Boronic acids as inhibitors of steroid sulfatase

Article abstract of DOI:10.1016/j.bmc.2006.08.033

Steroid sulfatase (STS) catalyzes the hydrolysis of steroidal sulfates such as estrone sulfate (ES1) to the corresponding steroids and inorganic sulfate. STS is considered to be a potential target for the development of therapeutics for the treatment of s

Full text of DOI:10.1016/j.bmc.2006.08.033

Bioorganic & Medicinal Chemistry 14 (2006) 8564–8573
Boronic acids as inhibitors of steroid sulfatase
Vanessa Ahmed, Yong Liu, Cassandra Silvestro and Scott D. Taylor*
Department of Chemistry, University of Waterloo, 200 University Avenue West, Waterloo, Ont., Canada N2L 3G1
Received 3 July 2006; revised 21 August 2006; accepted 22 August 2006
Available online 14 September 2006
Abstract—Steroid sulfatase (STS) catalyzes the hydrolysis of steroidal sulfates such as estrone sulfate (ES1) to the corresponding
steroids and inorganic sulfate. STS is considered to be a potential target for the development of therapeutics for the treatment
of steroid-dependent cancers. Two steroidal and two coumarin- and chromenone-based boronic acids were synthesized and exam-
ined as inhibitors of purified STS. The boronic acid analog of estrone sulfate bearing a boronic acid moiety at the 3-position in place
of the sulfate group was a good competitive STS inhibitor with a Ki of 2.8 lM at pH 7.0 and 6.8 lM at pH 8.8. The inhibition was
reversible and kinetic properties corresponding to the mechanism for slow-binding inhibitors were not observed. An estradiol deriv-
ative bearing a boronic acid group at the 3-position and a benzyl group at the 17-position was a potent reversible, non-competitive
STS inhibitor with a Ki of 250 nM. However, its 3-OH analog, a known STS inhibitor, exhibited an almost identical affinity for STS
and also bound in a non-competitive manner. It is suggested that these compounds prefer to bind in a hydrophobic tunnel close to
the entrance to the active site. The coumarin and chromenone boronic acids were modest inhibitors of STS with IC50s of 86 and
171 lM, respectively. Surprisingly, replacing the boronic acid group of the chromenone derivative with an OH group yielded a good
reversible, mixed type inhibitor with a Ki of 4.6 lM. Overall, these results suggest that the boronic acid moiety must be attached to a
platform very closely resembling a natural substrate in order for it to impart a beneficial effect on binding affinity compared to its
phenolic analog.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
famate is hydrolyzed by the enzyme resulting in the re-
lease of the phenolic portion of the inhibitor and
sulfamic acid which then irreversibly inhibits ES by a
yet unknown mechanism. EMATE (1),4 667-COU-
MATE (2)5, and chromenone 36 are examples of this
class of highly potent inhibitors. 667-COUMATE has
recently successfully completed phase I clinical trials
for the treatment of breast cancer.7
Steroid sulfatase (STS), also known as aryl sulfatase C
(ARSC) or estrone sulfatase, catalyzes the hydrolytic
desulfation of steroidal sulfates, such as dehydroepiand-
rosterone sulfate (DHEAS) or estrone sulfate (E1S), to
the corresponding steroids and inorganic sulfate.1 These
sulfated species are considered to be the storage form of
estrone (E1) and dehydroepiandrosterone (DHEA) in
breast tumor and other cells.1 Approximately one-third
of breast tumors in post-menopausal women require
stimulation by estrogens for optimal growth.1 Conse-
quently, considerable interest has arisen in designing
inhibitors of STS anticipating that such inhibitors could
be developed into therapeutics for the treatment of
estrogen-dependent breast cancer and other estrogen-
dependent diseases.2
In comparison to irreversible sulfamate inhibitors, far
fewer reversible STS inhibitors have been developed.2
Perhaps the most noteworthy are those developed by
Poirier and coworkers who reported that certain 17a-
benzyl-substituted estradiol derivatives, such as 4 and
5, are reversible STS inhibitors. Some of these com-
pounds, which were tested using homogenates of
JEG-3 cells, are the most potent reversible inhibitors
reported to date with IC50s in the low-nM region.8,9
The modality of inhibition was not reported. Several
non-steroidal competitive and non-competitive revers-
ible STS inhibitors have been obtained from high
throughput screening of compound libraries.10–12
Although some of these compounds exhibited good
potency in vitro, their potency in cell-based assays
was much lower.
Many inhibitors of STS have been reported.2,3 The vast
majority are aryl sulfamates (Ar–SO2NH2) which act as
irreversible suicide inhibitors.2 The S–O bond of the sul-
Keywords: Steroid sulfatase; Boronic acids; Inhibitors.
*
Corresponding author. Tel.: +1 519 888 4567x3325; fax: +1 519 746
0435; e-mail: s5taylor@sciborg.uwaterloo.ca
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.08.033

V. Ahmed et al. / Bioorg. Med. Chem. 14 (2006) 8564–8573
8565
O
O
O
H2NSO2O
H2NSO2O
O
O
H2NSO2O
3
2
1
HO
HO
HO
HO
5
4
Many reversible STS inhibitor inhibitors have been
obtained by replacing the sulfate group of estrone or
estradiol with O-, N-, or S-linked sulfate surrogates.2
However, for the most part, these have not proven to
be highly effective inhibitors with the vast majority
exhibiting Kis or IC50s in mid-to low lM range with
the better ones being around 10 lM. However, the vast
majority of these were never examined with pure enzyme
and the modality of inhibition was not determined.13
ases.21 On the basis of crystal structures of aryl sulfatase
A (ASA) as well as on kinetic studies on the wild type
and specific mutants, a mechanism has been proposed
by von Figura and coworkers that is now the most wide-
ly accepted mechanism for ARSs (Scheme 2).22,23 One of
the hydroxyls of the formylglycine hydrate attacks the
sulfur atom of the substrate resulting in cleavage of
the S–O bond, release of the hydroxyl or phenolic por-
tion of the substrate, and formation of a sulfated hy-
drate. The sulfate group is then eliminated from the
hydrate to give inorganic sulfate and formyl glycine
which is then rehydrated (Scheme 2). Several other ac-
tive site residues, including two conserved histidines,
are believed to function as general acids and bases dur-
ing the reaction. In light of this proposed mechanism, we
reasoned that boronic acids might act as potent inhibi-
tors of STS by forming reversible covalent adducts with
the active site hydrate and/or histidine residues in a
manner similar to serine proteases. Here, we report the
results of our studies on the inhibition of STS with ste-
roidal and non-steroidal boronic acids 6–9.
