The present study describes the biological in vitro and in vivo evaluation of 2-methoxy derivatives of estrogenic inhibitors of steroid sulfatase, namely 3-sulfamoyloxy-17α-p-tert-butylbenzyl(or benzyl)-1,3,5 (10)-estratrien-17β-ols. The addition of the 2-methoxy group conserves the potent inhibitory effect on steroid sulfatase activity (IC50s of 0.024 and 0.040 nm) while removing the estrogenic action. Using an ovariectomized mouse model, we show that the first generation of steroid sulfatase inhibitors tested, 3-sulfamoyloxy-17α-p-tert-butylbenzyl(or benzyl)estra-1,3,5 (10)-trien-17β-ols and estrone-3-O-sulfamate, are estrogenic compounds stimulating estrogen-sensitive uterine growth. Interestingly, the 2-methoxy-3-sulfamoyloxy-17α-benzylestra-1,3,5 (10)-trien-17β-ol (7) has no estrogenic activity but efficiently blocks (s.c. and p.o.) uterine growth induced by estrone sulfate, which is converted into estrone and then estradiol by steroid sulfatase and type 1 17β-hydroxysteroid dehydrogenase, respectively. This report clearly shows that a steroid sulfatase inhibitor can efficiently block estrogen action from the inactive precursor estrone sulfate, in vitro and in vivo.

E23 is the main steroid hormone supporting growth of tumors in patients with estrogen-sensitive breast cancer (1). In addition to the blockade of E2 action that can be achieved by the use of antiestrogens (2, 3), a complementary approach to reduce the effects of estrogens consists in the inhibition of steroidogenic enzymes involved in their synthesis (4). Thus, extensive work has focused over the years on the development of inhibitors of the steroidogenic enzymes, namely aromatase (5, 6, 7), 17β-HSDs (8, 9), and steroid sulfatase (10, 11, 12). Inhibition of aromatase has been shown to provide an efficient blockade of the synthesis of potent estrogens, estrone (E1), and E2(13, 14, 15, 16) but cannot block the effect of weak estrogen 5-diol (17, 18, 19). The enzymes steroid sulfatase and type 1 17β-HSD mediate the synthesis of 5-diol from the adrenal precursors DHEAS and dehydroepiandrosterone available in the circulation. In addition, transformation of sulfated steroid estrone sulfate (E1S), the most abundant C18 steroid in aged women, by steroid sulfatase and type 1 17β-HSD provides another major source of potent estrogens E1 and E2 in breast tissue (4, 20, 21, 22).

There is now evidence that an important amount of E2 in breast tumors originates from local transformation of E1S following the sulfatase pathway, whereas <10% results from the aromatization of androst-4-ene-3,17-dione and testosterone (23, 24). The same synthetic pathway accounts for the transformation of DHEAS into the estrogenic C19 steroid 5-diol. Thus, inhibition of steroid sulfatase, the enzyme responsible for the conversion of DHEAS to dehydroepiandrosterone and E1S to E1, may allow reduction of estrogen levels in tumors and could represent a promising approach for the treatment of estrogen-sensitive breast cancer.

Some of the most efficient steroid sulfatase inhibitors developed were sulfamate derivatives (10, 11, 12). The first reported inhibitor in this series, estrone sulfamate (EMATE; Ref. 25, 26) is a very potent irreversible inhibitor, but it is also an estrogenic compound (27), thus having less than optimal characteristics for the therapy of estrogen-sensitive cancers. Purohit et al.(28) then investigated various A-ring substituted analogues of EMATE and found that the 2-methoxy derivative was nonestrogenic and yet a potent inhibitor. Studies reported previously by other groups have in fact indicated that the E2 metabolite 2-methoxy-E2 has reduced ER binding affinity (29) as well as interesting antiangiogenic, antiproliferative, apoptotic, anticarcinogenic, and cytotoxic properties (30, 31, 32, 33, 34, 35). Our group has shown previously that the combined effects of a benzyl (or tert-butylbenzyl) group at C17α and a sulfamate group at C3 of E2 provide an improved inhibition of steroid sulfatase activity (36, 37). These inhibitors, however, induced the proliferation of estrogen-sensitive ZR-75-1 cells, thus suggesting an estrogenic activity (38). We then concluded that the 2-methoxy analogues of these inhibitors might be nonestrogenic and possibly promising therapeutic agents for estrogen-sensitive cancers by targeting important actions: (a) the inhibition of steroid sulfatase; (b) the inhibition of ER+ cell proliferation; and (c) the inhibition of tumor angiogenesis (Fig. 1). Herein, we present the chemical synthesis briefly, the steroid sulfatase in vitro and in vivo inhibitory activities, as well as an in vivo study on the estrogenic properties of these new steroidal inhibitors.

