Inhibition of estrone sulfatase by aromatase inhibitor-based estrogen 3-sulfamates

Article abstract of DOI:10.1016/j.steroids.2005.12.004

Our rationale is based on the finding that estrone 3-sulfamate (EMATE, 2d), a typical estrone sulfatase (ES) inhibitor, can be hydrolyzed and the pharmacological effect of the free estrogen contributes to the bioactivity of the sulfamate. A number of 3-su

Full text of DOI:10.1016/j.steroids.2005.12.004

steroids 7 1 ( 2 0 0 6 ) 371–379
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
Inhibition of estrone sulfatase by aromatase
inhibitor-based estrogen 3-sulfamates
∗
Mitsuteru Numazawa , Takako Tominaga, Yoko Watari, Yasue Tada
Tohoku Pharmaceutical University, 4-1 Komatsushima-4-chome, Aobaku, Sendai 981-9885, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Our rationale is based on the finding that estrone 3-sulfamate (EMATE, 2d), a typical estrone
sulfatase (ES) inhibitor, can be hydrolyzed and the pharmacological effect of the free estro-
gen contributes to the bioactivity of the sulfamate. A number of 3-sulfamoylated derivatives
of the good aromatase inhibitors, 2- and 4-halogeno (F, Cl, and Br) estrones and their estra-
diol analogs as well as 6␤-methyl and phenyl estrones, were synthesized and evaluated as
inhibitors of ES in human placental microsomes in comparison with the lead compound
EMATE. Among them, 2-chloro- and 2-bromoestrone 3-sulfamates (2b and 2c), along with
their estradiol analogs 3b and 3c, were powerful competitive inhibitors with Ki’s ranging
between 4.0 and 11.3 nM (Ki for EMATE, 73 nM). These four sulfamates as well as the 2-
fluoro analogs 2a and 3a inactivated ES in a time-dependent manner more efficiently than
EMATE, and 2-halogeno estrone sulfamates 2 also caused a concentration-dependent loss
of ES activity. The results may be useful for developing a new class of drugs having a dual
function, ES inhibition and aromatase inhibition, for the treatment of breast cancer.
© 2006 Elsevier Inc. All rights reserved.
Received 6 September 2005
Received in revised form 29
November 2005
Accepted 2 December 2005
Published on line 14 February 2006
Keywords:
Sulfamate
Halogeno estrogen
Esrone sulfatase
Aromatase
Inhibitor
Breast cancer
1
.
Introduction
in the treatment of post-menopausal patients. In addition,
inhibition of ES holds considerable potential for therapeu-
In post-menopausal women, despite the low levels of circu-
lating estrogens, concentrations of estrogens, estrone, estra-
diol, and their sulfates, in breast tumor tissue are several
times higher than those found in the plasma or in the area
of the breast considered as normal [1–4]. It is thought that the
high tissue and circulating levels of estrone sulfate can act
as a reservoir for the formation of biologically active estro-
gens via the action of steroid sulfatase (estrone sulfatase, ES).
The activity of this enzyme in breast tumors is much higher
than that of the aromatase complex [5–7]. As much as a 10-
fold greater amount of estrone may originate via the sulfatase
route than the aromatase pathway [8].
tic development. Several classes of ES inhibitors have been
reported in the past 10 years. Estrone 3-sulfamate (EMATE,
2d) is the first potent ES inhibitor that causes a time-
and concentration-dependent irreversible inactivation of the
enzyme [9,10]. This compound has been shown to possess
estrogenic properties and acts as a substrate of ES to release
estrone, according to its proposed mechanism of action
[11–13]. This has rendered EMATE unsuitable for use in the
treatment of breast cancer. Thus, the bioactivity of the free
scaffold is of greatest importance and has to be considered
in the design of sulfatase inhibitors for therapeutic appli-
cation. Sulfamoyloxy-substituted 2-phenylindoles have been
developed as antiestrogen-based inhibitors of ES [14]. These
compounds can be hydrolyzed to be potent antiestrogens to
offer the possibility of a dual mode of action, antiestrogen
Blockade of estrogen receptors by antiestrogens and inhi-
bition of estrogen biosynthesis by aromatase inhibitors are
therapeutic options, both of which have proved effective
∗
Corresponding author. Tel.: +81 22 234 4181; fax: +81 22 275 201.
E-mail address: numazawa@tohoku-pharm.ac.jp (M. Numazawa).
