Potent inhibition of steroid sulfatase activity by 3-O-sulfamate 17α- benzyl(or 4'-tert-butylbenzyl)estra-1,3,5(10)-trienes: Combination of two substituents at positions C3 and C17α of estradiol

Article abstract of DOI:10.1021/jm980677l

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 17α-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 17α-benzylestradiol (4) and 3-O- sulfamate 17α-(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 C17α-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.

Full text of DOI:10.1021/jm980677l

2280
J . Med. Chem. 1999, 42, 2280-2286
P oten t In h ibition of Ster oid Su lfa ta se Activity by 3-O-Su lfa m a te 17r-Ben zyl(or
4′-ter t-bu tylben zyl)estr a -1,3,5(10)-tr ien es: Com bin a tion of Tw o Su bstitu en ts a t
P osition s C3 a n d C17r of Estr a d iol
Liviu C. Ciobanu,† Roch P. Boivin,† Van Luu-The,‡ Fernand Labrie,†,‡ and Donald Poirier*,†
Medicinal Chemistry Division of LREM and MRC Group in Molecular Endocrinology, Laval University Medical Research
Center, Que´bec G1V 4G2, Canada
Received December 4, 1998
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 17R-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 17R-benzylestradiol (4) and 3-O-sulfamate 17R-(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 C17R-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.
In tr od u ction
in breast tumor tissues in comparison to normal tis-
sues.3 Since androgens and estrogens may be synthe-
sized inside cancer cells starting from DHEAS and E1S
available in blood circulation,1 the use of therapeutic
agents that inhibit steroid sulfatase activity may well
be a valuable approach to the treatment of hormone-
dependent diseases.
The enzyme steroid sulfatase is known to catalyze the
conversion of dehydroepiandrosterone sulfate (DHEAS)
and estrone sulfate (E1S) to the corresponding uncon-
jugated steroids DHEA and E1 and may be one of the
key enzymes involved in the regulation of the level of
active steroids in normal and malignant tissues (Figure
1). Indeed, DHEAS is the main sulfated steroid found
in human blood circulation and may thus be a potential
source of androgens and estrogens in peripheral tis-
sues.1 Upon hydrolysis by steroid sulfatase, DHEAS is
transformed to DHEA, which is further transformed to
4-androstenedione (∆4-dione), testosterone (T), dihy-
drotestosterone (DHT), 5-androstene-3â,17â-diol (∆5-
diol), and estradiol (E2) by steroidogenic enzymes:
namely, 3â-hydroxysteroid dehydrogenases, 17â-hy-
droxysteroid dehydrogenases, 5R-reductases, and aro-
matase. On the other hand, E1S is also the most
abundant C18-steroid in the circulation and could be
transformed to the estrogens E1 and E2.2 These andro-
gens and estrogens are produced locally in target tissues
and are probably responsible for the growth of androgen-
and estrogen-dependent tissues and associated diseases
such as prostate and breast cancers. Interestingly,
higher activity of steroid sulfatase has been detected
Many steroid sulfatase inhibitors have been developed
during the past few years.4 Most of them were obtained
from a C3-modification of E1S, the natural C18-steroid
substrate of the enzyme.4p In this series of inhibitors,
the 3-O-sulfamate estrone (1, EMATE) is well-known
and is one of the most potent inhibitors that has been
reported to date (Figure 2).4p,6 Recently, our group
identified for the first time the steroid sulfatase inhibi-
tory effect of an alkyl or substituted benzyl group
introduced at position 17R of 17â-estradiol.5 For this
new family of inhibitors, represented by 17R-benzylestra-
diol (2) and 17R-(tert-butylbenzyl)estradiol (3), we hy-
pothesized that they may inhibit steroid sulfatase
activity by a reversible interaction (probably hydropho-
bic) with a region within the enzymatic site. On the
contrary, EMATE (1) and its more potent 4-NO2 ana-
logue, as well as other steroidal or nonsteroidal sulfa-
mates, are active-site-directed irreversible inhibitors of
estrone sulfatase.4,6 Since 17R-benzyl(or tert-butylben-
zyl)estradiols (2 or 3) and 3-O-sulfamate estrone (1)
represent two families of inhibitors with substituents
that act at opposite positions on the steroid nucleus, and
most likely through different mechanisms, we designed
* Corresponding author: Dr. Donald Poirier, Laboratory of Molec-
ular Endocrinology, Laval University Medical Research Center, 2705
Laurier Blvd, Que´bec G1V 4G2, Canada. Tel: (418) 654-2296. Fax:
(418) 654-2761.