Chemical Synthesis.

Starting steroids 2-methoxyestrone (1) and 2-methoxy-3-O-benzylestrone (2) were purchased from Steraloids, Inc. (Newport, RI) or synthesized similarly as reported in literature (39). Target compounds 6 and 7, as well as intermediate compounds 35, were synthesized from 1 and 2 as exemplified in Fig. 2 with few modifications of the procedure used for the synthesis of 1114(37). Additional compounds 810 were synthesized using classical reduction or sulfamoylation reactions already reported in the preparation of similar compounds (37, 38, 40). The chemical structure of all compounds were fully characterized by infrared, NMR, and mass analysis, and the purity of tested compounds was determined by HPLC and found acceptable for biological assays with purities ranging from 97 to 99%. The next data reported for key compound 7 as well as the HPLC profile (Fig. 3) are representative of all compounds tested in our study. The full details of the experimental procedure and characterization will be available on request addressed to the corresponding author.

2-Methoxy-3-sulfamoyloxy-17α-benzylestra-1,3,5 (10)-trien-17β-ol (7). White solid; infrared (KBr): 3395 (OH and NH2); 1H NMR (CDCl3): 0.98 (s, 18-CH3) 1.3–2.4 (14H), 2.68 and 2.94 (2d of AB-system, J = 13.2 Hz, CH2Ph), 2.82 (m, 6-CH2), 3.89 (s, OCH3), 4.96 (s, OSO2NH2), 6.96 (s, 4-CH), 7.06 (s, 1-CH), 7.33 (m, CH2Ph); 13C NMR (CDCl3): 14.44 (18-C), 23.27 (15-C), 26.36 and 27.28 (7- and 11-C), 28.62 (6-C), 31.36 (12-C), 33.7 (16-C), 39.11 (8-C), 42.33 (1′-C), 44.34 (9-C), 46.69 (13-C), 49.53 (14-C), 56.35 (OCH3), 82.98 (17-C), 110.44 (1-C), 124.06 (4-C), 126.36 (5′’-C), 128.14 (3′’ and 5′’-C), 130.19 (5-C), 131 (2′’ and 6′’-C), 136.8 (10-C), 138.12 (1′’-C), 140.41 (3-C), 148.93 (2-C); LRMS for C26H33NO5S + NH4 [MNH4+]: 489.6 m/z; HPLC purity = 97.2% (C-18 Nova Pak column, H2O: methanol/30:70).

Steroid Sulfatase Assays (in Vitro Studies).

HEK-293 cells (American Type Culture Collection, Rockville, MD), transiently transfected with a sulfatase expression vector (pCMV-sulfa), were used as the source of steroid sulfatase activity. The pCMV-sulfa was constructed by insertion of a cDNA fragment, downstream of the CMV promoter of the pCMV vector, kindly provided by Dr. M. B. Mathews (Cold Spring Harbor Laboratories, Cold Spring Harbor, NY). The sulfatase cDNA fragment was obtained by screening of a human placenta cDNA library (Clontech Laboratories Inc., Palo Alto, CA) using the incomplete cDNA fragment kindly provided by Dr. L. J. Shapiro (Howard Hughes Medical Institute, Los Angeles, CA) as probe. Transfection of the expression vector was performed by the calcium phosphate procedure using 10 μg of recombinant plasmid/106 cells. The cells were initially plated at 104 cells/cm2 in Falcon culture flasks and grown in DMEM containing 10% (volume for volume) fetal bovine serum supplemented with 2 mm l-glutamine, 1 mm sodium pyruvate, 100 IU penicillin/ml, and 100 μg of streptomycin sulfate/ml. For assay, the HEK-293 cells transfected with steroid sulfatase activity were prepared by repeated freezing (−80°C), thawing (five times), and homogenization using a Dounce homogenizer. The enzymatic reaction was carried out at 37°C for the transformation of [3H]E1S into [3H]E1 in 1.25 ml of 100 μm Tris-acetate buffer (pH 7.4) containing 5 mm EDTA, 10% glycerol, 100 μm of radioactive substrate ([6,7-3H]estrone sulfate ammonium salt; New England Nuclear, Boston, MA), and an ethanolic solution of compound to test (at appropriate concentrations). About 2.2 mg protein were used in the tests. After 2 h of incubation, the reaction was stopped by the addition of 1.25 ml of xylene. The tubes were then stirred and centrifuged at 2500 rpm for 10 min to separate organic and aqueous phases. Radioactivities in 750 μl of each phase (organic: [3H]E1 and aqueous: [3H]E1S) were recorded by liquid scintillating counting with a Beckman LS3801 (Irvine, CA). The results are reported as a percentage (%) of inhibition at a fixed concentration of tested compounds (Table 1) or as an IC50 (Fig. 4).