Boronic acids have been used as inhibitors and probes of
enzymes and proteins, such as serine proteases, for many
years and recently, a highly potent and selective protease
inhibitor in the form of a peptidyl boronic acid has
recently been approved by the FDA for treatment of
relapsed and refractory multiple myeloma.16 These
inhibitors, some of which have Kis in the subnanomolar
range, function by forming reversible covalent adducts
with active site residues, such as the crucial serine resi-
due or an active site histidine residue as shown in
Scheme 1.16 Unlike serine proteases, ARSs do not have
an active site serine residue when in their catalytically
active form. Instead, all ARSs have an active site form-
ylglycine hydrate which is a result of a post-translational
enzymatic modification of a cysteine or serine residue.
Addition of water to the aldehyde yields the stable form-
ylglycine hydrate.17,18 The structures of STS19 and sever-
al other sulfatases20 have been elucidated by X-ray
crystallography. There is a very high degree of sequence
and structural homology at the active site such that their
active sites are almost superimposable hence it is be-
lieved that all ARSs function by a similar mechanism.
Several mechanisms have been proposed for aryl sulfat-
HO
O
HO
HO
B
B
OH
7
6
OH
O
O
HO
HO
B
B
O
O
OH
OH
HO
H
OH
-
9
8
B
R
O B
R
O
HO
OH
2. Results and discussion
HO
HO
OH
B
R
+
-
N
B R
HN
N
HN
2.1. Syntheses
OH
Two steroidal boronic acids were prepared. Steroid
derivative 6 in which the 3-OH of estrone was replaced
Scheme 1. Mechanism of inhibition of serine proteases by boronic
acids.

8566
V. Ahmed et al. / Bioorg. Med. Chem. 14 (2006) 8564–8573
R
R
+
+
+
H
N
HisB
+
Lys-NH2
H
O
+
O
S
Lys-NH2
H
+
O
Lys-NH3
H
N
HisB
Ca+2
H
N
HisB
Ca+2
Ca++
-
-O
ROSO3
S
O-
O-
O
O
O
O
O
H
H
-O AspC
HisA NH
O
H
HisA NH
O
O
FGly
HO Asp
-O Asp
H
H
HisA NH
FGly
FGly
X
H2O
ROH
+
Lys-NH2
H
+
N
HisB
Asp
O-
N
HisB
O
Lys-NH2
H
-
SO4
O
H
Ca+2
O
S
Ca+2
O
+
N
HisA
H
O
H
O
O
H
Asp
HisA NH2
H
FGly
FGly
Scheme 2. Proposed mechanism for aryl sulfatases.
O
O
O
O
BH
O
NaIO
4
O
B
O
Pd (dba) -CHCl
3
NH OAc/H O
2
3
4
2
HO
o
TfO
B
1,4-dioxane, 80 C
OH
10
6 (84 %)
11 (78 %)
Scheme 3. Synthesis of boronic acid 6.
O
O
O
1. TFAA, DMAP,
CH Cl
2. 1N HCl
EtOAc/MeOH
Pd(dppf)Cl -
2
CF3
2
2
CF3
HO
O
CH Cl , Et N,
2 2 3
o
dioxance, 93 C
3. Tf O, DMAP
2
O
o
BH
CH Cl , 0 C
2
2
O
O
B
HO
TfO
12 (67%)
4
O
13 (62%)
THF
0.8N NaOH
HO
HO
2N HCl
THF/MeOH
O
HO
B
B
OH
14 (84%)
B
7 (67%)
O
OH
OH
Scheme 4. Synthesis of compound 7.
with B(OH)2 was readily synthesized by first reacting tri-
flate 1024 with pinacolborane in the presence of a palla-
dium catalyst to give boronate ester 11 in 78% yield.
Reaction of the ester with NaIO4 in ammonium ace-
tate–water25 for 10 days gave compound 6 in 84% yield
(Scheme 3).
49 was reacted with trifluoroacetic anhydride which gave
the trifluoroacetate diester of 4 at 3- and 17-positions.
Selective hydrolysis of the ester at the 3-position with
aq 1 N HCl in EtOAc–MeOH followed by reaction with
triflic anhydride gave compound 12 in 67% yield (three
steps). Introduction of the pinacol borate ester as de-
scribed for compound 11 gave compound 13 in 62%
yield.26 Hydrolysis of the ester at the 17-position with
aq NaOH in THF gave compound 14 in 84% yield.
Hydrolysis of the pinacol boronate ester with 2 N aq
Since it has been shown that compound 4 is a very good
inhibitor of STS,8,9 we prepared the boronic acid analog
of 4, compound 7, as outlined in Scheme 4. Compound

V. Ahmed et al. / Bioorg. Med. Chem. 14 (2006) 8564–8573
8567
O
O
O
O
B
O
O
O
B
B
NH4OAc
NaIO4
8 (67%)
or
TfO
Tf2O, DMAP
CH2Cl2
O
O
HO
HO
O
O
O
19 (83%)
17 (97%)
15
acetone-water
16h
Et3N, dioxane
or
or
9 (77%)
or
Pd(dppf)Cl2-CH2Cl2
O
O
85 o
C
O
O
TfO
B
O
O
O
O
16
18 (50%)
20 (75%)
Scheme 5. Synthesis of compounds 8 and 9.
HCl in the presence of phenylboronic acid in THF–
MeOH27 gave the desired boronic acid 7 in 67% yield.
Compound 6 is a competitive STS inhibitor with a Ki of
2.8 lM at pH 7.0. Comparing the potency of 6 with
those of many other estrone derivatives bearing sulfate
surrogates that have appeared in the literature is difficult
since very few have been examined with pure enzyme
and the modality of inhibition was rarely determined.
Nevertheless, a comparison of 6 with other estrone
derivatives bearing sulfate surrogates whose Kis or
IC50s have been determined using pure enzyme or pla-
cental microsomes2 reveals that compound 6 is one of
the most potent inhibitors of this class. Moreover, com-
pound 6 is a purely competitive inhibitor which is in
contrast to many other estrone derivatives bearing sul-
fate surrogates which often exhibit noncompetitive or
mixed inhibition.30–33 Estrone phosphate is one of the
better estrone-based STS competitive inhibitors bearing
a sulfate surrogate.14,15 Under our assay conditions, we
determined estrone phosphate to have an IC50 of 5 lM
at pH 7.0. Thus, compound 6 is as potent as estrone
phosphate at pH 7.0.
Since the coumarin and chromenone platforms have
proven to be effective for obtaining highly potent sulfa-
mate inhibitors of STS, we also prepared coumarin
boronic acid 8 and chromenone boronic acid 9 as out-
lined in Scheme 5. Coumarin 1528 and chromenone
1629 were converted to triflates 17 and 18 in 97% and
50% yield, respectively. Attempts to convert these tri-
flates to the corresponding boronate esters 19 and 20
using the conditions described for 11 and 14 proved
unsuccessful. However, when bis(pinacolato)diboron
was used in place of pinacolborane the coumarin and
chromenone derivative 19 and 20 were readily prepared
in 83% and 75% yield, respectively. Removal of the pina-
col group as described above for 6 gave the desired bor-
onate esters 8 and 9 in 67% and 77% yield, respectively.