0039-128X/$ – see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2005.12.004

3
72
steroids 7 1 ( 2 0 0 6 ) 371–379
and ES inhibition. Purohit et al. sulfamoylated a number of
flavonoids, some of which are weak aromatase inhibitors, to
develop a new class of compound that can inhibit both aro-
matase and sulfatase, but the sulfatase inhibitory activities of
the flavonoid sulfamates were considerably less potent than
EMATE [15].
aration of 2-bromide 2c and 6␤-substituted compound 8 or
YMC R-Sil 5-06 column (250 mm × 4.6 mm i.d.; Kyoto, Japan)
in combination with hexane–EtOAc (75:25, v/v; 1 ml/min) as a
mobile phase for separation of 2-fluride 2a, and a UV detector
(280 nm).
We recently reported structure-activity relationships of
2.2.
General procedure for conversion of estrogens into
2
-, 4-, or 6-substituted estrones and their estradiol con-
geners as aromatase inhibitors, indicating that 2-halogeno-,
-methyl-, 6␣-aryl-, and 6␤-methylestrones are good com-
the 3-sulfamate derivatives
2
Sodium hydride (60% oil suspension, 130 mg, 3.2 mmol)
was separately added to stirred solutions of A- or B-ring-
substituted estrogens (1, 4, or 7) (0.32 mmol) in anhydrous
petitive inhibitors of aromatase with apparent K ’s ranging
between 100 and 660 nM [16]. In this paper, we synthesized
3
i
◦
-sulfamoylated derivatives of the good aromatase inhibitors
dimethylformamide (DMF) at 0 C. Subsequently, sulfamoyl
having an estrogenic steroid skeleton, which may have a dual
function, aromatase inhibition by the free steroid produced
by the hydrolysis and ES inhibition by the sulfamate. We also
evaluated the kinetics of their ES inhibitory activities in a
reversible or irreversible manner to develop a new class of drug
for the treatment of breast cancer. During the course of this
study, in a series of patent applications [17], 3-sulfamates of
chloride (300 mg, 2.6 mmol) was added carefully, and the
resulting mixtures were allowed to stand at room tempera-
ture with stirring for 9 h. The reaction mixtures were then
poured into a chilled 5% NaHCO3 solution (60 ml), extracted
with EtOAc (100 ml × 2), washed with saturated NaCl solu-
tion, and dried with Na2SO4. Evaporation of the solvent gave
oily substances that were purified by column chromatogra-
phy (hexane–EtOAc), preparative TLC (hexane–EtOAc), and/or
HPLC followed by recrystallization from an appropriate solvent
in each case.
2
-halogeno estrones, compounds 2a–c, were disclosed to be
potent inhibitors of ES; however, some of the physicochem-
ical data for the compounds as well as the kinetic data for
the reversible inhibition were not given. In addition, the irre-
versible inhibition was not examined.
2.3.
Conversion of A-ring-substituted estrone
3
-sulfamates (2 and 5a) into the estradiol derivatives (3
and 6a)
2
.
Experimental
Sodium borohydride (14 mg, 0.37 mmol) was separately added
2
.1.
Materials and general methods
to solutions of the estrone 3-sulfamates 2 and 5a (0.10 mmol)
◦
in MeOH (7 ml) at 0 C and the mixtures were stirred for 1 h at
◦
2
6
- and 4-Halogenoestrones (1 and 4) and 6␤-methyl- and
␤-phenylestrones (7) were synthesized by the previously
0 C. After this time, a few drops of acetic acid were added
to the mixture to stop the reaction. The mixture was then
extracted with EtOAc (70 ml), washed with 5% NaHCO3 solu-
tion and water, and dried with Na2SO4. Evaporation of the
solvent yielded solid products that were purified by recrys-
tallization from an appropriate solvent in each case.
reported methods [16]. Dextran-coated charcoal was obtained
from Sigma–Aldrich Corp. (St. Louis, MI, USA) and NADPH was
from Kohjin Co., Ltd. (Tokyo, Japan). [6,7- H2]Estrone sulfate
was purchased from Perkin-Elmer Japan Co., Ltd. (Yokohama,
Japan), and [1␤- H]androstenedione (25.4 Ci/mmol) ( H distri-
bution: ␤/␣-74.2/25.8) was from New England Nuclear Corp.
3
3
3
2.4.