Steroid Sulfatase and Estrogenicity Assays (in Vivo Studies).

Female BALB/c mice (BALB/cAnNCrlBR) weighing 18–20 grams were obtained from Charles River, Inc. (St-Constant, Québec, Canada) and housed four to five per cage in a temperature (22 ± 3°C) and light (12 h/day, lights on at 7h15) controlled environment. The mice were fed rodent chow and tap water ad libitum. The animals were OVX under isoflurane-anesthesia via bilateral flank incisions and randomly assigned to groups of 8–10 animals. Ten mice were kept intact (INT) as control. To exert an estrogenic effect and stimulate an estrogen-sensitive parameter as uterus, E1S is converted into E1 and then into E2, under the catalytic activities of the enzymes steroid sulfatase and type 1 17β-HSD, respectively. Mice in the intact and OVX control groups received the vehicle alone (8% ethanol-0.4% methylcellulose) during the 9-day period. The possible estrogenic activity (Fig. 5) of tested compounds was evaluated after their administration by subcutaneous (s.c.) injection [100 μg, s.c., once daily (ID)] alone to OVX female mice for 9 days. For the evaluation of the inhibition of steroid sulfatase activity (Figs. 6,7,8), the tested compounds were administered as suspension in 8% ethanol-0.4% methylcellulose to OVX mice for 9 days (1, 10, or 100 μg s.c. or oral (p.o.), ID, from day 2 to 10 of the study). The mice were simultaneously treated with E1S [2 μg, s.c., twice daily (BID)] from day 5 to 10 of the study. The tested compounds were also administered to OVX mice treated simultaneously with E1 (0.06 μg, s.c., BID) from day 5 to 10 of the study (Fig. 6). This was needed to ensure that the effect observed on the uterine weight does not result from an antiestrogenic effect instead of the inhibition of steroid sulfatase. On day 11, the mice were sacrificed by exsanguination followed by cervical dislocation. Uterus from mice were rapidly dissected, weighed, and kept in 10% buffered formalin for further histological examination. Data are expressed as the means ± SE, and statistical significance was determined according to the multiple range test of Duncan-Kramer (41).

In Vitro Study (Steroid Sulfatase Inhibitory Activity).

The inhibition of steroid sulfatase activity by 2-methoxylated compounds 3, 510 and reference compounds 1114 was investigated in homogenate of transfected HEK-293 cells transforming E1S to E1 (Table 1) following a procedure reported previously (37). In the phenol series, the 2-methoxy-E2 (8) does not inhibit steroid sulfatase activity. In the case of other 2-methoxy derivatives 3 and 5, the methoxy group at C2 is responsible for a reduced inhibitory action compared with that of the parent compounds 11 and 12. This is probably caused by the activating effect induced by the ortho-positioning of the methoxy group, resulting in lower acidity of the phenolic group at C3. Thus, at a concentration of 300 nm, compounds 3 and 5 with a tert-butylbenzyl or a benzyl group at C-17α inhibited 86 and 15% of the enzyme activity, respectively, whereas their analogues 11 and 12 without a 2-methoxy group were more potent with 97 and 66% inhibitions. These differences were even more pronounced in the test run with the same compounds at a concentration of 30 nm. In the sulfamate series, all of the sulfamate derivatives inhibited >80% of the enzyme activity at 30 nm. At this concentration, the use of a tert-butylbenzyl (or benzyl) group at C17α provided a stronger inhibition of the enzyme, and the potency of inhibitors 6 and 7 was found higher than that of 2-methoxy-EMATE (9) or 2-methoxy-E2-3-O-sulfamate (10). Thus, our first observation (37, 38) about the complementarity of the inhibitory effects of a sulfamate group at C3 and a suitable hydrophobic group at C17 was confirmed here again with the 2-methoxylated compounds. Moreover, compounds 6, 7, 13, and 14 with two inhibiting groups fully inhibited (99%) enzymatic activity and were more active compared with their analogues without the sulfamate group at C3, namely compounds 3, 5, 11, and 12.

