Thiosemicarbazones of the microbial metabolite madurahydroxylactone, a polysubstituted benzo[a]naphthacenequinone, have been previously reported by us as potent nonsteroidal inhibitors of the enzyme estrone sulfatase (cyclohexylthiosemicarbazone 1, IC50 0.46 microM). The active pharmacophore of 1 has now been identified to be 2-formyl-6-hydroxybenzoic acid cyclohexylthiosemicarbazone (25, IC50 4.2 microM). The active partial structure was derivatized in the search for novel agents against hormone-dependent breast cancer. Further substantial increases in activity were achieved by reversal of functional groups leading to the cyclohexylthiosemicarbazones of 5-formylsalicylic acid (35, IC50 0.05 microM) and 3-formylsalicylic acid (34, IC50 0.15 microM) as the most potent analogues identified to date. Both compounds were shown to be noncompetitive inhibitors of estrone sulfatase with Ki values of 0.13 microM and 0.12 microM, respectively. The compounds showed low acute toxicity in the hen's fertile egg screening test.