Bicyclic derivatives of the potent dual aromatase-steroid sulfatase inhibitor 2-bromo-4-{[(4-cyanophenyl)(4h-1,2,4-triazol-4-yl)amino]methyl}phenylsulfamate: synthesis, SAR, crystal structure, and in vitro and in vivo activities.

The design and synthesis of a series of bicyclic ring containing dual aromatase-sulfatase inhibitors (DASIs) based on the aromatase inhibitor (AI) 4-[(4-bromobenzyl)(4H-1,2,4-triazol-4-yl)amino]benzonitrile are reported. Biological evaluation with JEG-3 cells revealed structure-activity relationships. The X-ray crystal structure of sulfamate 23 was determined, and selected compounds were docked into the aromatase and steroid sulfatase (STS) crystal structures. In the sulfamate-containing series, compounds containing a naphthalene ring are both the most potent AI (39, IC(50 AROM)=0.25 nM) and the best STS inhibitor (31, IC(50 STS)=26 nM). The most promising DASI is 39 (IC(50 AROM)=0.25 nM, IC(50 STS)=205 nM), and this was evaluated orally in vivo at 10 mg kg(-1), showing potent inhibition of aromatase (93 %) and STS (93 %) after 3 h. Potent aromatase and STS inhibition can thus be achieved with a DASI containing a bicyclic ring system; development of such a DASI could provide an attractive new option for the treatment of hormone-dependent breast cancer.

Highly potent first examples of dual aromatase-steroid sulfatase inhibitors based on a biphenyl template.

Single agents against multiple drug targets are of increasing interest. Hormone-dependent breast cancer (HDBC) may be more effectively treated by dual inhibition of aromatase and steroid sulfatase (STS). The aromatase inhibitory pharmacophore was thus introduced into a known biphenyl STS inhibitor to give a series of novel dual aromatase-sulfatase inhibitors (DASIs). Several compounds are good aromatase or STS inhibitors and DASI 20 (IC(50): aromatase, 2.0 nM; STS, 35 nM) and its chlorinated congener 23 (IC(50): aromatase, 0.5 nM; STS, 5.5 nM) are examples that show exceptional dual potency in JEG-3 cells. Both biphenyls share a para-sulfamate-containing ring B and a ring A, which contains a triazol-1-ylmethyl meta to the biphenyl bridge and para to a nitrile. At 1 mg/kg po, 20 and 23 reduced plasma estradiol levels strongly and inhibited liver STS activity potently in vivo. 23 is nonestrogenic and potently inhibits carbonic anhydrase II (IC(50) 86 nM). A complex was crystallized and its structure was solved by X-ray crystallography. This class of DASI should encourage further development toward multitargeted therapeutic intervention in HDBC.

A new therapeutic strategy against hormone-dependent breast cancer: the preclinical development of a dual aromatase and sulfatase inhibitor.

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.

Synthesis of aromatase inhibitors and dual aromatase steroid sulfatase inhibitors by linking an arylsulfamate motif to 4-(4H-1,2,4-triazol-4-ylamino)benzonitrile: SAR, crystal structures, in vitro and in vivo activities.

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.

Anticancer steroid sulfatase inhibitors: synthesis of a potent fluorinated second-generation agent, in vitro and in vivo activities, molecular modeling, and protein crystallography.

