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4-(((4-Cyanophenyl)(4H-1,2,4-triazol-4-yl)amino)methyl)phenyl sulfamate (6 a) was the first dual aromatase-sulfatase inhibitor (DASI) reported. Several series of its derivatives with various linker systems between the steroid sulfatase (STS) and the aromatase inhibitory pharmacophores were synthesised and evaluated in JEG-3 cells. The X-ray crystal structures of the aromatase inhibitors, DASI precursors 42 d and 60, and DASI 43 h were determined. Nearly all derivatives show improved in vitro aromatase inhibition over 6 a but decreased STS inhibition. The best aromatase inhibitor is 42 e (IC(50)=0.26 nM) and the best DASI is 43 e (IC(50 aromatase)=0.45 nM, IC(50 STS)=1200 nM). SAR for aromatase inhibition shows that compounds containing an alkylene- and thioether-based linker system are more potent than those that are ether-, sulfone-, or sulfonamide-based, and that the length of the linker has a limited effect on aromatase inhibition beyond two methylene units. Compounds 43 d-f were studied in vivo (10 mg kg(-1), single, p.o.). The most potent DASI is 43 e, which inhibited PMSG-induced plasma estradiol levels by 92 % and liver STS activity by 98 % 3 h after dosing. These results further strengthen the concept of designing and developing DASIs for potential treatment of hormone-related cancers.

Purpose: The production of E2 is paramount for the growth of estrogen receptor-positive breast cancer. Various strategies have been used, including the use of enzyme inhibitors against either aromatase (AROM) or steroid sulfatase (STS), in an attempt to ablate E2 levels. Both these enzymes play a critical role in the formation of estrogenic steroids and their inhibitors are now showing success in the clinic., Experimental Design: We show here, in a xenograft nude mouse model, that the inhibition of both enzymes using STX681, a dual AROM and STS inhibitor (DASI), is a potential new therapeutic strategy against HDBC. MCF-7 cells stably expressing either AROM cDNA (MCF-7(AROM)) or STS cDNA (MCF-7(STS)) were generated. Ovariectomized MF-1 female nude mice receiving s.c. injections of either androstenedione (A(4)) or E2 sulfate and bearing either MCF-7(AROM) or MCF-7(STS) tumors were orally treated with STX64, letrozole, or STX681. Treatment was administered for 28 days. Mice were weighed and tumor measurements were taken weekly., Results: STX64, a potent STS inhibitor, completely blocked MCF-7(STS) tumor growth but failed to attenuate MCF-7(AROM) tumor growth. In contrast, letrozole inhibited MCF-7(AROM) tumors but had no effect on MCF-7(STS) tumors. STX681 completely inhibited the growth of both tumors. AROM and STS activity was also completely inhibited by STX681, which was accompanied by a significant reduction in plasma E2 levels., Conclusions: This study indicates that targeting both the AROM and the STS enzyme with a DASI inhibits HDBC growth and is therefore a potentially novel treatment for this malignancy.

By introducting the steroid sulfatase inhibitory pharmacophore into aromatase inhibitor 1 (YM511), two series of single agent dual aromatase-sulfatase inhibitors (DASIs) were generated. The best DASIs in vitro (JEG-3 cells) are 5, (IC50(aromatase) = 0.82 nM; IC50(sulfatase) = 39 nM), and 14, (IC50(aromatase) = 0.77 nM; IC50(sulfatase) = 590 nM). X-ray crystallography of 5, and docking studies of selected compounds into an aromatase homology model and the steroid sulfatase crystal structure are presented. Both 5 and 14 inhibit aromatase and sulfatase in PMSG pretreated adult female Wistar rats potently 3 h after a single oral 10 mg/kg dose. Almost complete dual inhibition is observed for 5 but the levels were reduced to 85% (aromatase) and 72% (sulfatase) after 24 h. DASI 5 did not inhibit aldosterone synthesis. The development of a potent and selective DASI should allow the therapeutic potential of dual aromatase-sulfatase inhibition in hormone-dependent breast cancer to be assessed.

Aromatase inhibitors in clinical use block the biosynthesis of estrogens. Hydrolysis of estrone 3-sulfate by steroid sulfatase is an important additional source of tumor estrogen, and blockade of both enzymes should provide a more effective endocrine therapy. Sulfamoylated derivatives of the aromatase inhibitor YM511 inhibited sulfatase and aromatase in JEG-3 cells with respective IC(50) values of 20-227 and 0.82-100 nM (cf. letrozole, 0.89 nM). One dual inhibitor was potent against both enzymes in vivo, validating the concept.