Enzyme preparation
(
Boston, MA, USA).
Melting points were measured on a Yanagimoto melting
point apparatus (Kyoto, Japan) and are uncorrected. Infrared
IR) spectra were recorded in KBr pellets on a Perkin-Elmer
Human placental microsomes (sedimenting at 105,000 × g for
60 min) were obtained as described by Ryan [18]. They were
washed once with 0.05 mM dithiothreitol solution, lyophilized,
and stored at −20 C until use.
(
◦
FT-IR 1725X spectrophotometer (Norwalk, CT, USA), and ultra
violet (UV) spectra were determined in 95% ethanol on a
Hitachi 150-UV spectrophotometer (Tokyo, Japan). 1H Nuclear
magnetic resonance (NMR) spectra were obtained in CDCl3
or in CDCl3–CD3OD with a JEOL EX 270 (270 MHz) spectrom-
eter (Tokyo, Japan) using tetramethylsilane as an internal
standard. High resolution mass spectra (HRMS) were deter-
mined using a JEOL JMS-DX 303 spectrometer. Thin-layer
chromatography (TLC) was performed on E. Merck precoated
plates (silica gel 60F-254, layer thickness 0.25 and 0.5 mm
for analytical and preparative use, respectively; Darmstadt,
Germany). Column chromatography was conducted with sil-
ica gel 60, 70–230 mesh (E. Merck). High-performance liq-
uid chromatography (HPLC) was carried out using a Waters
2.5.
Estrone sulfatase assay
For the biochemical evaluation, the standard literature
method [10] was used. All enzymatic studies were carried out
in 67 mM phosphate buffer, pH 7.5, at a final incubation vol-
3
ume of 0.5 ml. The incubation mixture contained [ H]estrone
sulfate (20 ␮M for IC50 experiments or 2.0, 3.0, 5.0, and 10 ␮M
5
for kinetic experiments, 2 × 10 dpm), 20 ␮g of protein of the
lyophilized microsomes, various concentrations of inhibitors,
◦
and 25 ␮l of MeOH. Incubations were carried out at 37 C for
5 min and terminated by addition of 3 ml of toluene, followed
by vortexing for 45 s. After centrifugation at 2500 rpm for 5 min
◦
5
10 pump (Milford, MA, USA), Symmetry PrepTM C18 7 ␮m
at 4 C, an aliquot (1.0 ml) in duplicate was removed from the
column (250 mm × 7.8 mm i.d.; Waters) in combination with
organic layer and added to scintillation mixture for deter-
mination of [ H]estrone production. Control samples with
3
MeCN–H2O (40:60, v/v, 3.5 ml/min) as a mobile phase for sep-

steroids 7 1 ( 2 0 0 6 ) 371–379
373
no inhibitor were incubated simultaneously. Blank samples
◦
were obtained by incubating with boiled microsomes (95 C,
0 min). The basal sulfatase activity (without the inhibitor)
for the IC50 assay was found to be 3.80 nmol/min/mg protein
approximately 3.8% conversion of the used substrate). Kinetic
1
(
evaluation of the results was performed graphically by the
Lineweaver–Burk plot as well as by a computer program uti-
lizing non-linear least-squares curve fitting [19].
2
.6.
Irreversible estrone sulfatase assay
The irreversible inhibition study was essentially conducted
according to the previously described method [13]. Various
concentrations of inhibitors were incubated with placental
microsomes (2.5 mg of protein) and 500 ␮l of MeOH in 67 mM
phosphate buffer, pH 7.5, in a total volume of 5 ml. Aliquots
Fig. 2 – Synthesis of 3-sulfamates of 6-substituted estrones.
Reagents: (i) ClSO2NH2, dimethylacetamide.
(
1
0.5 ml) in duplicate were removed at various time periods (0,
3
5
H]androstenedione (3 × 10 dpm), 20 ␮g of protein from the
5, 30, and 45 min) and added to a suspension of dextran-
◦
lyophilized microsomes, and a 20 min-incubation time were
used in this study and the assay was carried out in 67 mM
phosphate buffer, pH 7.5, in the presence of NADPH (180 ␮M)
under air.
coated charcoal (2 mg) in the phosphate buffer (0.25 ml) at 0 C.
The mixture was vortexed for 30 s and allowed to stand at
0
Aliquots (0.25 ml) of the supernatant (equivalent to 67 ␮g of
protein) were added to a solution of [ H]estrone sulfate (20 ␮M,
2
0
[
◦
C for 10 min and, then, centrifuged at 3000 rpm for 5 min.