The methoxy derivatives 3, 6, 7, and 9 and the reference inhibitor 13(37) were next tested at various concentrations to determine the IC50s for inhibition of steroid sulfatase activity. The results presented in Fig. 4 illustrate that all of these compounds are potent inhibitors of the enzyme. In our test, the IC50 of 2-methoxy-EMATE (9) was 1.7 nm, whereas compounds 6, 7, and 13 with the two inhibiting groups tert-butylbenzyl (or benzyl) and sulfamate were much more potent (IC50 = 0.04, 0.024, and 0.03 nm). The nonsulfamoylated inhibitor 3 was also a good inhibitor with an IC50 in the range of 30 nm. Although the introduction of the methoxy group at C2 resulted in a decrease of the inhibitory activity, this decrease can however be considered as small. Indeed, the IC50s of the reference inhibitor 13 and its 2-methoxy analogue 6 were 0.03 and 0.04 nm, respectively. In a previous test performed by Purohit et al.(28), the IC50 of 2-methoxy-EMATE (9) in placental microsomes was found to be ≥7-fold beyond that of EMATE. Rather interestingly, with an IC50 of 0.024 nm, compound 7 was apparently the best inhibitor of steroid sulfatase within those tested in our assay. Compound 7 was also tested on type 1 17β-hydroxysteroid dehydrogenase, and no inhibition of E1 into E2 transformation was observed at the two concentrations of 0.1 and 1 μm.4 After a preliminary evaluation of the biodisponibility of the tested compounds, the benzyl derivative 7 was selected for additional in vivo studies focusing on antisulfatase and estrogenic activities.

In Vivo Studies.

Estrogenic, antiestrogenic, and steroid sulfatase inhibitory (antisulfatase) activities of tested compounds were investigated in vivo using the OVX mouse model. As shown in Figs. 5,6,7,8, ovariectomy induces a 70% decrease of uterine weight because of deprivation of estrogens. When administered to OVX mice, E1S is converted in the uterine tissue into E1 by the steroid sulfatase and then into E2 by type 1 17β-hydroxysteroid dehydrogenase. The increase of uterine weight, an estrogen-sensitive tissue, then reflected the formation of active estrogens from E1S.

To discriminate between the estrogenic or nonestrogenic activities of a series of steroid sulfatase inhibitors, compounds 7, 12, 14, EMATE, and ICI 164,384 were injected s.c. to OVX female mice in the absence of treatment with E1S (Fig. 5). No significant response or estrogenic stimuli of the uterus was observed with the pure antiestrogen ICI 164,384 as well as with our selected 2-methoxylated inhibitor, compound 7, at the high dose of 100 μg. In contrast, the parent inhibitors 12, 14, and EMATE showed full estrogenic activity as reported previously in vitro(38, 27). Thus, none of these three later inhibitors can be used as therapeutic agents for the treatment of estrogen-sensitive breast cancer.

In the experiment reported in Fig., 6E1S or E1 were injected s.c. to OVX mice simultaneously to the administration of the selected inhibitor 7 at the daily dose of 100 μg. The uterine weight increase induced by E1S was strongly inhibited (84%) by the nonestrogenic steroid sulfatase inhibitor 7, whereas the uterine weight induced by E1 was not significantly decreased (14%, nonsignificant). These results obtained with E1 and E1S clearly demonstrate that compound 7 does not act as an antiestrogen (by blocking ER) but rather is a potent steroid sulfatase inhibitor efficiently blocking the transformation of E1S into active estrogens.