2.2. Inhibition studies
Inhibition studies were carried out using purified STS in
Tris buffer containing 5% DMSO with 4-methylumbel-
liferyl-6-O-sulfate (MUS) as substrate. Kis or IC50s
determined at pH 7.0 for boronic acids 6–9 as well as
for estrone, steroid derivative 4, and chromenone 16
are given in Table 1.
As mentioned earlier, boronic acids are competitive
inhibitors of serine proteases and function by forming
covalent adducts with active site residues such as serine
and histidine.16 Simple aryl boronic acids are generally
modest serine protease inhibitors with Kis ranging from
the millimolar to low micromolar depending on the en-
zyme and the substituents on the boronic acid. However,
peptide boronic acids that have the primary specificity
requirement for the protease are much more potent
inhibitors with Kis sometimes extending into the sub-
nanomolar range and exhibit an affinity for the protease
that is many orders of magnitude greater than the corre-
sponding substrate. Moreover, these inhibitors show
kinetic properties corresponding to the mechanism for
slow-binding inhibitors.34 Compound 6 can be consid-
ered the boronic acid analog of estrone sulfate, a natural
substrate of STS. The Km for estrone sulfate in 0.1 M
Tris–HCl, 0.1% Triton X-100, at pH 7.5 is 95 lM.35
Dibbelt et al. have shown, using [35S]-labeled DHEAS
as substrate, that estrone sulfate is an inhibitor of STS
exhibiting mixed inhibition with a Ki of about 1 lM in
0.1 M Tris–acetate at pH 7.0.32 Thus, it appears that
compound 6 has an affinity for STS that is about equal
to estrone sulfate. Estrone itself is mainly a non-compet-
itive inhibitor with a Ki of 63 lM and aKi of 110 lM.
Thus, replacing the 3-OH of estrone with a boronic acid
Table 1. Kis or IC50s for compounds 4, 6–9, 16, and estrone
Inhibitor
pH
Ki or IC50 (lM)
aKi (lM)e
6
7.0
7.5
8.0
8.5
8.8
7.0
7.0
7.0
7.0
7.0
7.0
2.8 0.4a
2.1 0.3a
3.8 0.5a
7.0 0.6a
6.8 0.8a
0.25 0.02a
171 9b
NAc
6
NAc
6
NAc
6
NAc
6
NAc
7
0.30 0.02
NDd
NDd
8
9
Estrone
86 4b
63 2a
0.25 0.02a
4.6 0.8a
111 14
0.42 0.01
4
16
40
4
a Ki.
b IC50
.
c NA, not applicable.
d ND, not determined.
e Dissociation constant from ES complex.

8568
V. Ahmed et al. / Bioorg. Med. Chem. 14 (2006) 8564–8573
moiety resulted in about 23-fold increase in potency and
changes the modality of inhibition from noncompetitive
to competitive. STS assays performed in the presence of
compound 6 exhibited a linear increase in product with
time during the entire time course of the assays (10 min).
Preincubation of 6 with STS for up to 60 min did not re-
sult in an increase in potency. These results suggest that
compound 6 is not a slow-binding inhibitor. Moreover,
the inhibition was found to be reversible as demonstrat-
ed by experiments in which the activity was recovered by
dilution into a solution containing a large excess of
substrate.
Since boronic acid inhibitor 6 was considerably more
potent than estrone, compound 7 was prepared in the
anticipation that substitution of the 3-OH moiety in 4
with a boronic acid would also result in a significant in-
crease in inhibitory potency. Compound 7 is a 10-fold
more potent inhibitor than compound 6 with a Ki of
252 nM. However, compounds 7 and 4 exhibited almost
identical Kis and both were mainly non-competitive
inhibitors of STS with both inhibitors exhibiting similar
affinities for both the free and substrate bound forms of
the enzyme. These results suggest that both 7 and 4 pref-
erably bind in a region outside the active site. It has been
suggested that the high affinity of estrone derivatives
bearing benzyl groups at the 17-position for STS is a re-
sult of the benzyl group extending into a hydrophobic
channel between the two antiparallel helices that are be-
lieved to insert into the membrane of the endoplasmic
reticulum.2a Since our results with compounds 4 and 7
suggest that these compounds bind in an area outside
the active site, it is possible that they bind in the channel
between the helices and block the entrance to the active
which is a small tunnel at the top of the two helices.19
In some instances, the inhibition of serine proteases with
boronic acids is very pH-dependent. Bender has report-
ed that the inhibition of subtilisin with benzene boronic
acid increases significantly between pH 6.0 and 8.0, and
then rapidly decreases at pH > 8.0.36 The increase in
inhibition from pH 6.0 to 8.0 was attributed to the in-
crease in the concentration of a reactive residue in the
active site such as histidine or serine while the decrease
was attributed to a decrease in the concentration of
the reactive trigonal form of the boronic acid inhibitor.
We have observed a modest decrease in inhibitor poten-
cy as the pH increases from pH 7.5 to 8.5 then levels off
as the pH increases to 8.8. The inhibition remains com-
petitive throughout this pH range and even at pH 8.8
compound 6 is still a good inhibitor with a Ki of
6.8 lM. It is unlikely that it is the tetrahedral hydrated
form of 6 that binds since the concentration of this spe-
cies should increase with increasing pH and one would
expect an increase in inhibition as the pH increases.
Moreover, this decrease in inhibition is not due to a de-
crease in the trigonal form of the inhibitor since its pKa
should be similar to that of 4-methylphenyl boronic acid
which is 9.3.37 The slight increase in Ki with pH found
with compound 6 is in contrast to the large increase in
Ki with pH exhibited by estrone phosphate whose Ki
increases over 50-fold between pH 7.0 and 8.5
(52 lM).15 This large increase is a result of the enzyme’s
preference to bind the monoanionic form of estrone
phosphate.
IC50s were determined for coumarin and chromenone
boronic acids 8 and 9 at pH 7.0. Both of these com-
pounds were relatively poor STS inhibitors with the cou-
marin having an IC50 of 171 lM and the chromenone
having an IC50 of 86 lM. We also determined the IC50
of chromenone 16 to see if the substitution of the pheno-
lic group in 16 with a boronic acid group had any effect
on inhibitor potency as found with compound 6.38 Sur-
prisingly, compound 16 exhibited an IC50 of 6 lM which
is 14 times less than its boronic acid analog 9. A more
detailed kinetic analysis revealed that it exhibits mixed
inhibition with a Ki of 4.6 lM and an aKi of 40 lM
and the inhibition is reversible. Thus, this compound is
much more potent than its boronic acid analog and al-
most as potent as steroid boronic acid 6. Like the com-
pounds reported by Poirier and coworkers (such as 4
and 5), chromenone 16 does not require a sulfate mimic
to exhibit good potency. Horvath and coworkers have
demonstrated the potential of the chromenone platform
for obtaining reversible STS inhibitors.39 These workers
examined a series of 6-substituted 2-(1-adamantyl)-4-
(thio)chromenones as STS inhibitors.39 Those bearing
non-ionizable or very weakly acidic moieties at the 6-po-
sition were modest reversible inhibitors of STS with Kis
generally greater than 50 lM. The 6-COOH derivatives
were the most potent of the series having Kis of about
500 nM. The 6-OH derivative was not examined.