3
5
× 10 dpm) in 67 mM phosphate buffer, pH 7.5 (total volume,
3.
Results
◦
.5 ml), and the mixture was incubated at 37 C for 20 min.
3H]Estrone release was determined as described above. The
3.1.
Synthesis of estrogen 3-O-sulfamates
basal sulfatase activity (without the inhibitor) for the irre-
versible assay was 1.23 nmol/min/mg protein (approximately
A- or B-ring-substituted estrones (1, 4, and 7), good to excellent
1
5% conversion of the used substrate).
.7. Aromatase assay
Aromatase activity was measured using a radiometric method
aromatase inhibitors, were converted into the corresponding
3
-O-sulfamate derivatives 2, 5, and 8 by treatment with sul-
famoyl chloride in the presence of NaH in anhydrous DMF [22]
Figs. 1 and 2). The products were purified principally by silica
2
(
gel column chromatography, followed by recrystallization. In
some cases, preparative TLC and HPLC were also employed for
the purification. The estrone sulfamate analogs 2 and 5 were
then treated with NaBH4 in MeOH to give the 17␤-hydroxy
steroids 3 and 6, estradiol 3-O-sulfamate analogs, respectively
3
3
[
20] in which H2O released from [1␤- H]androstenedione into
the incubation medium during aromatization was used as an
index of enzyme activity. The inhibition assay was principally
the same as the previous method [21]. Briefly, 300 nM of [1␤-
Fig. 1 – Synthesis of 3-sulfamates of 2- or 4-halogeno estrogens. Reagents: (i) ClSO2NH2, dimethylacetamide and (ii) NaBH4,
MeOH.

Table 1 – Physicochemical properties of 3-sulfamates of 2-, 4-, 6␤-substituted estrones and estradiols
◦
−1
1H NMR (CDCl3), ␦
2-H (J, Hz) 4-H (J, Hz)
Compound
X
Yield (%)
mp ( C)
Recryst. solvent
UV (EtOH; nm) (ε)
IR (KBr) (cm
)
18-Me
1-H (J, Hz)
17␣-H (J, Hz)
2
4
2
-Substituted estrones 2
a
b
c
F
Cl
Br
55.7
68.7
18.9
197–200
212–215
219–222
MeOH
Acetone
MeOH
1978 (273)
1188 (275), 1188 (283)
1716 (275), 1716 (283)
1185, 1392, 1716
1192, 1383, 1712
1192, 1382, 1720
0.93 (s)
0.94 (s)
0.94 (s)a
7.09 (d, 12.2)
7.37 (s)
7.52 (s)
–
–
–
7.15 (d, 8.0)
7.23 (s)
7.24 (s)
–
–
–
-Substituted estrones 5
a
b
c
F
Cl
Br
18.9
8.5
19.1
183–186
178–182
192–196
MeOH
MeOH
MeOH
507 (265)
746 (278)
3531 (273)
1184, 1384, 1720
1261, 1341, 1732
1248, 1340, 1755
0.93 (s)
0.93 (s)
0.90 (s)
7.09 (d, 8.8)
7.25 (d, 8.0)
7.29 (d, 8.1)
7.23 (d, 8.3)
7.04 (d, 8.4)
7.02 (d, 8.6)
–
–
–
–
–
–
-Substituted estradiols 3
a
b
c
F
Cl
Br
50.5
81.0
35.5
191–193
177–180
191–193
Acetone
MeOH
MeOH
532 (272)
1286 (274), 1202 (282)
4612 (276)
1198, 1373, 3358
1189, 1361, 3518
1189, 1359, 1525
0.77 (s)
0.77 (s)
0.78 (s)a
7.09 (d, 9.8)
7.39 (s)
7.51 (s)
–
–
–
7.12 (d, 5.6)
7.35 (s)
7.21 (s)
3.69 (t, 8.5)
3.69 (t, 8.6)
3.65 (t, 8.5)
b
4
6
-Substituted estradiols 6
77.0
a
F
204–206
MeOH
540 (263)
1183, 1369, 3525
0.78 (s)
7.09 (d, 8.9)
7.21 (t, 8.2)
–
3.69 (t, 7.1)
␤-Substituted estrones 8
b
a
b
Me
Ph
34.8
10.2
174–177
177–179
MeOH
MeOH
426 (267), 394 (274)
846 (264), 813 (270)
1184, 1376, 1733
1188, 1382, 1722
0.92 (s)
0.82 (s)
7.31 (d, 8.5)
7.41 (d, 8.5)
7.21 (dd, 2.4, 8.4)
7.01 (d, 8.5)
7.14 (d, 2.4)
6.77 (d, 2.3)
–
b
4.31 (m) (6␤-H)
E2MATE, 3d
H
97.1
202–204
MeOH
461 (268)
1183, 1363, 3522
0.89 (s)c
7.31 (d, 8.4)
7.19 (dd, 2.4, 8.2)
7.02 (s)
3.68 (t, 8.7)
a
Solvent: CDCl3:CD3OD = 1:1.