† Medicinal Chemistry Division of LREM.
‡ MRC Group in Molecular Endocrinology.
10.1021/jm980677l CCC: $18.00 © 1999 American Chemical Society
Published on Web 05/29/1999

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2281
F igu r e 1. Steroid sulfatase transformation of sulfated steroids DHEAS and E1S to hormones interacting with androgen (AR)
and estrogen (ER) receptors. Other enzymes: 3â-dehydrogenase ∆5,∆4-isomerases (I), 17â-hydroxysteroid dehydrogenases (II),
5R-reductases (III), aromatase (IV).
compounds that contain both substituents on the same
molecule: namely, 3-O-sulfamate 17R-benzylestradiol
(4) and 3-O-sulfamate 17R-(tert-butylbenzyl)estradiol
(5). Herein, we report the chemical synthesis and the
inhibitory activity of the first two members (compounds
4 and 5) of this new series of steroid sulfatase inhibitors.
We also report the type of inhibition for the most potent
inhibitor, compound 5, and its unsulfamoylated ana-
logue, compound 3.
synthesized by a simple chemical pathway (Scheme 1).
The intermediates 2 and 3 were first obtained by a
stereoselective addition of Grignard reagents to the C17-
carbonyl of E1. The 18-methyl on the â-face of steroids
directs the nucleophile attack on the less hindered
R-face, giving the 17R-alkylated compound. An excess
of commercially available benzylmagnesium bromide
Ch em istr y
Compounds 4 and 5, each with two different kinds of
substituents that do not act in the same way, were
F igu r e 2. Two families of known inhibitors (1-3) and newly proposed inhibitors (4 and 5) of steroid sulfatase.

2282 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Brief Articles
Sch em e 1a
a
Reagents: (a) BnMgBr, THF, 0 °C-rt, overnight; (b) tert-butylbenzyl bromide, Mg, diethyl ether, rt, overnight; (c) H2NO2S-Cl, DBMP,
CH2Cl2, rt, 1.5 h.
(2.0 M in THF) or in situ generated (tert-butylbenzyl)-
magnesium bromide (tert-butylbenzyl bromide, magne-
sium turnings, diethyl ether, 0 °C) was used for the
Grignard reaction (0 °C to room temperature, over-
night). In addition to alkylated compound 2 or 3,
unreacted E1 was obtained because of the low reactivity
of such hindered 17-ketosteroids7 and the low solubility
of the phenolate species generated. To facilitate the
chromatographic separation of the product (2 or 3) from
the starting material (E1), a quantitative carbonyl
reduction of E1 to E2 was carried out with NaBH4
(MeOH, 1-2 h, 0 °C to room temperature). After silica
gel chromatography, compounds 2 and 3 were obtained
in yields of 64% and 77%, respectively, in addition to
E1S to E1 (A) and DHEAS to DHEA (B) by the steroid sulfatase
F igu r e 3. Lineweaver-Burk plots for the transformation of
30% and 17% of E2. Although higher yields were
obtained for the Grignard reaction when the phenolic
group was protected as a tert-butyldimethylsilyl ether,
this protection/deprotection approach added two more
steps and was found to be too time-consuming for the
improvement of overall yield.
activity of homogenized transfected (293) cells.
2-5 and reference compound 1 (EMATE) were then
tested for their ability to inhibit steroid sulfatase
activity transforming E1S to E1. As reported in Table 1
and in agreement with the literature, EMATE strongly
inhibited the steroid sulfatase activity (IC50 ) 2.1 nM).