Although chromenone 16 is 20-fold less potent than
compound 4 it is a smaller, non-steroidal compound
and could be used as a lead to the development of yet
more potent reversible STS inhibitors.
Our results do not allow us to determine whether a
reversible covalent adduct is formed between 6 and
STS. Although it is a good inhibitor, it does not exhibit
an affinity for STS that is orders of magnitude greater
than that of estrone sulfate. If it does form a reversible
adduct this adduct is not as stable as that observed
with the peptide boronic acid inhibitors of serine pro-
teases. Boronic acid inhibitors of serine proteases are
considered to be transition state analog inhibitors in
that they mimic the tetrahedral transition state formed
during the reaction. The reaction of STS with ES does
not proceed by a tetrahedral transition state. The tran-
sition state for the cleavage of the S–O bond of the
substrate probably resembles a trigonal bipyramidal
intermediate X as shown in Scheme 2. This may ex-
plain why compound 6 is not as potent an inhibitor
of STS as the peptide boronic acids are of serine pro-
teases. Nevertheless, compound 6 is still one of the best
estrone-based STS inhibitors bearing a sulfate surro-
gate ever reported.
In summary, we have prepared several novel steroidal
and non-steroidal boronic acids and examined them as
inhibitors of STS. The potency of these compounds
and their mode of inhibition varied dramatically. It ap-
pears that the boronic acid moiety must be attached to a
platform very closely resembling a natural substrate in
order for it to impart any beneficial effect on binding
affinity. Thus, estrone boronic acid 6 is a good compet-

V. Ahmed et al. / Bioorg. Med. Chem. 14 (2006) 8564–8573
8569
itive inhibitor of STS even at basic pH. The fact that this
compound exhibits a good affinity for STS even at basic
pHs may be significant in terms of obtaining the crystal
structure of an inhibitor-STS complex. The structure of
an STS-inhibitor complex has not yet been reported.
Such a structure would be very useful for rational inhib-
itor design. However, one of the potential difficulties in
obtaining the structure of an STS-inhibitor complex is
that the enzyme is crystallized at pH 8.5 where its affin-
ity for ligands is often far from optimal.30 However,
since 6 binds to STS with a good affinity at basic pHs,
this compound may enable one to obtain the structure
of an STS inhibitor complex and such studies are in pro-
gress.40 Compound 4, an estradiol derivative which, in
addition to the boronic acid group at the 3-position also
bears a benzyl group at the 17-position, was a potent
non-competitive STS inhibitor. However, the fact that
compound 7 and its phenolic precursor 4 exhibit a sim-
ilar affinity and are non-competitive inhibitors suggests
that these compounds bind in a region outside the active
site, possibly in the hydrophobic channel between the
two membrane-spanning helices. The coumarin and
chromenone boronic acids 8 and 9 were not good inhib-
itors. However, the chromenone precursor to 8, com-
pound 16, is a remarkably good STS inhibitor and
may be useful as a lead to the development of yet more
potent reversible STS inhibitors.
trometer. Chemical ionization (CI) mass spectra were
run in the positive mode using ammonia as ionizing
agent. Positive ion electrospray (ESI) mass spectra were
obtained with a Waters/Micromass QTOF Ultima
Global mass spectrometer. Infrared spectra were ob-
tained on a Perkin-Elmer Spectrum RX Fourier trans-
form spectrophotometer. Melting points were
determined on a Fisher–Johns melting point apparatus
and are uncorrected. Steroid sulfatase from human pla-
centa was purified as previously described.41
3.2. Syntheses
3.2.1. 3-Pinacolatoboroestra-1,3,5-(10)-triene-17-one (11).
To a mixture of estrone triflate 1024 (4.02 g, 10 mmol)
and Pd(dppf)Cl2–CH2Cl2 (320 mg, 0.39 mmol, 3.9 mol
%) in dioxane (50 ml) under argon were added Et3N
(8.4 ml, 60 mmol) and 4,4,5,5-tetramethyl-1,3,2-dioxa-
borolane (4.2 ml, 28.9 mmol). The reaction mixture
was heated at 100 °C for 16 h. The reaction was cooled
to room temperature and concentrated. Purification of
the residue by flash chromatography (elution with ethyl
acetate/hexane 1:3) gave compound 11 (2.969 g, 78%) as
a white solid: mp 193 °C; 1H NMR (300 MHz, CDCl3) d
7.58 (d, J = 8.0 Hz, 1H), 7.55 (s, 1H), 7.29 (d,
J = 7.8 Hz, 1H), 2.95–2.88 (m, 2H), 2.65–2.40 (m, 2H),
2.40–2.25 (m, 1H), 2.20–1.90 (m, 4H), 1.70–1.35 (m,
6H), 1.32 (s, 12 H), 0.89 (s, 3H); 13C NMR (75 MHz,
CDCl3) d 220.6, 143.1, 135.8, 135.6, 132.2, 124.7,
83.6, 50.6, 47.9, 44.7, 38.0, 35.8, 31.6, 29.1, 26.5, 25.6,
24.8, 21.6, 13.8; LRMS (EI) m/z (%) 381 (M+1, 25),
380 (M+, 100), 379 (24), 323 (13), 294 (30), 281 (24);
HRMS (EI) calcd for C24H33BO3 380.2523; found
380.2531.
3. Experimental
3.1. General methods
All starting materials and reagents were obtained from
Aldrich Chemical Company. THF and dioxane were
distilled from sodium-benzophenone. CH2Cl2 was dis-
tilled from calcium hydride under nitrogen. Silica-gel
3.2.2. Estra-1,3,5-(10)-triene-17-one-3-boronic acid (6).
To a mixture of estrone boronate 11 (1.14 g, 3 mmol) in
acetone (450 ml) was added a solution of ammonium
acetate (924 mg, 12 mmol) and sodium periodate
(2.568 g, 12 mmol) in H2O (360 ml). The reaction mix-
ture was stirred for 10 days at room temperature. After
removal of acetone by rotary evaporation, the remain-
ing aqueous solution was extracted with ethyl acetate.
