Curcumin is a natural product widely used as a spice in food. It has been shown to inhibit cyclooxygenase (COX)-1 and -2 and to suppress lipopolysaccharide-induced COX-2 and iNOS gene expression. In the present study, curcumin and 22 of its derivatives were evaluated for their chemopreventive potential. Based on COX-2 inhibition, curcumin (IC50=15.9 microM), 1,7-bis(3-fluoro-4-hydroxyphenyl)-1,6-heptadiene-3,5-dione (19) (IC50=23.7 microM) and 2,6-bis(3-fluoro-4-hydroxybenzylidene)cyclohexanone (23) (IC50=5.5 microM) were found to be most potent. Tricyclic derivatives 2,6-bis(4-hydroxy-3-methoxybenzylidene)cyclohexanone (10), 2,6-bis(4-hydroxy-3,5-dimethoxybenzylidene)cyclohexanone (13) and 2,5-bis(4-hydroxy-3,5-dimethoxybenzylidene)cyclopentanone (21) inhibited LPS-induced COX-2 and iNOS gene expression in murine macrophages with potency equal to curcumin. RT-PCR experiments demonstrated suppression of COX-2 and iNOS gene expression occurred at the transcriptional level. The most active compounds in the macrophage assays, 13 and 23, were also the most cytotoxic, however. Topical application of curcumin, 10, 13, 21, and 6, a methoxy derivative of curcumin, showed strong inhibition of 12-O-tetradecanoyl-13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity in mouse skin. These data suggest that structural elements responsible for COX-1 and COX-2 inhibition do not correlate well with those responsible for inhibiting COX-2 and iNOS gene expression, but elements capable of inhibiting COX-2 and iNOS gene expression also contribute to inhibition of TPA-induced ODC activity. The most potent compounds in these assays, 10, 13 and 21, as well as curcumin, were further evaluated for inhibition of 7,12-dimethylbenz(a)anthracene (DMBA)-induced preneoplastic lesion formation in a mouse mammary organ culture model, and dose-dependent responses were observed. Most potent effects were at concentrations between 1 and 5 microM for 10, 13 and 21, and at 10 microM for curcumin. These data demonstrate the substitution pattern on the aromatic moiety is especially crucial for activity.