On the other hand, compounds 2 and 3, which contain
only a 17R-benzyl or a 17R-tert-butylbenzyl group, also
strongly inhibited the enzyme activity (IC50 ) 230 and
8.3 nM, respectively). Interestingly, compounds 4 and
5 (IC50 ) 0.39 and 0.15 nM, respectively), which bear
both inhibiting groups (sulfamate and benzyl/tert-bu-
tylbenzyl) on the same molecule, were better inhibitors
than the preceding compounds 1-3 that contain a single
inhibitory group. In accordance with the results ob-
tained for compounds 2 and 3, the tert-butylbenzyl
analogue 5 was a better inhibitor than the benzyl
analogue 4. When compared to EMATE (1), compounds
4 and 5 were found to be 5- and 14-fold more potent for
the transformation of E1S to E1. Compounds 1-5 were
also tested for their ability to inhibit the transformation
of C19-steroid substrate DHEAS to DHEA by steroid
sulfatase (Table 1). The results were similar to those
obtained above with E1S as enzyme substrate. Indeed,
compounds 4 and 5 (IC50 ) 4.1 and 1.4 nM) bearing two
inhibiting groups were more potent inhibitors than
compounds 2 and 3 (IC50 ) 325 and 14 nM) that have
only a C17R-substituent and EMATE (IC50 ) 5.6 nM)
that has only a C3-sulfamate group. Thus, compounds
4 and 5 were respectively 1.4- and 4-fold more potent
than EMATE for the transformation of DHEAS to
DHEA. Interestingly, the data in Table 1 suggest an
additive-like inhibitory effect of the two kinds of sub-
stituents (17R-benzyl or substituted benzyl and 3-O-
sulfamate) added on the estradiol nucleus.
The next step consisted of regioselectively introducing
the sulfamoyl group at position C3. The sulfamoyl
chloride needed for the sulfamoylation was first syn-
thesized from chlorosulfonyl isocyanate following a
modified procedure described by Peterson et al.8 There-
after, sulfamoylated derivatives 4 and 5 were obtained
in yields of 59% and 79%, respectively, by adding
sulfamoyl chloride (6 equiv) to a solution of 3-hydrox-
ysteroids 2 and 3 in dichloromethane and 2,6-di-tert-
butyl-4-methylpyridine (DBMP).9 Compounds 4 and 5
were isolated using an aqueous workup and purified by
silica gel chromatography with hexane/acetone (75:25)
as eluent. Compound 4 was also obtained in a 70% yield
by the addition of 5 equiv of sulfamoyl chloride to a
solution of 3-hydroxysteroid 2 and THF in the presence
of sodium hydride at 0 °C.10 The inhibitors with mono
(2 and 3) and bis (4 and 5) substituents were fully
characterized by IR, 1H NMR, 13C NMR,11 MS, and
elemental analysis.
Resu lts a n d Discu ssion
We used a homogenate of human embryonal kidney
(293) cells transiently transfected with a sulfatase
expression vector as our source of steroid sulfatase
activity. As illustrated in Figure 3, the enzyme catalyzed
efficiently the transformation of E1S and DHEAS to E1
and DHEA, respectively, but E1 was the preferred
substrate. Lineweaver-Burk plot analysis indicates
that the Vmax values for E1S and DHEAS were 6.25 and
1.5 nmol/mg of protein/min, respectively, while the Km
values were 15 and 19 µM, respectively. Compounds
Because a washing assay is easier using intact trans-
fected cells in culture, this method could be a convenient

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2283
Ta ble 1. Inhibition of Steroid Sulfatase Activity by EMATE (1) and Newly Synthesized Compounds (2-5)
IC50 (nM)
a
compd
substituents
E1S f E1
DHEAS f DHEAa
1 (EMATE)
3-O-sulfamate
17R-benzyl
2.1 ( 0.2
230 ( 56
8.3 ( 1.6
5.6 ( 0.3
2
3
4
5
325 ( 61
17R-tert-butylbenzyl
14 ( 1
3-O-sulfamate and 17R-benzyl
3-O-sulfamate and 17R-tert-butylbenzyl
0.39 ( 0.02 [5]b
0.15 ( 0.01 [14]b
4.1 ( 0.3 [1.4]b
1.4 ( 0.1 [4]b
a
b
Substrate concentration: [3H]E1S (100 µM) or [14C]DHEAS (100 µM). The numbers in brackets are the potency of 4 and 5 versus
EMATE (1).