As shown in Fig. 7, the inhibitors 12, 14, and EMATE (all without a methoxy group) reported previously did not reduce uterine weight when compared with control (OVX + E1S), thus indicating their estrogenic properties. The administration of a nonestrogenic steroid sulfatase inhibitor should block estrogen synthesis from E1S, and, correspondingly, the uterine weight increase induced by E1S will be prevented. Nevertheless, an estrogenic inhibitor of the steroid sulfatase can itself exert an inhibitory action on steroid sulfatase (reducing uterine weight), but, simultaneously, it stimulates the growth of the uterus. In the same experiment, the antiestrogen ICI 164,384 induces a dose-dependent decrease of the uterine weight. Santner and Santen have reported previously the inhibition of estrone sulfatase by the antiestrogen ICI 164,384 in rat mammary tumors (42). However, with a Ki of 11 μm, this was a weak inhibitor. The effect of ICI 164,384 in OVX mice simultaneously treated with E1S (30 and 91% at 10 and 100 μg) is predominantly attributable to its antiestrogenic activity, rather than to antisulfatase activity. In contrast to other tested inhibitors, compound 7, the 2-methoxy derivative of 14, reduced by >84% the uterine growth induced by E1S at both doses of 10 and 100 μg. A similar inhibitory effect (72%) was also observed following the administration of compound 7 at a lower dose of 1 μg, s.c. (Fig. 8). This later compound was also tested at a 100-μg dose administered p.o., and the inhibition achieved was the same as obtained with a 10-μg dose injected s.c. (83 and 80%, respectively). In summary, the in vivo experiments with the uterine weight model clearly prove that the synthesized compound 7 has no antiestrogenic activity and is a nonestrogenic potent inhibitor of steroid sulfatase activity, both following parenteral and p.o. administration.

The results obtained from our in vivo study are in agreement with previous reports indicating that EMATE is an estrogenic compound that could not be used in the treatment of estrogen-sensitive cancers (27, 28). Our results also demonstrate that the reversible inhibitor 17α-benzyl-E2 (12) and the irreversible inhibitor 3-O-sulfamate-17α-benzyl-E2 (14) are both estrogenic in the OVX mouse model (uterine weight). At the opposite and more interestingly, the 3-O-sulfamate-2-methoxy-17α-benzyl-E2 (7), which is the 2-methoxylated analogue of 14, was found to be a potent steroid sulfatase inhibitor, nonestrogenic, and without antiestrogenic activity (no effect mediated by ER). This compound reverses very efficiently the uterine weight stimulation normally induced by E1S after its transformation into active estrogens by blocking the steroid sulfatase. Compound 7 is also a potent in vitro inhibitor of steroid sulfatase with an IC50 of 0.024 nm for the transformation of E1S to E1 in homogenate of transfected HEK-293 cells. Because of the combined effects of the sulfamate and benzyl (or tert-butylbenzyl) groups introduced at C3 and C17α (37), the potency of 6 and 7 are comparable with that of the parent compound without the 2-methoxy group. Because it was shown previously that estrogen metabolite 2-methoxy-E2 (8) has low binding affinity for the estrogen receptor, exerts cytotoxic action in cancer cell cultures, and inhibits tumor angiogenesis (29, 30, 31, 32, 33, 34, 35, 43), we thus expect that the targeted 2-methoxylated inhibitors reported above might conserve some of these properties in addition to their very potent inhibition of steroid sulfatase. Additional studies will however be necessary to confirm this point as well as to focus on the use of these inhibitors in the therapy of estrogen-sensitive cancers.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported by the Medical Research Council of Canada (now the Canadian Institutes of Health Research) and Endorecherche. D. P. is a recipient of the Fonds de la Recherche en Santé du Québec fellowship.

3

The abbreviations used are: E2, estradiol; HSD, hydroxysteroid dehydrogenase; NMR, nuclear magnetic resonance; 5-diol, androst-5-ene-3β,17β-diol; ER, estrogen receptor; OVX, ovariectomized; HPLC, high-performance liquid chromatography; DHEAS, dehydroepiandrosterone sulfate; CMV, cytomegalovirus; HEK, human embryonic kidney.

