whereas its methyl ester afforded 14-nitro derivatives 9a,b as chief products. The observed patterns of reactivity along with mechanistic experiments would suggest that nitrite-induced nitration of 1 proceeds through complex reaction pathways set in motion by attack of NO(2) to the 12- and 14-positions. Separate experiments showed that 1 can inhibit nitrite-induced N-nitrosation of 2,3-diaminonaphthalene at pH values of 4 and 5.5, as well as decomposition of caffeic acid under similar conditions. Overall, these results provide the first detailed insight into the reaction behavior of a retinoid toward reactive nitrogen species and shed light on previously overlooked nitrite scavenging properties of 1 of potential relevance to the mechanism of its antiinflammatory, antimutagenic, and cancer chemopreventive action.