F igu r e 5. Time-dependent inactivation of steroid sulfatase
activity ([3H]E1S to [3H]E1) by inhibitors 1 (EMATE), 3, and
5, in the absence or presence of a large excess of enzyme
substrate E1S (100 µM). The enzyme activity of the control
(100%) was 0.49 nmol/h/mg of protein. The assay was per-
formed as described in the Experimental Section.
F igu r e 4. Effect of the new inhibitor 5 and the known
inhibitor 1 (EMATE) on steroid sulfatase activity. Inhibitor
(5 µM) or control was incubated for 1 h at 37 °C with intact
transfected (293) cells (500 000 cells/well) and thereafter
washed three times (or not) with DMEM culture medium. The
enzymatic activity was then evaluated by incubating [3H]E1S
(650 000 dpm/well) for 18 h and measuring the [3H]E1 formed.
The enzyme activity of the control (100%) was 0.33 nmol/
1 000 000 cells/h.
C3-sulfamate group), these compounds, which are rep-
resented by 4 and 5, are 5- and 14-fold more potent
inhibitors for the transformation of E1S to E1 and 1.4-
and 4-fold more potent inhibitors for the transformation
of DHEAS to DHEA. Compound 5 also inactivated
steroid sulfatase activity in intact (293) transfected cells
or in microsomal preparation. We propose that inhibi-
tors such as 4 and 5 act by two different mechanisms,
taking into account the nature of the substituents: 3-O-
sulfamate and 17R-substituted benzyl. After the inhibi-
tion of steroid sulfatase by the sulfamate group of 4 and
5, the released phenolic compounds 2 and 3 were still
able to inhibit a neighboring free enzyme molecule. In
the situation when the inhibitor concentration is satu-
rating, it is conceivable that the majority of the enzyme
molecules will be inactivated by the sulfamate group,
since the molecule bearing the sulfamate group (com-
pound 5) possesses higher affinity than phenolic com-
pound 3 (without sulfamate), and thus the liberated
phenolic compound 3 does not have a significant inhibi-
tory effect. In such a case, the 17R-substituent of
compound 5 facilitates probably the rate of binding,
leading to a more potent inactivation of the enzyme.
However, in the situation when the inhibitor concentra-
tion is not yet saturating, it is likely that one molecule
of 5 could act on two molecules of enzymes, because the
hydrolysis of the inactivating sulfamate group generates
the compound 3 that exerts its inhibitory effect on the
neighboring free enzyme and thus shows an overal
better efficacy. An apparent additive inhibitory effect
could thus be observed because one molecule of com-
pound 5 could act on two molecules of the enzyme in
two different manners: one enzyme molecule is inacti-
vated by covalently binding to the sulfamoyl group and
way to assess the reversibility/irreversibility of an
inhibitor in the case that the inhibitor could be washed
out efficiently. As illustrated in Figure 4, compound 5
and EMATE exhibit potent inhibitory effect on steroid
sulfatase activity in the experiment using intact cells,
and washing does not reverse significantly the inhibi-
tory effect of compound 5 and EMATE. In this assay,
however, compound 5 seems to have slightly less potent
inhibitory effect than EMATE. This could be due to
differential solubility of the compounds or to washing
inefficiency. To further analyze the inactivation capabil-
ity of compound 5 and its unsulfamoylated analogue,
compound 3, we performed a time-dependent inactiva-
tion experiment using microsomes of (293) cells trans-
fected with sulfatase expression vector. As illustrated
in Figure 5, compound 3, which does not possess a
sulfamoylated group, does not inactivate the sulfatase
activity, whereas preincubation with compound 5, which
possesses a sulfamoylated group, inactivates microsomal
sulfatase activity at a concentration 100-fold lower than
EMATE. Interestingly, the inactivation of both EMATE
and compound 5 is reversed by an excess of the
substrate E1S. The result strongly suggests that in the
experiment using intact cells (Figure 4) the apparent
stronger inhibitory effect of EMATE is probably due to
the inefficiency of washing.