4

V. Luu-The, unpublished data.

Fig. 1.

The use of 2-methoxyestradiol nucleus to develop nonestrogenic steroid sulfatase inhibitors [R1 = H or SO2NH2 and R2 = H or C(CH3)3].

Fig. 1.

The use of 2-methoxyestradiol nucleus to develop nonestrogenic steroid sulfatase inhibitors [R1 = H or SO2NH2 and R2 = H or C(CH3)3].

Close modal
Fig. 2.

Chemical synthesis of 2-methoxyestradiol (or estrone) derivatives 310 and chemical structures of reference compounds 1114. The reagents are: (a) Mg, p-tert-butyl-benzyl-Br, diethyl ether, 0°C, 12 h; (b) benzyl-MgCl, THF, 0°C, 12 h; (c) Pd(OH)2/C (20%), rt, 12 h; (d) 2,6-DBMP, H2NSO2Cl, CH2Cl2, rt, 4 h; and (e) NaBH4, methanol, 0°C, 1 h.

Fig. 2.

Chemical synthesis of 2-methoxyestradiol (or estrone) derivatives 310 and chemical structures of reference compounds 1114. The reagents are: (a) Mg, p-tert-butyl-benzyl-Br, diethyl ether, 0°C, 12 h; (b) benzyl-MgCl, THF, 0°C, 12 h; (c) Pd(OH)2/C (20%), rt, 12 h; (d) 2,6-DBMP, H2NSO2Cl, CH2Cl2, rt, 4 h; and (e) NaBH4, methanol, 0°C, 1 h.

Close modal
Fig. 3.

HPLC profile of key compound 7. The experiment was performed with a C18 Nova Pak reversed phase column (150 × 3.9 mm), a mixture of H2O:methanol/30:70 as eluent, and a UV detector (205 nm).

Fig. 3.

HPLC profile of key compound 7. The experiment was performed with a C18 Nova Pak reversed phase column (150 × 3.9 mm), a mixture of H2O:methanol/30:70 as eluent, and a UV detector (205 nm).

Close modal
Fig. 4.

Effects of increasing concentration of compounds 3, 6, 7, 9, 13, and E1S on enzymatic activity of steroid sulfatase transfected in HEK-293 cells (see “Materials and Methods” for details). The results are expressed as a percentage of transformation of [3H]E1S into [3H]E1. Data points represent the mean values ± SE from triplicate incubations.

Fig. 4.

Effects of increasing concentration of compounds 3, 6, 7, 9, 13, and E1S on enzymatic activity of steroid sulfatase transfected in HEK-293 cells (see “Materials and Methods” for details). The results are expressed as a percentage of transformation of [3H]E1S into [3H]E1. Data points represent the mean values ± SE from triplicate incubations.

Close modal
Fig. 5.

Effect of estrone sulfate (E1S), steroid sulfatase inhibitors (7, 12, 14, and EMATE), and a pure antiestrogen (ICI 164,384) on the uterine weight of OVX mice (∗∗P < 0.01, experimental versus OVX control animals). INT, intact mice. CTL, control group.

Fig. 5.

Effect of estrone sulfate (E1S), steroid sulfatase inhibitors (7, 12, 14, and EMATE), and a pure antiestrogen (ICI 164,384) on the uterine weight of OVX mice (∗∗P < 0.01, experimental versus OVX control animals). INT, intact mice. CTL, control group.

Close modal
Fig. 6.

Effect of steroid sulfatase inhibitor 7 on uterine weight of OVX mice from groups treated with estrone sulfate (E1S) and estrone, respectively. (∗∗P < 0.01, experimental versus OVX + E1S control animals). INT, intact mice; CTL, control group.

Fig. 6.

Effect of steroid sulfatase inhibitor 7 on uterine weight of OVX mice from groups treated with estrone sulfate (E1S) and estrone, respectively. (∗∗P < 0.01, experimental versus OVX + E1S control animals). INT, intact mice; CTL, control group.

Close modal
Fig. 7.