Solvent: CDCl3:CD3OD = 5:1.
Solvent: CDCl3:CD3OD = 3:5.
b
c

steroids 7 1 ( 2 0 0 6 ) 371–379
375
Table 2 – HRMS data for 3-sulfamates of 2-, 4-, 6␤-substituted estrones and estradiols
Compound
Formula
Calculated (%)
Found (%)
2
4
2
-Substituted estrones 2
2-Fluoro, a
2-Chloro, b
2-Bromo, c
C18H22SNO4F
C18H22SNO4Cl
C18H22SNO4Br
367.1254
383.0958, 385.0929
427.0453, 429.0432
367.1252
383.0944, 385.0894
427.0428, 429.0423
-Substituted estrones 5
4
4
4
-Fluoro, a
-Chloro, b
-Bromo, c
C18H22SNO4F
C18H22SNO4Cl
C18H22SNO4Br
367.1254
383.0958, 385.0929
427.0453, 429.0432
367.1276
383.0914, 385.0348
427.0418, 429.0260
-Substituted estradiols 3
2
2
2
-Fluoro, a
-Chloro, b
-Bromo, c
C18H24SNO5F
C18H24SNO5Cl
C18H24SNO5Br
369.1411
385.1115, 387.1086
429.0690, 431.0589
369.1407
385.1114, 387.1046
429.0626, 431.0619
4
6
-Substituted estradiols 6
-Fluoro, a
4
C18H24SNO5F
369.1411
369.1406
␤-Substituted estrone 8
6
6
␤-Methyl, a
␤-Phenyl, b
C19H25SNO4
C24H27SNO4
363.1505
425.1660
363.1530
425.1680
(
Fig. 1). The structures of all of the synthesized sulfamates
were determined by the spectrometric and HRMS analyses
Tables 1 and 2).
the substrate essentially for 15, 13, and 45 min, after this
time, the remaining sulfatase activity was measured using the
tritiated estrone sulfate as a substrate. The time-dependent
inactivation was observed when all of the inhibitors exam-
(
3
.2.
Estrone sulfatase inhibition assay
Reversible inhibition of ES activity in human placental micro-
somes by the A- or B-substituted estrogen sulfamates 2, 3, 5,
Table 3 – Sulfatase inhibition by 3-sulfamates of 2-, 4-,
or 6␤-substituted estrogens
6
, and 8, synthesized in this study, was initially examined, and
Sulfamates
IC50, nMa
Apparent
Ki, nMb
Inhibitionc
the results are shown in Table 3. In addition to the above com-
pounds, estrone 3-sulfamate (EMATE, 2d) [9] and its estradiol
analog (E2MATE, 3d) [11], typical estrone sulfatase inhibitors
previously reported, are listed for comparison. ES activity in
the placental microsomes was determined by the radiomet-
2-Substituted estrones 2
2-Fluoro, a
-Chloro, b
2-Bromo, c
57.2 ± 0.72
18.2 ± 1.5
4.0 ± 0.4
5.5 ± 0.6
Competitive
Competitive
Competitive
2
15.0 ± 0.58
15.4 ± 0.72
3
ric method in which the amount of [ H]estrone released from
3
the substrate [ H]estrone 3-sulfate during the incubation was
4-Substituted estrones 5
4-Fluoro, a
used as an index of the sulfatase activity. IC50 values for all
of the inhibitors were first obtained, and then the inhibitors,
except for 6-phenyl steroid 8b, were further studied to char-
acterize the nature of their interactions with the active site
under initial velocity conditions. Hydrolysis of the labeled
estrone sulfate was measured at several concentrations of
the inhibitor in the presence of increasing concentrations of
the sulfate substrate. The results of the studies were plotted
in a typical Lineweaver–Burk plot. All of the inhibitors pre-
vented ES activity in a competitive manner, and the apparent
80.2 ± 2.6
20.1 ± 0.9
1900 ± 100
1500 ± 100
Competitive
Competitive
Competitive
4-Chloro, b
-Bromo, c
3600 ± 100
2500± 200
4
6␤-Substituted estrones 8
6
6
␤-Methyl, a
␤-Phenyl, b