The combined extracts were dried over sodium sulfate
and concentrated. Purification of the residue by flash
chromatography (elution with acetone/hexane 1:3) gave
boronic acid 6 (839 mg, 84%) as a white solid: mp 181–
˚
chromatography was performed using silica gel 60 A
(230–400 mesh) obtained from Silicycle (Laval, Quebec,
Canada). 1H, 13C, 11B, and 19F NMR spectra were
recorded on a Bruker Avance 300 spectrometer. NMR
spectra are reported in parts per million (ppm) relative
to internal standards or solvent peaks. For NMR spec-
tra run in CDCl3, chemical shifts (d) for 1H NMR spec-
tra are reported relative to internal Me4Si (d 0.0 ppm),
chemical shifts for 13C spectra are relative to the residual
solvent peak (d 77.0 ppm, central peak), 11B NMR are
relative to external triethylborate (d 0.0 ppm), and 19F
NMR relative to an external fluoroform (d 0.0 ppm).
For NMR spectra run in DMSO-d6, chemical shifts
for 1H NMR spectra are reported relative to the residual
solvent peak (d 2.49 ppm, central peak), chemical shifts
for 13C spectra are relative to the residual solvent peak
(d 39.5 ppm, central peak), and 11B NMR are relative
to external triethylborate (d 0.0 ppm). For NMR spectra
run in CD3OD, chemical shifts for 1H NMR spectra are
reported relative to the residual solvent peak (d 3.31,
central peak), chemical shifts for 13C spectra are relative
to the residual solvent peak (d 49.00, central peak), and
11B NMR are relative to external triethylborate (d
0.0 ppm). Low-resolution (LRMS) and high-resolution
(HRMS) electron impact (EI) mass spectra were ob-
tained on a JEOL HX110 double focusing mass spec-
1
182 °C; H NMR (300 MHz, DMSO-d6) d 7.77 (s, 2H,
B(OH)2), 7.48 (d, J = 7.5 Hz, 1H), 7.45 (s, 1H), 7.18
(d, J = 7.5 Hz, 1H), 2.80 (br s, 2H), 2.40–1.85 (m, 6H),
1.74 (br s, 1H), 1.65–1.20 (m, 6H), 0.78 (s, 3H); 13C
NMR (75 MHz, DMSO-d6) d 220.0, 141.9, 135.4,
135.3, 131.2, 124.6, 50.2, 47.7, 44.5, 38.1, 35.8, 31.8,
29.3, 26.5, 25.7, 21.6, 13.9; LRMS (ESI) m/z (%) 299
(M+1, 100), 298 (M+, 25), 281 (27), 231 (82); HRMS
(ESI) calcd for C18H24BO3 298.1855; found 298.1862.
3.2.3. 3-O-[(Trifluoromethylsulfonyl)oxy]-17a-benzyl-17b-
trifluoroacetyloxyestra-1,3,5-(10)-triene (12). To
a
solution of estradiol 49 (2.888 g, 8 mmol) and 4-(dimeth-
ylamino)pyridine (2.44 g, 20 mmol) in methylene chlo-
ride (120 ml) at 0 °C was added trifluoroacetic
anhydride (2.7 ml) over 30 min. The mixture was stirred

8570
V. Ahmed et al. / Bioorg. Med. Chem. 14 (2006) 8564–8573
for 7 h at room temperature before quenching with
water. The layers were separated and the aqueous phase
was extracted with methylene chloride and the combined
organics were dried over Na2SO4. After removal of sol-
vent, the residue was dissolved in ethyl acetate (150 ml)
and methanol (30 ml). 40 ml of 1 N HCl was added and
the resulting mixture was stirred 1 h at room tempera-
ture. Reaction mixture was diluted with water and
extracted with ethyl acetate. The combined extracts were
dried over Na2SO4 and concentrated. Flash chromatog-
raphy (elution with ethyl acetate/hexane 1:8 to 1:6) affor-
ded crude 17-trifluoroacetoxy estrone as a white foam.
This material was dissolved in methylene chloride
(120 ml) and N,N-dimethylaminopyridine (1.22 g,
10 mmol) was added. The resulting mixture was cooled
to 0 °C and triflic anhydride (1.5 ml, 8.9 mmol) was add-
ed over 10 min. After stirring for 1 h at 0 °C, the reac-
tion mixture was quenched with ice-cold water and
extracted with methylene chloride. The combined ex-
tracts were washed with water, brine, dried over
Na2SO4, and concentrated. Purification of the residue
by flash chromatography (elution with ethyl acetate/hex-
ane 1:8) gave compound 12 (3.13 g, 67%) as a white sol-
id: mp 112–113 °C; 1H NMR (300 MHz, CDCl3) d 7.35–
7.24 (m, 4 H), 7.11 (d, J = 7.1 Hz, 2H), 7.02
(d, J = 8.8 Hz, 1H), 6.97 (s, 1H), 3.90 (d, J = 14.6 Hz,
1H), 2.96–2.85 (m, 2H), 2.77 (d, J = 14.6 Hz, 1H),
2.40–2.20 (m, 4H), 2.00–1.70 (m, 4H), 1.70–1.35 (m,
5H), 0.92 (s, 3H); 13C NMR (75 MHz, CDCl3) d 156.8
(q, J = 38 Hz, CF3C@O), 147.6, 140.3, 139.2, 135.8,
130.4, 128.4, 127.2, 126.9, 121.2, 118.8 (q, J = 333 Hz,
CF3), 118.2, 114.6 (q, J = 285 Hz, CF3), 99.9, 50.5,
48.2, 43.6, 38.9, 37.8, 33.0, 32.7, 29.4, 26.9, 26.1, 23.0,
14.2; 19F NMR (282 MHz, CDCl3) d À72.7, À75.1;
LRMS (CI) m/z (%) 608 (M+18, 25), 494 (40), 477
(87), 476 (100), 461 (29), 385 (70), 329 (41); HRMS
CDCl3) d 33.2; LRMS (EI) m/z (%) 568 (M+, 8), 478
(9), 454 (100), 439 (28), 363 (62), 267 (32); HRMS (EI)
calcd for C33H40BF3O4 568.2972; found 568.2972.
3.2.5. 3-O-Pinacolatoboro-17a-benzyl-17b-hydroxyestra-
1,3,5-(10)-triene (14). To a solution of 13 (480 mg,
0.845 mmol) in THF (240 ml) at room temperature
was added 0.8 N NaOH (24 ml) slowly over 10 min.