Effect of steroid sulfatase inhibitors (7, 12, 14, and EMATE) and a pure antiestrogen (ICI 164,384) on the uterine weight of OVX mice after a simultaneous administration of estrone sulfate (E1S). Results allow to compare the antisulfatase potency of the tested inhibitors at two different doses (∗∗P < 0.01, experimental versus OVX + E1S control animals). INT, intact mice; CTL, control group. Data in brackets represent the reduction (%) of induced uterine weight (CTL + E1S + OVX).

Fig. 7.

Effect of steroid sulfatase inhibitors (7, 12, 14, and EMATE) and a pure antiestrogen (ICI 164,384) on the uterine weight of OVX mice after a simultaneous administration of estrone sulfate (E1S). Results allow to compare the antisulfatase potency of the tested inhibitors at two different doses (∗∗P < 0.01, experimental versus OVX + E1S control animals). INT, intact mice; CTL, control group. Data in brackets represent the reduction (%) of induced uterine weight (CTL + E1S + OVX).

Close modal
Fig. 8.

Effect of steroid sulfatase inhibitor 7, injected s.c. or administered p.o., on the uterine weight of OVX mice receiving simultaneously estrone sulfate (E1S). ∗∗P < 0.01, experimental versus OVX + E1S control animals. INT, intact mice; CTL, control group. Data in brackets represent the reduction (%) of induced uterine weight (CTL + E1S + OVX).

Fig. 8.

Effect of steroid sulfatase inhibitor 7, injected s.c. or administered p.o., on the uterine weight of OVX mice receiving simultaneously estrone sulfate (E1S). ∗∗P < 0.01, experimental versus OVX + E1S control animals. INT, intact mice; CTL, control group. Data in brackets represent the reduction (%) of induced uterine weight (CTL + E1S + OVX).

Close modal
Table 1

In vitro inhibition of steroid sulfatase activity (homogenates of transfected HEK-293 cells) by 2-methoxy derivatives and reference inhibitorsa

CompoundbC3-groupC2-groupC17α-groupInhibition (%) at 30 nmcInhibition (%) at 300 nmcInhibition (%) at 3000 nmcIC50 (nm)
3 Phenol Methoxy t-Butyl-benzyl 52 86 95 28 ± 9 
5 Phenol Methoxy Benzyl 15 63  
6 Sulfamate Methoxy t-Butyl-benzyl 99 99 99 0.040 ± 0.002 
7 Sulfamate Methoxy Benzyl 99 99 99 0.024 ± 0.008 
8 Phenol Methoxy  
9 Sulfamate Methoxy Ketone 89 98 99 1.70 ± 0.16 
10 Sulfamate Methoxy 80 97 99  
11 Phenol t-Butyl-benzyl 91 97 97  
12 Phenol Benzyl 19 66 92  
13 Sulfamate t-Butyl-benzyl 99 99 99 0.030 ± 0.003 
14 Sulfamate Benzyl 99 99 99  
CompoundbC3-groupC2-groupC17α-groupInhibition (%) at 30 nmcInhibition (%) at 300 nmcInhibition (%) at 3000 nmcIC50 (nm)
3 Phenol Methoxy t-Butyl-benzyl 52 86 95 28 ± 9 
5 Phenol Methoxy Benzyl 15 63  
6 Sulfamate Methoxy t-Butyl-benzyl 99 99 99 0.040 ± 0.002 
7 Sulfamate Methoxy Benzyl 99 99 99 0.024 ± 0.008 
8 Phenol Methoxy  
9 Sulfamate Methoxy Ketone 89 98 99 1.70 ± 0.16 
10 Sulfamate Methoxy 80 97 99  
11 Phenol t-Butyl-benzyl 91 97 97  
12 Phenol Benzyl 19 66 92  
13 Sulfamate t-Butyl-benzyl 99 99 99 0.030 ± 0.003 
14 Sulfamate Benzyl 99 99 99  
a

Inhibition of the transformation of [3H]E1S (100 μm) to [3H]E1.

b

See Fig. 2 for the chemical structures of tested compounds.

c

Error estimated at ± 5%.

We thank Richard Labrecque and Yvon Fréchette for useful discussions about some aspects of the chemical synthesis. We also thank Mei Wang and Guy Reimnitz for their contribution in performing the enzymatic assays.

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