In summary, we have developed a new family of
potent steroid sulfatase inhibitors by combining the
inhibiting actions of two kinds of substituents, a 17R-
benzyl (or tert-butylbenzyl) and a 3-O-sulfamate, on the
estradiol nucleus. When compared to EMATE (only a

2284 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
Brief Articles
activated by heating. The system was kept at room tempera-
ture, a solution of tert-butylbenzyl bromide (2.7 mL, 14.8 mmol)
in dry diethyl ether (15 mL) was added dropwise (about 15
min), and the reaction mixture was allowed to stir for 3 h.
The in situ generated Grignard reagent was then added slowly
to a solution of estrone (400 mg, 1.5 mmol) in dry THF (40
mL) at room temperature, and the reaction was allowed to stir
overnight. The reaction mixture was poured into a saturated
solution of NH4Cl, extracted with EtOAc, dried over MgSO4,
and evaporated under reduced pressure. The crude mixture
of 3 and remaining estrone was dissolved in MeOH (50 mL)
and treated at 0 °C with NaBH4 in excess (112 mg) for 30 min.
After the usual workup, purification by chromatography
(hexane/EtOAc, 9:1) afforded estradiol (68 mg, 17%) and
alkylated compound 3 (475 mg, 77% yield): white solid; IR υ
(film) 3395 (OH); 1H NMR δ (CDCl3) 0.97 (s, 3H, 18-CH3), 1.33
(s, 9H, tert-butyl), 2.65 and 2.90 (2d of AB system, J ) 13.2
Hz, 2H, CH2Ph-t-Bu), 2.84 (m, 2H, 6-CH2), 4.68 (br, 1H, OH
phenol), 6.58 (d, J ) 2.4 Hz, 1H, 4-CH), 6.63 (dd, J 1 ) 2.7 Hz,
and J 2 ) 8.3 Hz, 1H, 2-CH), 7.17 (d, J ) 8.6 Hz, 1H, 1-CH),
7.22 (d, J ) 8.2 Hz, 2H, 2′ and 6′-CH), 7.35 (d, J ) 8.2 Hz, 2H,
3′ and 5′-CH); 13C NMR δ (acetone-d6) 15.25 (C18), 24.03 (C15),
27.45 (C11), 28.52 (C7), 30.51 (C6; under solvent peaks), 31.87
(C(CH3)3), 32.21 (C12), 33.61 (C16), 34.92 (C(CH3)3), 41.11 (C8),
42.92 (C1′), 44.88 (C9), 48.02 (C13), 50.38 (C14), 83.76 (C17),
113.69 (C2), 116.08 (C4), 125.29 (C3′′ and C5′′), 127.13 (C1),
131.88 (C2′′ and C6′′), 132.25 (C10), 137.35 (C1′′), 138.58 (C5),
149.07 (C4′′), 156.05 (C3); EI-LRMS m/e 418 (M+, 1.8), 400 (M+
- H2O, 18), 385 [M+ - (H2O + CH3), 10], 270 [M+ - (CH2Ph-
t-Bu), 100], 253 (56), 159 (54), 147 (82), 133 (83), 57 (75). Anal.
Calcd for C29H38O2: C, 83.21; H, 9.15. Found: C, 82.85; H,
9.53.
one is inhibited by the liberated phenolic compound 3.
This situation is unique for inhibitors such as sulfa-
mates 4 and 5, in which the active group bind covalently
to the enzymes generating a free compound (2 or 3) that
could possesses additional reversible inhibitory effect.
Since the C17R-substituent is not limited to a benzyl
or a tert-butylbenzyl group,5 the synthesis of other
analogues of 4 and 5 can be considered to optimize this
new family of promising inhibitors. Furthermore, it is
probable that the concept illustrated here of using two
kinds of inhibiting substituents on the same molecule
can be applied to other steroid sulfatase inhibitors
previously reported in the literature.