An improved steroid sulfatase inhibitor was prepared by replacing the N-propyl group of the second-generation steroid-like inhibitor (2) with a N-3,3,3-trifluoropropyl group to give (10). This compound is 5-fold more potent in vitro, completely inhibits rat liver steroid sulfatase activity after a single oral dose of 0.5 mg/kg, and exhibits a significantly longer duration of inhibition over (2). These biological properties are attributed to the increased lipophilicity and metabolic stability of (10) rendered by its trifluoropropyl group and also the potential H-bonding between its fluorine atom(s) and Arg(98) in the active site of human steroid sulfatase. Like other sulfamates, (10) is expected to be sequestered, and transported by, erythrocytes in vivo because it inhibits human carbonic anhydrase II (hCAII) potently (IC(50), 3 nmol/L). A congener (4), which possesses a N-(pyridin-3-ylmethyl) substituent, is even more active (IC(50), 0.1 nmol/L). To rationalize this, the hCAII-(4) adduct, obtained by cocrystallization, reveals not only the sulfamate group and the backbone of (4) interacting with the catalytic site and the associated hydrophobic pocket, respectively, but also the potential H-bonding between the N-(pyridin-3-ylmethyl) group and Nepsilon(2) of Gln(136). Like (2), both (10) and its phenolic precursor (9) are non-estrogenic using a uterine weight gain assay. In summary, a highly potent, long-acting, and nonestrogenic steroid sulfatase inhibitor was designed with hCAII inhibitory properties that should positively influence in vivo behavior. Compound (10) and other related inhibitors of this structural class further expand the armory of steroid sulfatase inhibitors against hormone-dependent breast cancer.

17beta-hydroxysteroid dehydrogenase Type 1, and not Type 12, is a target for endocrine therapy of hormone-dependent breast cancer.

Oestradiol (E2) stimulates the growth of hormone-dependent breast cancer. 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyse the pre-receptor activation/inactivation of hormones and other substrates. 17beta-HSD1 converts oestrone (E1) to active E2, but it has recently been suggested that another 17beta-HSD, 17beta-HSD12, may be the major enzyme that catalyses this reaction in women. Here we demonstrate that it is 17beta-HSD1 which is important for E2 production and report the inhibition of E1-stimulated breast tumor growth by STX1040, a non-oestrogenic selective inhibitor of 17beta-HSD1, using a novel murine model. 17beta-HSD1 and 17beta-HSD12 mRNA and protein expression, and E2 production, were assayed in wild type breast cancer cell lines and in cells after siRNA and cDNA transfection. Although 17beta-HSD12 was highly expressed in breast cancer cell lines, only 17beta-HSD1 efficiently catalysed E2 formation. The effect of STX1040 on the proliferation of E1-stimulated T47D breast cancer cells was determined in vitro and in vivo. Cells inoculated into ovariectomised nude mice were stimulated using 0.05 or 0.1 microg E1 (s.c.) daily, and on day 35 the mice were dosed additionally with 20 mg/kg STX1040 s.c. daily for 28 days. STX1040 inhibited E1-stimulated proliferation of T47D cells in vitro and significantly decreased tumor volumes and plasma E2 levels in vivo. In conclusion, a model was developed to study the inhibition of the major oestrogenic 17beta-HSD, 17beta-HSD1, in breast cancer. Both E2 production and tumor growth were inhibited by STX1040, suggesting that 17beta-HSD1 inhibitors such as STX1040 may provide a novel treatment for hormone-dependent breast cancer.

Efficacy of three potent steroid sulfatase inhibitors: pre-clinical investigations for their use in the treatment of hormone-dependent breast cancer.

Estrogenic steroids, such as estradiol, are known to play a crucial role in the development and growth of hormone-dependent breast cancer. Steroid sulfatase (STS) inhibitors that can prevent the biosynthesis of these steroids via the sulfatase pathway offer therapeutic potential. We show here the in vivo profile, including the efficacy in a xenograft breast cancer model and pharmacokinetics, of three potent STS inhibitors. MCF-7 cells stably over-expressing STS cDNA (MCF-7STS) were generated. Ovariectomised, MF-1, female nude mice receiving subcutaneous injections of estradiol sulfate (E2S) and bearing MCF-7STS xenografts, were orally treated with the STS inhibitors STX64, STX213, and STX1938. Treatment was administered once weekly at a dose of 1 mg/kg for 35 days during which animals received E2S thrice weekly. Mice were weighed and tumor measurements taken weekly. Furthermore, the pharmacokinetics for STX213 was determined in rats. STX213 and STX1938 exhibited potent STS inhibition in vivo. However, STX1938 demonstrated a greater duration of activity. In vehicle treated nude mice receiving E2S, tumor volumes increased by 260% after 35 days compared to day zero. STX64 (1 mg/kg) failed to reduce tumor growth when given once weekly. STX213 and STX1938 (once weekly, 1 mg/kg) significantly inhibited (P < 0.05) tumor growth over this same time period. These compounds completely inhibited liver and tumor STS activity and significantly reduced the levels of plasma E2. This study indicates that the STS inhibitor, STX213, exhibits excellent efficacy and pharmacokinetics and therefore offers a potentially novel treatment for hormone-dependent breast cancer.

