Structure-activity relationships for the formation of secondary radicals and inhibition of keratinocyte proliferation by 9-anthrones

The biological properties of tumor-promoting and antipsoriatic 9-anthrones have been hypothesized to be mediated by free radical products such as the corresponding 9-anthron-10-yl radicals or by O2-, OH, and other persistent secondary radicals that are formed in the skin after topical treatment with 9-anthrones. To gain additional insights into the possible role of reactive oxygen or secondary radicals in mediating the biological effects of 9-anthrones, we have used EPR spectroscopy to investigate the formation of these species by a series of 9-anthrones or 9-anthrone dimers with known tumor-promoting and antipsoriatic activities. The effect of the 9-anthrones on keratinocyte proliferation in vitro was also investigated. 5,5-Dimethyl-1-pyrroline N-oxide was used as a spin trap to detect reactive oxygen-centered radicals in aqueous buffer/dimethylsulfoxide solutions. Super-radicals in aqueous buffer/dimethylsulfoxide solutions. Superoxide was trapped during the autoxidation of most of the 9-anthrones. For 9-anthrones that generated no detectable superoxide, evidence of anthronyl-peroxyl radical formation was found instead. In the presence of Fe3+ complexed to EDTA, but not diethylenetriaminepentaacetic acid, the hydroxyl radical was produced by all of the 9-anthrones. 9-Anthrone dimers produced oxygen-centered radicals only weakly or not at all. Direct EPR was used to detect 9-anthrone-derived secondary radicals in keratinocyte suspensions or in dimethysulfoxide solutions. These radicals were similar to those previously reported to occur in skin after topical treatment with the antipsoriatic drug anthralin (1,8-dihydroxy-9-anthrone). In contrast to the ubiquitous ability of the 9-anthrones to generate reactive oxygen radicals, only the hydroxy-substituted 9-anthrones or their dimers possessed significant secondary radical-forming ability. The ability of the 9-anthrones or dimers to form secondary radicals in keratinocytes was found to correlate with their in vitro inhibition of keratinocyte proliferation. The data suggest the possible importance of reactive dimeric intermediates in mediating the biological effects of the 9-anthrones.