Exp er im en ta l Section
A. Ch em ica l Syn th esis. Analytical thin-layer chromatog-
raphy (TLC) was performed on Merck 60 F254 silica gel plates
(0.20 mm), and compounds were visualized using UV light or
ammonium molybdate/sulfuric acid/water (with heating). Flash
column chromatography was performed with 230-400 mesh
ASTM silica gel 60 (E. Merck). Infrared spectra (IR) are
expressed in cm-1 and were obtained on a Perkin-Elmer 1600
(series FTIR) spectrophotometer. Nuclear magnetic resonance
spectra (NMR) were recorded with a Bruker AC/F 300 spec-
trometer at 300 MHz (1H) or 75 MHz (13C), and the chemical
shifts (δ) are expressed in ppm. Assignment of 13C NMR
signals was made easier by distortionless enhancement by
polarization transfer (DEPT) experiments, heteronuclear shift
correlation (HSC) experiments, and data from the literature.11
Low-resolution mass spectra (LRMS) were obtained from
electron impact (EI) and recorded with a V.G. Micromass 16F
spectrometer. High-resolution mass spectra (HRMS) obtained
from fast-atom bombardment (FAB) with a NBA matrix were
provided by Le Centre Re´gional de Spectrome´trie de Masse
(Universite´ de Montre´al, Montre´al, Canada). Elemental analy-
ses (CHNS) were carried out by Le Laboratoire d′Analyse
EÄ le´mentaire de l′Universite´ de Montre´al (Montre´al, Canada).
Syn th esis of 3,17â-Dih yd r oxy-17r-ben zylestr a -1,3,5-
(10)-tr ien e (2). Commercially available estrone (500 mg, 1.85
mmol) in dry THF (50 mL) was stirred under argon atmo-
sphere and treated at 0 °C with benzylmagnesium bromide
(2.0 M in THF) (5.55 mL, 11.10 mmol). The reaction mixture
was allowed to return to room temperature overnight. Then,
a saturated aqueous solution of NH4Cl was added and the
solution extracted with EtOAc. The combined organic layer
was washed with brine, dried over MgSO4, and filtered, and
solvent was evaporated to dryness. Thereafter, the crude
mixture of 2 and remaining estrone was dissolved in MeOH
(50 mL), and NaBH4 (140 mg, 3.70 mmol) was added at 0 °C.
After complete reduction of estrone to estradiol (1-2 h), the
reaction was quenched with H2O, MeOH was evaporated under
vacuum, and the mixture was extracted with EtOAc and
treated as above. Purification by chromatography (hexane/
EtOAc, 8:2) afforded estradiol (152 mg, 30%) and alkylated
compound 2 (428 mg, 64% yield): white solid; IR υ (film) 3415
Syn th esis of 3-O-Su lfa m a te 17â-Hyd r oxy-17r-ben zyl-
estr a -1,3,5(10)-tr ien e (4). To a stirred solution of 17R-
benzylestradiol (2) (95 mg, 0.26 mmol) in CH2Cl2 (30 mL) was
added 2,6-di-tert-butyl-4-methylpyridine (DBMP) (160 mg, 0.78
mmol), and the mixture was stirred under argon atmosphere
for 15 min at room temperature. Sulfamoyl chloride (182 mg,
1.56 mmol) was then added in portions. After 1.5 h, the
solution was washed with water and dried over MgSO4. The
solvent was evaporated under vacuum, and the crude mixture
was purified by chromatography (hexane/acetone, 75:25) to
give unreacted phenol 3 (13 mg, 14%) and sulfamoylated
compound 4 (92 mg, 79%): white solid; IR υ (KBr) 3508, 3283
and 3195 (OH and NH2), 1371 and 1178 (SdO); 1H NMR δ
(DMSO-d6) 0.83 (s, 3H, 18-CH3), 1.20-1.75 (m, 10H), 1.84 (m,
1H), 2.23 (m, 1H), 2.36 (m, 1H), 2.59 and 2.77 (2d of AB system,
J ) 13.4 Hz, 2H, CH2Ph), 2.82 (m, 2H, 6-CH2), 4.14 (s, OH),
6.96 (d, J ) 2.3 Hz, 1H, 4-CH), 7.01 (dd, J 1 ) 2.4 Hz and J 2
)
8.4 Hz, 1H, 2-CH), 7.15-7.30 (m, 5H, CH2Ph), 7.35 (d, J )
8.6 Hz, 1H, 1-CH), 7.89 (s, 2H, NH2); 13C NMR δ (acetone-d6)
15.15 (C18), 23.97 (C15), 27.23 (C11), 28.14 (C7), 30.31 (C6;
under solvent peaks), 32.13 (C12), 33.52 (C16), 40.61 (C8),
43.44 (C1′), 45.00 (C9), 47.98 (C13), 50.35 (C14), 83.72 (C17),
120.24 (C2), 123.07 (C4), 126.61 (C4′′), 127.46 (C1), 128.45 (C3′′
and C5′′), 132.18 (C2′′ and C6′′), 139.37 (C10), 139.90 (C5),
140.52 (C1′′), 149.45 (C3); FAB-HRMS calcd for C25H30O4NS
(M+ - H) 440.18954, found 440.19120. Anal. Calcd for C25H31O4-
NS: C, 68.00; H, 7.08; N, 3.17; S, 7.26. Found: C, 68.43; H,
7.02; N, 3.23; S, 7.81.