Dual aromatase-sulfatase inhibitors based on the anastrozole template: synthesis, in vitro SAR, molecular modelling and in vivo activity.

The synthesis and biological evaluation of a series of novel Dual Aromatase-Sulfatase Inhibitors (DASIs) are described. It is postulated that dual inhibition of the aromatase and steroid sulfatase enzymes, both responsible for the biosynthesis of oestrogens, will be beneficial in the treatment of hormone-dependent breast cancer. The compounds are based upon the Anastrozole aromatase inhibitor template which, while maintaining the haem ligating triazole moiety crucial for enzyme inhibition, was modified to include a phenol sulfamate ester motif, the pharmacophore for potent irreversible steroid sulfatase inhibition. Adaption of a synthetic route to Anastrozole was accomplished via selective radical bromination and substitution reactions to furnish a series of aromatase inhibitory pharmacophores. Linking these fragments to the phenol sulfamate ester moiety employed SN2, Heck and Mitsunobu reactions with phenolic precursors, from where the completed DASIs were achieved via sulfamoylation. In vitro, the lead compound, 11, had a high degree of potency against aromatase (IC50 3.5 nM), comparable with that of Anastrozole (IC50 1.5 nM) whereas, only moderate activity against steroid sulfatase was found. However, in vivo, 11 surprisingly exhibited potent dual inhibition. Compound 11 was modelled into the active site of a homology model of human aromatase and the X-ray crystal structure of steroid sulfatase.

Dual aromatase-sulfatase inhibitors based on the anastrozole template: synthesis, in vitro SAR, molecular modelling and in vivo activity.

The synthesis and biological evaluation of a series of novel Dual Aromatase-Sulfatase Inhibitors (DASIs) are described. It is postulated that dual inhibition of the aromatase and steroid sulfatase enzymes, both responsible for the biosynthesis of oestrogens, will be beneficial in the treatment of hormone-dependent breast cancer. The compounds are based upon the Anastrozole aromatase inhibitor template which, while maintaining the haem ligating triazole moiety crucial for enzyme inhibition, was modified to include a phenol sulfamate ester motif, the pharmacophore for potent irreversible steroid sulfatase inhibition. Adaption of a synthetic route to Anastrozole was accomplished via selective radical bromination and substitution reactions to furnish a series of inhibitory aromatase pharmacophores. Linking these fragments to the phenol sulfamate ester moiety employed S(N)2, Heck and Mitsunobu reactions with phenolic precursors, from where the completed DASIs were achieved via sulfamoylation. In vitro, the lead compound, 11, had a high degree of potency against aromatase (IC(50) 3.5 nM), comparable with that of Anastrozole (IC(50) 1.5 nM) whereas, only moderate activity against steroid sulfatase was found. However, in vivo, 11 surprisingly exhibited potent dual inhibition. Compound 11 was modelled into the active site of a homology model of human aromatase and the X-ray crystal structure of steroid sulfatase.

Dual aromatase-steroid sulfatase inhibitors.

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.

In vivo efficacy of STX213, a second-generation steroid sulfatase inhibitor, for hormone-dependent breast cancer therapy.

