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 (<BO>1</BO>), a cancer chemopreventive agent and a pluripotent morphogen, was found to react efficiently with nitrite ions in a biphasic system consisting of CH<INF>2</INF>Cl<INF>2</INF>/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 <BO>3a</BO>,<BO>b </BO>and the decarboxylated 12,14-dinitro and 5,6-epoxy-14-nitro derivatives <BO>4 </BO>and <BO>5a</BO> by spectral analysis. Use of ¹⁵NO<INF>2</INF>^- followed by extensive 2D NMR analysis, including ¹H,¹⁵N heteronuclear multiple bond correlation experiments, allowed identification of nitronitrate derivatives as additional constituents of the mixture. Under similar conditions, <BO>1</BO> methyl ester gave mainly <BO>3a</BO>,<BO>b</BO>. 5,6-Epoxyretinoic acid (<BO>2</BO>) reacted smoothly with acidic nitrite to give mainly <BO>5a</BO> and its isomer <BO>5b</BO> whereas its methyl ester afforded 14-nitro derivatives <BO>9a</BO>,<BO>b</BO> as chief products. The observed patterns of reactivity along with mechanistic experiments would suggest that nitrite-induced nitration of <BO>1 </BO>proceeds through complex reaction pathways set in motion by attack of NO<INF>2</INF> to the 12- and 14-positions. Separate experiments showed that <BO>1</BO> 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 <BO>1 </BO>of potential relevance to the mechanism of its antiinflammatory, antimutagenic, and cancer chemopreventive action.