5900 ± 2300
6100 ± 37
Competitive
–
40% inhibition at 100 ␮M
2-Substituted estradiols 3
2-fluoro, a
2-chloro, b
2-bromo, c
34.8 ± 1.7
16.0 ± 0.9
22.6 ± 2.8
27.7 ± 1.0
11.3 ± 0.6
10.6 ± 0.5
Competitive
Competitive
Competitive
inhibition constants (K ) were obtained by analysis of Dixon
4-Substituted estradiols 6
i
4
-fluoro, a
88.3 ± 1.0
38.2± 2.3
Competitive
plots. The Lineweaver–Burk plot of sulfatase inhibition by 2-
chloroestrone sulfamate 2b is shown in Fig. 3. In these studies,
the apparent Km for estrone sulfate was found to be approxi-
mately 15 nM.
For comparison
EMATE, 2d
E2MATE, 3d
320 ± 6.2
73 ± 5.8
Competitive
Competitive
251 ± 11.4
133 ± 5.1
Sulfamates of 2-halogeno- and 4-fluoroestrones and 2-
chloroestradiol, compounds 2, 5a, and 3b, which were pow-
erful competitive inhibitors of ES, as well as 6␤-methylestrone
analog 8a, which was a fair competitive inhibitor, were then
tested for their abilities to cause a time-dependent inactiva-
tion of the enzyme. The placental microsomes were prein-
cubated with the various inhibitors prior to the addition of
a
3
Concentrations of 20 ␮M of [ H]estrone sulfate and 20 ␮g protein
from human placental microsomes were used.
Inhibition constant was obtained by Dixon plot. In these exper-
iments, the apparent Km for the substrate estrone sulfate was
found to be about 15 nM.
b
c
Inhibition type was determined by Lineweaver–Burk plot. Results
are shown as the mean ± S.E.

3
76
steroids 7 1 ( 2 0 0 6 ) 371–379
Fig. 5 – Time- and concentration-dependent inactivation of
human placental ES by 2-fluoroestrone 3-sulfamate (2a).
Concentrations of inhibitor: control (0 nM) (ꢃ); 100 nM (ꢁ);
Fig. 3 – Lineweaver–Burk plot of estrone sulfatase inhibition
by 2-chloroestrone 3-sulfamate (2b). Concentrations of
inhibitor: control (0 nM) (᭹); 2 nM (ꢀ); 4 nM (ꢁ); 8 nM (ꢂ). The
plots intersect on the y-axis (1/V axis), 0.092 (pmol/min/␮g
3
00 nM (ꢁ); 400 nM (ꢀ); 500 nM (ꢀ); control plus estrone
−
1
sulfate (500 nM) (᭹). Each point represents the mean of two
protein) , indicative of a simple competitive inhibitor. The
determinations, which varied by less than 5% of the mean.
K value was determined by plotting the slopes of each plot
i
against inhibitor concentration and the −K value equalling
i
the negative intercept on the x-axis (inhibitor concentration
axis). Each point represents the mean of two
determinations, which varied by less than 5% of the mean.
The inhibition experiments with the other sulfamates
examined gave essentially similar plots to that shown in
Fig. 3 (data not shown).
was protected by adding the substrate estrone sulfate to
the preincubation medium in a dose-dependent manner
(
Fig. 4).
3
.3.
Aromatase inhibition assay
To determine whether the estrogen sulfamates described
above still had the ability to inhibit aromatase, reversible inhi-
bition of aromatase activity in the placental microsomes by
the sulfamates 2, 3, 5, and 8 was next studied at a concen-
tration of 100 nM of each sulfamate (Table 4). The aromatase
ined were preincubated with estrone sulfatase in the pla-
cental microsomes (Figs. 4 and 5). The time-dependent inac-
tivation by EMATE was also carried out for comparison.
