Steroid sulfates are precursors of hormones that stimulate androgen- and estrogen-dependent cancers. Thus, steroid sulfatase, the enzyme that catalyzes conversion of DHEAS and E1S to the corresponding unconjugated steroids DHEA and E1, appears to be one of the key enzymes regulating the level of active androgenic and estrogenic steroids. Since 17alpha-substituted benzylestradiols and 3-O-sulfamate estrone (EMATE) represent two families of steroid sulfatase inhibitors that probably act through different mechanisms, we synthesized compounds 3-O-sulfamate 17alpha-benzylestradiol (4) and 3-O-sulfamate 17alpha-(tert-butylbenzyl)estradiol (5) that contain two kinds of substituents on the same molecule. In our enzymatic assay using a homogenate of human embryonal (293) cells transfected with steroid sulfatase, compounds 4 and 5 were found to be more potent inhibitors than already known steroid sulfatase inhibitors that have only a C17alpha-substituent or only a C3-sulfamate group (EMATE). The IC50 values of 4 and 5 were, respectively, 0.39 and 0.15 nM for the transformation of E1S to E1 and 4.1 and 1.4 nM for the transformation of DHEAS to DHEA. Compound 5 inhibited the steroid sulfatase activity in intact transfected (293) cell culture assays by inactivating the enzyme activity. Compound 5 also inactivates the steroid sulfatase activity at lower concentration than EMATE in microsomes of transfected (293) cells. In this assay, an excess of natural substrate E1S protects enzyme against inactivation by 5 or EMATE. Furthermore, the unsulfamoylated analogue of 5, compound 3, did not inactivate the steroid sulfatase.