PURPOSE: Steroid sulfatase (STS) inhibitors that can decrease or prevent the biosynthesis of estrogenic steroids via the sulfatase route may play an important role in the treatment of breast cancer. We compare the in vivo efficacy of two potent STS inhibitors, STX64 and STX213, in a xenograft breast cancer model. EXPERIMENTAL DESIGN: MCF-7 cells stably expressing STS cDNA (MCF-7STS) were generated. Ovariectomized MF-1 female nude mice receiving s.c. injections of estradiol sulfate (E2S) and bearing both MCF-7STS and wild-type MCF-7 (MCF-7WT) tumors were orally treated with STX64 and STX213. Treatment was given for 49 days followed by a recovery period of 35 days in which animals received only E2S. Mice were weighed, and tumor measurements were taken weekly. RESULTS: STX64 and STX213 exhibited potent STS inhibition in vivo. However, STX213 showed a greater duration of activity. In vehicle-treated nude mice receiving E2S, tumor volumes increased 5.5-fold for MCF-7WT and 3.8-fold for MCF-7STS after 49 days compared with day 0. MCF-7WT tumor growth was reduced by 56% by STX213 over the dosing period, and subsequent growth was retarded during the recovery period. All treatments fully inhibited growth of MCF-7STS tumors, and recovery of these tumors was significantly retarded (P<0.01). All compounds completely inhibited liver and tumor STS activity. Additionally, STS mRNA expression in the MCF-7STS tumors directly correlated with the corresponding STS enzyme activity. CONCLUSIONS: This study indicates that STS inhibitors attenuate hormone-dependent human breast cancer growth and therefore offer a potentially novel treatment for this condition.

A letrozole-based dual aromatase-sulphatase inhibitor with in vivo activity.

The role of aromatase inhibitors in the treatment of hormone-dependent breast cancer is well established. However, it is now recognised that steroid sulphatase (STS) inhibitors represent a new form of endocrine therapy. To explore the potential advantage of dual inhibition by a single agent, we recently developed a series of dual aromatase-sulphatase inhibitors (DASIs) based on the aromatase inhibitor YM511. We report here a new structural class of DASI obtained by obtained introducing the pharmacophore for STS inhibition, i.e. a phenol sulphamate ester into another established aromatase inhibitor letrozole. Hence, the bis-sulphamate 9 was synthesised which exhibited IC(50) values of 3044 nM for aromatase and >10 microM for STS in JEG-3 cells. However, at a single oral dose of 10mg/kg, 9 inhibited aromatase and rat liver STS by 60% and 88%, respectively, 24h after administration. A proposed metabolite of 9, carbinol 10, was synthesised. Despite also showing weak STS inhibition in JEG-3 cells, 10 inhibited rat liver STS activity to the same extent as 9 at a single oral dose of 10mg/kg. Thus, the concept of a letrozole-based DASI has been validated and could be further developed and modified for therapeutic exploitation.

The role of steroid sulphatase in regulating the oestrogenicity of oestrogen sulphamates.

Oestrogen sulphamates have increased systemic, but reduced hepatic oestrogenicity which results from their sequestration and transport through the liver by red blood cells. Oestrogen sulphamates act as prodrugs for the release of natural oestrogens but, as yet, there is little information as to how the sulphamoyl moiety is cleaved from the steroid nucleus. In the present investigation we have used the potent steroid sulphatase (STS) inhibitor, 667 COUMATE, to explore the possibility that STS might be responsible for the hydrolysis of oestrogen sulphamates. Administration of oestrone sulphamate (10 microg/day, s.c., for 5 days) to ovariectomised rats resulted in a 3.5-fold increase in the uterine weights of treated animals. Co-administration of oestrone sulphamate and 667 COUMATE (2 mg/kg) completely blocked STS activity in treated animals and completely abrogated the ability of oestrone sulphamate to stimulate uterine growth. In vitro studies, using [(3)H]oestrone sulphamate or [(3)H]oestrone, revealed that the uptake of the sulphamate derivative (95.9+/-2.4%) by red blood cells was considerably higher than that for the non-sulphamoylated oestrogen (25.1+/-1.9%). Results from these studies demonstrate convincingly that STS is the enzyme responsible for the removal of the sulphamoyl group from oestrogen sulphamates. This enzyme therefore has a crucial role in regulating the oestrogenicity associated with this class of drug.

First dual aromatase-steroid sulfatase inhibitors.

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.

Development of a sensitive high-performance liquid chromatography method for the detection of 667 COUMATE in vivo.