1
(OH); H NMR δ (CDCl3) 0.97 (s, 3H, 18-CH3), 2.68 and 2.94
(2d of AB system, J ) 13.3 Hz, 2H, CH2Ph), 2.83 (m, 2H,
6-CH2), 4.52 (br, 1H, OH phenol), 6.58 (d, J ) 2.3 Hz, 1H,
4-CH), 6.63 (dd, J 1 ) 2.5 Hz and J 2 ) 8.4 Hz, 1H, 2-CH), 7.18
(d, J ) 8.3 Hz, 1H, 1-CH), 7.25 to 7.35 (m, 5H, CH2Ph); 13C
NMR δ (acetone-d6) 15.12 (C18), 23.87 (C15), 27.29 (C11), 28.37
(C7), ∼30 (C6; under solvent peaks), 32.04 (C12), 33.41 (C16),
40.92 (C8), 43.34 (C1′), 44.70 (C9), 47.90 (C13), 50.22 (C14),
83.66 (C17), 113.57 (C2), 115.88 (C4), 126.45 (C4′′), 126.99 (C1),
128.29 (C3′′ and C5′′), 132.05 (C10, C2′′, and C6′′), 138.43 (C5),
140.38 (C1′′), 155.88 (C3); EI-LRMS m/e 362 (M+, 18), 344 (M+
- H2O, 8.4), 271 (M+ - CH2Ph, 100), 253 (64), 228 (15), 213
(38), 159 (61), 133 (61), 91 (63). Anal. Calcd for C25H30O2: C,
82.83; H, 8.34. Found: C, 82.40; H, 8.50.
Syn th esis of 3-O-Su lfa m a te 17â-Hyd r oxy-17r-(ter t-bu -
tylben zyl)estr a -1,3,5(10)-tr ien e (5). As described above for
the synthesis of 4, 17R-(tert-butylbenzyl)estradiol (3) (37 mg,
0.088 mmol) was treated in CH2Cl2 (30 mL) with DBMP (54
mg, 0.26 mmol) and sulfamoyl chloride (62 mg, 0.53 mmol).