After stirring for 10 min, water (60 mL) was added
and the mixture was extracted with Et2O. The combined
extracts were washed with water, brine, dried over
Na2SO4, and concentrated. Purification of the residue
by flash chromatography (elution with ethyl acetate/hex-
ane, 1:6 to 1:5) gave 14 (335 mg, 84%) as a white solid:
mp 211–213 °C; 1H NMR (300 MHz, CDCl3) d 7.63
(d, J = 7.7 Hz, 1H), 7.59 (s, 1H), 7.37–7.24 (m, 6H),
3.00–2.90 (m, 3H), 2.95 (d, J = 12.8 Hz, 1H), 2.71–2.66
(m, 1H), 2.30–2.20 (m, 1H), 2.05–1.91 (m, 2H), 1.80–
1.50 (m, 7H), 1.50–1.25 (m, 2H, overlapping), 1.36
(s, 12H, overlapping), 0.97 (s, 3H); 13C NMR
(75 MHz, CDCl3) d 143.8, 138.5, 136.0, 135.7, 132.2,
131.1, 128.4, 126.3, 126.0 (br, C–B), 124.8, 83.6, 83.0,
49.8, 46.9, 44.7, 42.5, 39.4, 33.7, 31.5, 29.4, 27.6, 26.1,
25.0, 23.4, 14.6; 11B NMR (96 MHz, CDCl3) d 33.7;
LRMS (EI) m/z (%) 472 (M+, 8), 454 (7), 381 (96), 380
(100), 363 (79), 323 (18), 237 (22); HRMS (EI) calcd
for C31H41BO3 472.3149; found 472.3151.
3.2.6. 17a-Benzyl-17b-hydroxyestra-1,3,5-(10)-triene-3-
boronic acid (7). To a solution of 14 (47 mg, 0.1 mmol)
and phenylboronic acid (13 mg, 0.105 mg) in THF
(8 ml)/MeOH (3 ml) was added 2 N HCl (2 ml). The
resulting mixture was stirred overnight and then
quenched with water. The mixture was extracted with
ethyl acetate and the combined extracts were washed
with water, brine, dried over Na2SO4, and concentrated.
Purification of the residue by flash chromatography
(elution with acetone/hexane 1:4 to 1:2) gave 7 (26 mg,
67%) as a white solid: mp 216–218 °C; 1H NMR
(300 MHz, DMSO-d6 with one drop of D2O) d 7.46
(d, J = 7.4 Hz), 7.41 (s, 1H), 7.20–7.05 (m, 6H), 3.10
(s, 2H, B(OH)2), 2.80–2.75 (m, 3H), 2.54 (d,
J = 13.2 Hz, 1H), 2.35–2.25 (m, 1H), 2.25–2.15 (m,
1H), 1.85–1.75 (m, 1H), 1.70–1.20 (m, 10H), 0.78 (s,
3H); 13C NMR (75 MHz, DMSO-d6 with one drop of
D2O) d 142.6, 139.9, 135.5, 135.3, 131.8, 131.6, 131.0
(br, C-B), 127.8, 126.0, 124.7, 82.6, 49.5, 47.2, 44.4,
42.4, 39.7, 32.2, 31.3, 29.5, 27.6, 26.2, 23.3; LRMS
(ESI, +LiOAc) 787 (2M+Li, 100), 397 (M+Li, 47);
HRMS (ESI) calcd for C25H31LiBO3 396.2563; found
396.2549.
+
(ESI) calcd for C28H28F6O5S+NH4 608.1905; found
608.1927.
3.2.4. 3-O-Pinacolatoboro-17a-benzyl-17b-trifluoroace-
tyloxyestra-1,3,5-(10)-triene (13). To a mixture of triflate
12 (1.60 g, 2.71 mmol) and Pd(dppf)Cl2–CH2Cl2
(110 mg, 0.135 mol, 5 mmol %) in dioxane (15 ml) under
argon was added Et3N (3.4 ml, 24 mmol) and 4,4,5,5-
tetramethyl-1,3,2-dioxaborolane (1.7 ml, 11.7 mmol).
The reaction mixture were heated at 92–96 °C for 3 h,
cooled to room temperature, diluted with water, and
extracted with ethyl acetate. The combined extracts were
dried over Na2SO4 and concentrated. Purification of the
residue by flash chromatography (elution with ethyl ace-
tate/hexane, 1:10) gave compound 13 (952 mg, 62%) as a
white solid: mp 143–144 °C ; 1H NMR (300 MHz,
CDCl3) d 7.59 (d, J = 7.8 Hz, 1H), 7.54 (s, 1H), 7.36–
7.27 (m, 4H), 7.11 (d, J = 6.5 Hz, 2H), 3.87
(d, J = 14.7 Hz, 1H), 2.92–2.88 (m, 2H), 2.78
(d, J = 14.7 Hz, 1H), 2.55–2.20 (m, 4H), 2.00–1.75
(m, 4H), 1.70–1.40 (m, 5H), 1.32 (s, 12H), 0.90 (s, 3H);
13C NMR (75 MHz, CDCl3) d 156.7 (q, J = 41 Hz,
C@O), 143.2, 136.0, 135.7, 135.7, 132.3, 130.5, 128.4,
126.9, 126.2 (br, C–B), 124.8, 114.8 (q, J = 286 Hz,
CF3), 100.1, 83.6, 50.7, 48.3, 44.2, 39.2, 38.0, 33.2,
32.8, 29.3, 27.4, 26.1, 24.9, 23.0, 14.3; 19F NMR
(282 MHz, CDCl3) d À75.1; 11B NMR (96 MHz,
3.2.7. 3-[(trifluoromethylsulfonyl)oxy]-6-oxo-8,9,10,11-
tetrahydro-7H-cylohepta[c][1]-benzopyran (17). To
a
solution of coumarin 1527 (3.00 g, 13.0 mmol) and
4-(dimethylamino)pyridine (400 mg, 3.28 mmol) in
methylene chloride (100 ml) at 0 °C were added 2,6-luti-
dine (3.05 ml, 26 mmol) then triflic anhydride (2.65 ml,
15.6 mmol) over 10 min. The reaction mixture was stir-
red for 1 h at 0 °C then quenched with ice and 0.5 N
HCl (30 ml). The layers were separated and the aqueous
phase was extracted with methylene chloride. The com-
bined organics were washed with 0.5 N HCl, 5% NaH-

V. Ahmed et al. / Bioorg. Med. Chem. 14 (2006) 8564–8573
8571
CO3, dried over Na2SO4, and then concentrated. Purifi-
cation of the residue by flash chromatography (elution
with ethyl acetate/hexane 1:4) gave triflate 17 (4.562 g,
97%) as a white solid: mp 86–87 °C; 1H NMR
(300 MHz, CDCl3) d 7.73 (d, J = 8.9 Hz, 1H), 7.21 (d,
J = 2.0 Hz, 1H), 7.18 (dd, J = 8.9 Hz, 2.0 Hz, 1H),
2.95–2.87 (m, 4H), 1.94–1.85 (m, 2H), 1.70–1.55 (m,
4H); 13C NMR (75 MHz, CDCl3) d 160.9, 152.9,
152.4, 149.8, 129.8, 125.8, 119.9, 118.6 (q, J = 315 Hz,
CF3), 117.0, 110.1, 31.7, 28.1, 26.8, 25.3, 24.7; 19F
NMR (282 MHz, CDCl3) d À72.3; LRMS (EI) m/z
(%) 362 (M+, 60), 347 (7), 230 (15), 229 (100), 201
(18); HRMS (EI) calcd for C15H13F3O5S 362.0436;
found 362.0438.