Steroid sulphatase inhibitors which decrease or prevent the biosynthesis of oestrogens, potentially have an important role in the treatment of breast cancer in postmenopausal women. The non-steroidal sulphatase inhibitor 667 COUMATE has been shown to be active both in vitro and in vivo. The pharmacokinetics of this drug have not been investigated. In preparation for the clinical evaluation of this agent, a sensitive and robust reversed phase high-performance liquid chromatography (HPLC) method was developed for the detection of 667 COUMATE in biological fluids. The sulphatase inhibitor was extracted from plasma with diethyl ether and separated from putative metabolites and endogenous plasma components with a C3-phenyl column. Using this method an extraction efficiency of 76+/-5% and a limit of detection of less than 0.1 ng/ml was achieved. The stability of this agent was investigated under different pH conditions and during storage in plasma at room temperature or -20 degrees C. 667 COUMATE was found to be stable when stored in acidified plasma (pH 4.5) at -20 degrees C. In conclusion, the HPLC method developed is a reproducible and sensitive assay that will enable quantitation of the potent non-steroidal sulphatase inhibitor 667 COUMATE in biological fluids in the forthcoming Phase I clinical trial.

Novel D-ring modified steroid derivatives as potent, non-estrogenic, steroid sulfatase inhibitors with in vivo activity.

In pursuit of novel steroid sulfatase (STS) inhibitors devoid of estrogenicity, several D-ring modified steroid derivatives were synthesised. In vitro evaluation of the compounds identified two highly potent inhibitors, 4a and 4b, which were 18 times more active than estrone-3-O-sulfamate (EMATE), both having IC(50) values of ca. 1nM. These 16,17-seco-estra-1,3,5(10)-triene-16,17-imide derivatives were synthesised from estrone, via the intermediate 1, which was easily alkylated, deprotected and sulfamoylated affording the final compounds in high yields. In order to assess their biological profile, the selected inhibitors were tested for their in vivo inhibitory potency and estrogenicity in ovariectomised rats. After an oral dose of 10mg/kg per day for 5 days, 4a and 4b were found to inhibit rat liver steroid sulfatase by 99%. They were also devoid of estrogenic activity in the uterine weight gain assay, indicating that these two leads have therapeutic potential for the treatment of hormone-dependent breast cancer.

Inhibition of MCF-7 breast cancer cell proliferation and in vivo steroid sulphatase activity by 2-methoxyoestradiol-bis-sulphamate.

The endogenous oestrogen metabolite, 2-methoxyoestradiol (2-MeOE2) inhibits the growth of breast cancer cells and is also a potent anti-angiogenic agent. We have previously shown that the 3-sulphamoylated derivatives of 2-methoxyoestrogens are more potent than the non-sulphamoylated compounds. In this study, we have compared the abilities of 2-methoxyoestradiol-bis-sulphamate (2-MeOE2bisMATE) and 2-MeOE2 to inhibit the growth of MCF-7 breast cancer cells. Both compounds inhibited cell growth with the IC(50) for 2-MeOE2bisMATE (0.4 microM) being six-fold lower than that for 2-MeOE2 (2.5 microM). Oestrogen sulphamates are potent inhibitors of steroid sulphatase (STS) activity. 2-MeOE2bisMATE was found to retain its STS inhibitory activity and in a placental microsome assay system it was equipotent with oestrone-3-O-sulphamate (EMATE). An in vivo study was also carried out to compare the potency of 2-MeOE2bisMATE with that of EMATE and the non-steroidal STS inhibitor, 667 coumarin sulphamate (667 COUMATE). After a single oral dose (10mg/kg) some recovery of STS activity was detected by day 3 (10%) with activity partially restored (55%) by day 7 after administration of 667 COUMATE. For the other two steroidal compounds, STS activity remained almost completely inactivated for up to 5 days with complete restoration of activity occurring by day 15. The anti-proliferative and STS inhibitory properties of 2-MeOE2bisMATE suggest that it has considerable potential for development as a novel anti-cancer drug.