The irreversible inactivation caused by the 2-bromide 2c
Table 4 – Aromatase inhibition by 3-sulfamates of 2-, 4-
or 6␤-substituted estrogens
Sulfamate
IC50, ␮Ma
2
-Substituted estrones, 2
2
2
2
-Fluoro, a
-Chloro, b
-Bromo, c
34% Inhibition at 100 ␮M
25% Inhibition at 100 ␮M
11% Inhibition at 100 ␮M
4
-Substituted estrones, 5
4
4
4
-Fluoro, a
-Chloro, b
-Bromo, c
35% Inhibition at 100 ␮M
Not determined
37% Inhibition at 100 ␮M
6
2
␤-Substituted estrones, 8
␤-Methyl, a
6␤-Phenyl, b
6
40% Inhibition at 100 ␮M
41.8 ± 2.3 ␮M
Fig. 4 – Time-dependent inactivation of human placental ES
by 3-sulfamates of halogeno estrogens. Control, no
inhibitor (᭹); 2-chloroestrone 2b, 100 nM (ꢃ);
-Substituted estradiols, 3
2
-Fluoro, a
33% Inhibition at 100 ␮M
40% Inhibition at 100 ␮M
30% Inhibition at 100 ␮M
2
-bromoestrone 2c, 100 nM (ꢁ); 2-bromoestrone 2c, 100 nM
plus estrone sulfate, 200 nM (ꢄ); 4-fluoroestrone 5a, 400 nM
ꢀ); 2-chloroestradiol 3b, 100 nM (ꢄ), 200 nM (ꢁ);
-bromoestradiol 3c, 100 nM (ꢄ), 200 nM (ꢀ). Each point
2-Chloro, b
2-Bromo, c
(
2
E2MATE, 3d
24% Inhibition at 100 ␮M
a
3
represents the mean of two determinations, which varied
by less than 5% of the mean.
300 nM of [1␤- H]androstenedione and 20 ␮g of protein from pla-
cental microsomes were used.

steroids 7 1 ( 2 0 0 6 ) 371–379
377
inhibitory activities of all of these compounds were extremely
low.
tion was reduced in the presence of excess substrate estrone
3-sulfate, indicating that the inactivation was directed to the
active site. Although the charcoal-trapping method was used
to separate enzyme from unbound inhibitor, direct evidence
that the inactivation was irreversible is not available. How-
ever, based on the results, it is likely that the time-dependent
inactivation would proceed in an irreversible manner. Inter-
estingly, the time-dependent inactivation follows a biphasic
course, with a fast initial phase and a slower later phase; this
biphasic loss of the activity was previously seen for the inacti-
vation by not only other steroidal sulfamates including EMATE
and E2MATE, but also non-steroidal sulfamates, including
flavonoids, coumarins, and chromenones [10,13,27–30]. The
biphasic loss is most probably a result of metabolic depen-
dent elimination of the inhibitor from the incubate because
the chloro steroid 2b was stable in phosphate buffer at
4
.
Discussion
ES inhibition by a series of 3-sulfamates of the good aro-
matase inhibitors 2- and 4-halogeno estrones, compounds
2
and 5, and their estradiol analogs 3 and 6 as well as 6␤-
methyl- and 6␤-phenylestrone 3-sulfamates (8) were tested
using human placental microsomes as the enzyme source.
The sulfamates of 2-halogeno- and 4-fluoroestrogens, 2, 3,
5
a, and 6a, were very potent competitive inhibitors of ES
with apparent Ki values ranging from 4.0 to 38.2 nM. On
the other hand, the inhibitory activities of the 4-chloro-
and 4-bromoestrone derivatives 5b and 5c along with 6␤-
◦
substituted estrone analogs 8 were very low with the K val-
37 C for up to 60 min (data not shown). Alternative explana-
i
ues in the micromolar range. Introduction of a fluoro, chloro,
or bromo moiety at the C-2 position of EMATE (2d) and
E2MATE (3d) and that of a fluoro moiety at the C-4 position of
the parent sulfamates markedly increased the ES inhibitory
activity.
The structural modifications, 2-methoxylation, 2- or 4-
nitration, 2- or 4-alkylation, and 2- or 4-allylation, of the lead
steroid EMATE have been reported to lead to more potent irre-
versible ES inhibitors lacking the estrogenic effect displayed by
the lead compound [23–25]. 2-Halogeno derivatives of EMATE
were potent inhibitors of ES with IC50 values between 0.8 and
tions include the existence of two different enzyme species
in the microsomes used for the experiments; for exam-
ple, two different isozymes or two different conformations
of ES.