(305 mg, 2.5 mmol) in methylene chloride (80 ml) at
0 °C were added 2,6-lutidine (2.33 ml, 20 mmol) then tri-
flic anhydride (2.02 ml, 12 mmol) over 10 min. After
addition, the reaction mixture was stirred for 1.5 h at
0 °C then quenched with ice and 0.5 N HCl (30 ml).
The layers were separated and the aqueous phase was
extracted with methylene chloride. The combined organ-
ics were washed with 0.5 N HCl, 5% NaHCO3, dried
over Na2SO4, and then concentrated. Purification of
the residue by flash chromatography (elution with ethyl
acetate/hexane 1:2 to 1:1) gave chromenone triflate 18
(1.76 g, 50%) as a white solid and recovered starting
material (350 mg, 16%): mp 88–89 °C; 1H NMR
(300 MHz, CDCl3) d 7.98 (s, 1H), 7.52 (t, J = 9.3 Hz,
2H), 6.24 (s, 1H), 1.29 (s, 9H); 13C NMR (75 MHz,
CDCl3) d 177.2, 176.8, 155.5, 145.8, 126.7, 124.5,
120.5, 118.6 (q, J = 315 Hz, CF3), 117.9, 106.5, 36.5,
27.7; 19F NMR (282 MHz, CDCl3) d À72.5; MS (EI)
m/z (%) 350 (M+, 65), 217 (100); HRMS (EI) calcd for
C14H13F3O5S 350.0436; found 350.0442.
3.2.8. 3-Pinacolatoboro-6-oxo-8,9,10,11-tetrahydro-7H-
cylohepta-[c][1]benzopyran (19). To a mixture of couma-
rin triflate 17 (1.81 g, 5 mmol), bis(pinacolato)diboron
(1.435 g, 5.65 mmol), Pd(dppf)Cl2–CH2Cl2 (123 mg,
0.15 mmol, 3 mol%), and dry KOAc (735 mg, 7.5 mmol)
under argon was added dioxane (30 ml). The resulting
mixture was heated at 85–90 °C overnight. After cooling
to room temperature, the reaction mixture was loaded
directly onto a silica column. Purification by flash chro-
matography (elution with 100% hexane, then ethyl ace-
tate/hexane 1:4) afforded boronate 19 (1.41 g, 83%) as a
white solid: mp 119–120 °C; 1H NMR (300 MHz,
CDCl3) d 7.63 (s, 1H), 7.59 (d, J = 8.6 Hz, 1H), 7.55
(d, J = 8.6 Hz, 1H), 2.90–2.82 (m, 4H), 1.83 (quint,
J = 5.5 Hz, 2H), 1.62–1.49 (m, 4H); 13C NMR
(75 MHz, CDCl3) d 161.8, 153.1, 151.9, 129.8, 129.7,
123.0, 122.8, 121.8, 84.2, 31.9, 27.8, 26.8, 25.5, 24.9,
24.8; 11B NMR (96 MHz, CDCl3) d 30.4; LRMS (EI)
m/z (%) 340 (M+, 100), 339 (30), 325 (19), 312 (17),
254 (10), 241 (18); HRMS (EI) calcd for C20H25BO4
340.1846; found 340.1838.
3.2.11. 2-tert-Butyl-6-pinacolatoboro-4H-1-benzopyran-
4-one (20). To a mixture of chromenone triflate 18
(1.044 g, 3 mmol), bis(pinacolato)diboron (861 g,
3.39 mmol), Pd(dppf)Cl2–CH2Cl2 (74 mg, 0.09 mmol,
3 mol%) and dry potassium acetate (441 mg, 4.5 mmol)
under argon was added dioxane (18 ml). The resulting
mixture was heated at 85–90 °C for 16 h. After cooling
to room temperature, the reaction mixture was loaded
onto a silica column. Purification by flash chromatogra-
phy (elution with 100% hexane, then ethyl acetate/hex-
ane 1:4) afforded chromenone boronate 20 (725 mg,
1
75%) as a light yellow solid: mp 135–136 °C; H NMR
(300 MHz, CDCl3) d 8.61 (s, 1H), 7.98 (d, J = 8.4 Hz,
1H), 7.36 (d, J = 8.4 Hz, 1H), 6.22 (s, 1H), 1.29 (s,
21H); 13C NMR (75 MHz, CDCl3) d 178.7, 175.8,
158.3, 139.1, 133.2, 122.6, 117.0, 107.0, 84.0, 36.4,
27.8, 24.8; 11B NMR (96 MHz, CDCl3) d 30.4; MS
(EI) m/z (%) 328 (M, 100), 327 (95), 313 (75), 285(48),
271 (39), 229 (75); HRMS (EI) calcd for C19H25BO4
328.1846; found 328.1852.
3.2.9. 6-Oxo-8,9,10,11-tetrahydro-7H-cylohepta-[c][1]ben-
zopyran-3-boronic acid (8). To a mixture of coumarin
boronate 19 (342 mg, 1 mmol) in acetone (150 ml) was
added a solution of ammonium acetate (308 mg,
4 mmol) and sodium periodate (856 mg, 4 mmol) in
H2O (120 ml). The reaction mixture was stirred over-
night at room temperature. After removal of acetone
by rotary evaporation, the residue was extracted with
ethyl acetate and then concentrated. Purification of the
residue by flash chromatography (elution with ethyl ace-
tate/hexane 1:1 to 100% ethyl acetate) gave coumarin
boronic acid 8 (172 mg, 67%) as a white solid:
3.2.12.
2-tert-Butyl-4H-1-benzopyran-4-one-6-boronic
acid (9). To a mixture of chromenone boronate 20
(328 mg, 1 mmol) in acetone (150 ml) was added a solu-
tion of ammonium acetate (308 mg, 4 mmol) and sodi-
um periodate (856 mg, 4 mmol) in H2O (120 ml). The
reaction mixture was stirred overnight at room temper-
ature. After removal of acetone by rotary evaporation,
the residue was extracted with ethyl acetate and then
concentrated. Purification of the residue by flash chro-
matography (elution with ethyl acetate/hexane 1:1 to
100% ethyl acetate) gave chromenone boronic acid 9
(189 mg, 77%) as a white solid: mp 176–178 °C; 1H
NMR (300 MHz, DMSO-d6 with one drop of D2O) d
8.47 (s, 1 H), 8.28 (s, 2 H, partially exchanged with
D2O, B(OH)2), 8.09 (d, J = 8.2 Hz, 1H), 7.53 (d,
J = 8.3 Hz, 1H), 6.07 (s, 1H), 1.27 (s, 9H); 13C NMR
(75 MHz, DMSO-d6 with one drop of D2O) d 178.0,
176.0, 157.6, 140.0, 131.8, 122.3, 117.4, 106.7, 36.5,
27.8; 11B NMR (96 MHz, DMSO-d6 with one drop of
D2O) d 28.6; LRMS (ESI) m/z (%) 248 (M + 1, 12),
1
mp > 260 °C; H NMR (300 MHz, DMSO-d6 with one
drop of D2O) d 8.32 (s, 2H, B(OH)2), 7.82 (d,
J = 7.7 Hz, 1 H), 7.70–7.60 (m, 2H), 3.00 (m, 2H), 2.78
(m, 2H), 1.80 (m, 2H), 1.56 (m, 2H), 1.47 (m, 4H); 13C
NMR (75 MHz, DMSO-d6 with one drop of D2O) d
161.4, 153.9, 151.8, 130.0, 128.8, 123.9, 121.9, 120.9,
31.7, 27.5, 26.6, 25.5, 25.0; 11B NMR (96 MHz,
DMSO-d6 with one drop of D2O) d 30.3; LRMS (ESI)
m/z (%) 259 (M+1, 100), 258 (M+, 29); HRMS (ESI)
calcd for C14H15BO4 258.1178; found 258.1177.