After workup, the crude mixture was purified by chromatog-
raphy (hexane/acetone, 75:25) to afford unreacted phenol 3 (6
mg, 16%) and sulfamoylated compound 5 (26 mg, 59%): white
solid; IR υ (KBr) 3538, 3367 and 3196 (OH and NH2), 1371
1
and 1186 (SdO); H NMR δ (DMSO-d6) 0.83 (s, 3H, 18-CH3),
1.26 (s, 9H, tert-Butyl), 1.20-1.75 (m, 11H), 1.85 (m, 1H), 2.23
(m, 1H), 2.36 (m, 1H), 2.54 and 2.73 (2d of AB system, J )
13.3 Hz, 2H, CH2Ph-t-Bu), 2.82 (m, 2H, 6-CH2), 4.11 (s, OH),
Syn th esis of 3,17â-Dih yd r oxy-17r-(ter t-bu tylben zyl)-
estr a -1,3,5(10)-tr ien e (3). Magnesium (1.1 g, 45.3 mmol) was
added in a dry three-neck flask under argon atmosphere and
6.96 (d, J ) 2.0 Hz, 1H, 4-CH), 7.01 (dd, J 1 ) 2.2 Hz and J 2
)

Brief Articles
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12 2285
8.4 Hz, 1H, 2-CH), 7.20 and 7.26 (2d of AB system, J ) 8.2
Hz, 2H, CH2Ph-t-Bu), 7.35 (d, J ) 8.6 Hz, 1H, 1-CH), 7.88 (s,
NH2); 13C NMR δ (acetone-d6) 15.18 (C18), 23.99 (C15), 27.22
(C11), 28.13 (C7), 30.30 (C6; under solvent peaks), 31.85
(C(CH3)3), 32.13 (C12), 33.58 (C16), 34.90 (C(CH3)3), 40.90 (C8),
42.89 (C1′), 44.98 (C9), 47.94 (C13), 50.36 (C14), 83.72 (C17),
120.23 (C2), 123.06 (C4), 125.30 (C3′′ and C5′′), 127.44 (C1),
131.86 (C2′′), 137.28 (C1′′), 139.36 (C10), 139.91 (C5), 149.10
tions of a sonicated cell suspension at 10000g for 15 min and
100000g for 30 min. The microsomal fraction (100000g pellet)
was resuspended in 100 mM phosphate buffer (pH 7.4)
containing 1 mM EDTA and 20% glycerol. Time-dependent
inactivation of the enzyme was performed by preincubation
with inhibitor 3 (0.1 µM), 5 (1 nM), or EMATE (0.1 µM) for
0-20 min at 37 °C, followed by incubation with 0.5% dextran-
coated charcoal for 15 min. The concentrations indicated have
been determined by preliminary experiments. Dextran-coated
charcoal was sedimented by centrifugation (2000g), and the
supernatant was used to determine the steroid sulfatase
activity as described above for the incubation with homog-
enized cells using [3H]E1S as substrate, except that phosphate
buffer was used. In the substrate protection assays, 100 µM
E1S was added along with the inhibitor in the preincubation
period.
(C4′′), 149.46 (C3); FAB-HRMS calcd for C29H38O4NS (M+
+
H) 496.25217, found 496.24930. Anal. Calcd C29H39O4NS: C,
69.99; H, 7.90; N, 2.80; S, 6.44. Found: C, 70.31; H, 8.20; N,
2.85; S, 6.51.
B. Ster oid Su lfa ta se Assa ys. Human embryonal kidney
(293) cells (American Type Culture Collection, Rockville, MD),
transiently transfected with a sulfatase expression vector
(pCMV-sulfa), were used as the source of 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 (Kingston, R. E.; Chen,
C. A.; Okayama, H. In Current Protocols in Molecular Biology;
Ausubel, E. M., Brent, R., Kingston, R. E., Moore, D. D.,
Seidman, J . G., Smith, J . A., Struhl, K., Eds.; J ohn Wiley and
Sons: New York, 1991; pp 9.1.1-9.1.9). The cells were initially
plated at 104 cells/cm2 in Falcon culture flasks and grown in
Dulbecco’s modified Eagle’s medium containing 10% (v/v) fetal
bovine serum supplemented with 2 mM L-glutamine, 1 mM
sodium pyruvate, 100 IU penicillin/mL, and 100 µg of strep-
tomycin sulfate/mL.
Ack n ow led gm en t. The authors thank the Medical
Research Council of Canada, Fonds de Recherche en
Sante´ du Que´bec, and Endorecherche for financial
support. The technical support of Guy Reimnitz and Mei
Wang is also appreciated.
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freezing (-80 °C) and thawing (five times) and homogenization
using a Dounce homogenizer. The reaction was carried out at
37 °C in 1.25 mL of 100 mM Tris-acetate buffer (pH 7.4)
containing 5 mM ethylenediaminetetraacetic acid (EDTA), 10%
glycerol, 100 µM [3H]E1S or [14C]DHEAS as substrate, and an
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transformation of E1S and DHEAS, respectively. After 2 h of
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Rever sibility or Ir r ever sibility of In h ibitor s (h om og-
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steroid sulfatase were prepared by two successive centrifuga-

2286 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 12
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