Although a mechanism for irreversible inactivation of ES
by EMATE has not been definitely proved so far, inactivation
of ES by transfer of the 3-sulfamate moiety from the inhibitor
to the active site, producing sulfamoylated ES, appears likely
[13]. On the basis of this mechanism, it is reasonable that the 3-
sulfamoyl moiety of the halo estrogens would be transferred
more effectively to an amino acid residue of the active site
than that of EMATE because of their inherent physicochem-
ical property, electron-withdrawing property of the halogeno
group, and/or their binding geometry in the active site, which
is more proper for the transfer than that involved in the EMATE
binding.
Recently, it was reported that one of sulfamoylated deriva-
tives of the powerful non-steroidal aromatase inhibitor YM511
analogs directly inhibited both ES and aromatase to a great
extent in JEG-3 cells [31] without hydrolysis of the sulfamoyl
moiety. All of the potent ES inhibitors synthesized in this study
did not show any significant inhibitory activity against aro-
matase, indicating that a sulfamoyl function at C-3 should
prevent access of the inhibitors to the active site of aromatase
in vitro. However, it is predicted that the inhibitors act as a sub-
strate of ES during the irreversible inactivation of the enzyme
or during circulation in vivo to release an estrogen scaffold,
which would be expected to play a role in aromatase inhibition
[11–13]. Although antiestrogen based inhibitors of ES have pre-
viously been reported [14,15,27], the sulfamates described in
this study are the first aromatase inhibitor-based ES inhibitors
with an estrogen scaffold. The present findings would be use-
ful for the development of a novel anti-breast cancer agent
having a dual function, as both aromatase inhibitor and sulfa-
tase inhibitor. It should be noted that 2-halogeno estrogens
[32–34] and 6-alkylestradiols [35] have very low affinity for
the estrogen receptor (ER) in uterine cytosol; some of the 2-
halides have agonistic properties [32–34] whereas it is cur-
rently uncertain whether 6-substituted estrones 7 are antie-
strogenic or estrogenic ligands for ER. However, if the free
estrogens are antiestrogenic ligands, their 3-sulfamates may,
possibly, become to have antiestrogenic function along with
the above dual functions.
6
.1 nM in the placental micosomal system in patent appli-
cations [17]. It was recently reported that the 2-chloro com-
pound 2b was superior to the parent steroid 2d by a fac-
tor of 2.7 [26]. However, there was no kinetic data for the
reversible inhibition; moreover, it was not shown whether
or not this chloride causes a time-dependent inactivation of
ES.
The relative inhibitory activities among 2-halogeno estrone
sulfamates 2 on the basis of the kinetic data (K : 18.2 versus
i
4
.0 versus 5.5 nM for 2a versus 2b versus 2c) were roughly
comparable to those obtained previously on the basis of IC50
values [17,26]. In addition, a series of the estradiol derivatives
3
were also potent competitive inhibitors of ES with K val-
i
ues ranging from 11.3 to 27.7 nM where the relative inhibitory
activities were similar to those observed in a series of estrone
analogs. It is noted that 4-fluoro compounds 5a and 6a among
the 4-halogeno estrone sulfamates 5 and 6 showed power-
ful inhibitory activities, which were equivalent to those of
the corresponding 2-fluoro steroids 2a and 3a, but 4-chloro
and 4-bromoestrone sulfamates 5b and 5c were poor compet-
itive inhibitors. 3-Sulfamates of another powerful aromatase
inhibitor 6␤-methyl- and 6␤-phenylestrones (7), compound
8
6
, were poor inhibitors of ES with Ki values of more than
.1 ␮M.
-Halogeno estrogen sulfamate 2 and 3 as well as 4-fluoro-
2
and 6␤-methylestrone sulfamates 5a and 8a caused a time-
and concentration-dependent inactivation of ES in placen-
tal microsomes. Among them, 2-chloro- and 2-bromoestrone
analogs 2b and 2c were the most powerful time-dependent
inactivators where ES activity was completely lost at a con-
centration of 100 nM during 15 and 30 min of preincubation
of compounds 2c and 2b, respectively. The rate of inactiva-

3
78
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Sendai, Japan, for supplying us with human term placenta.
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