3.2.10. 2-tert-Butyl-6-[(trifluoromethylsulfonyl)oxy]-4H-
1-benzopyran-4-one (18). To a solution of chromenone
1628 (2.16 g, 10.0 mmol) and 4-(dimethylamino)pyridine

8572
V. Ahmed et al. / Bioorg. Med. Chem. 14 (2006) 8564–8573
247 (M+, 100); HRMS (ESI) calcd for C13H15BO4
246.1178; found 246.1185.
tion of [I]; Vo is the velocity in the absence of inhibitor;
B is background and s is the slope factor.
3.3. Inhibition studies
References and notes
3.3.1. Determination of Ki and aKi values for compounds
4, 6, 7, and 16. An appropriate volume of a MUS stock
solution in 0.1 M Tris–HCl of desired pH was added to
the wells of a 96-well microtiter plate containing 0.1 M
Tris–HCl buffer of the same pH such that the total vol-
ume was 80 ll. To the wells was added 10 ll of a stock
solution of inhibitor in 50% DMSO. For a control, 10 ll
of 50% DMSO was added instead. The assay was initi-
ated by the addition of 10 ll STS (115 nM stock solu-
tion in 20 mM Tris–HCl, pH 7.4, 0.1% Triton X-100).
To detect non-enzymatic hydrolysis of the substrate
10 ll of enzyme storage buffer was added instead. The
final volume of the assay was 100 ll. The final concen-
tration of buffer was 92 mM TrisHCl, 0.01% Triton
X-100, and 5% DMSO. The final enzyme concentration
was 11.5 nM. For studies at pH 7.0, 7.5, and 8.0, the fi-
nal concentration of MUS was 83.3–500 lM, or approx-
imately 0.5–3 times the Km value at pH 7 and 7.5
(145 lM at pH 7.0; 170 lM at pH 7.4) and 0.25–1.5
times the Km value at pH 8.0 (338 lM). For studies at
pH 8.5 and 8.8, the final concentration of MUS ranged
between 100 and 2500 lM or approximately 0.1–3 times
the Km value (830 lM, pH 8.5; 980 lM, pH 8.8). The
final concentration of inhibitor was 0.5–4 times Ki.
The reactions were followed by detection of fluorescent
product, 4-methylumbelliferone (excitation 360 nm,
emission, 460 nm), over 10 min using a Spectramax
GeminiXS plate reader (Molecular Devices, CA) at
30 °C. Each reaction was performed in triplicate. Con-
trols were performed in an identical manner but did
not contain STS. Initial rates (v) were determined by
taking the slopes of plots of the change in relative fluo-
rescence units with time. These data were plotted as
Lineweaver–Burk graphs and Ki values were calculated
from replots of the slopes or intercepts of the Linewe-
aver–Burk graphs according to the equations for mixed
and competitive inhibition.
1. Reed, M. J.; Purohit, A.; Woo, L. W.; Newman, S. P.;
Potter, B. V. Endocr. Rev. 2005, 26, 171–202.
2. For recent reviews see (a) Nussbaumer, P.; Billich, A.
Med. Res. Rev. 2004, 24, 529–576; (b) Winum, J. Y.;
Scozzafava, A.; Montero, J. L.; Supuran, C. T. Med. Res.
Rev 2005, 25, 186–228.
3. Caution must be exercised when comparing the potency of
STS inhibitors with one another since the conditions under
which STS inhibitors are examined vary tremendously
from study to study in terms of quality of the ES
preparation, the type of assay and the specific assay
conditions. Moreover, the modality of inhibition (com-
petitive, noncompetitive, etc.) was rarely determined.
4. Purohit, A.; Williams, G. J.; Howarth, N. M.; Potter, B.
V.; Reed, M. J. Biochemistry 1995, 34, 11508–11514.
5. Woo, L. W. L.; Purohit, A.; Malini, B.; Reed, M. J.;
Potter, B. V. L. Chem. Biol. 2000, 7, 773–791.
6. Nussbaumer, P.; Lehr, P.; Billich, A. J. Med. Chem. 2002,
45, 4310–4320.
7. Stanway, S. J.; Purohit, A.; Woo, L. W.; Sufi, S.; Vigushin,
D.; Ward, R.; Wilson, R. H.; Stanczyk, F. Z.; Dobbs, N.;
Kulinskaya, E.; Elliott, M.; Potter, B. V.; Reed, M. J.;
Coombes, R. C. Clin. Cancer Res. 2006, 12, 1585–1592.
8. Poirier, D.; Boivin, R. P. Bioorg. Med. Chem. Lett. 1998,
8, 1891–1896.
9. Boivin, R. P.; Luu-The, V.; Lachance, R.; Labrie, F.;
Poirier, D. J. Med. Chem. 2000, 43, 4465–4478.
10. Nussbaumer, P.; Horvath, A.; Lehr, P.; Wolff, B.; Geyl,
D.; Billich, A. Bioorg. Med. Chem. Lett 2003, 13, 3673–
3677.
11. Jutten, P.; Schumann, W.; Har¨tl, A.; Heinisch, L.; Grafe,
U.; Werner, W.; Ulbricht, H. Bioorg. Med. Chem. Lett.
2002, 12, 1339–1342.
12. Lee, W.; DeRome, M., DeBear, J.; Noell, S., Epsatein, D.,
Mahle, C., DeCarr, L., Woodruff, K., Huang, Z., Dumas,
J., Aryl piperazines: A new class of steroid sulfatase
inhibitors for the treatment of hormone-dependent breast
cancer. Abstract of Papers, 226th ACS National Meeting,
New York, NY, USA, Sept. 7–11, 2003, MEDI-301.
13. An exception is estrone phosphate which has been shown
to be a reversible competitive inhibitor with pure STS and
partially purified STS. It exhibits a Ki of about 1 lM at pH
7.0 and 52 lM at pH 8.5. See Refs. 14 and 15.
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3.3.2. Determination of IC50s for compounds 8, 9, and 16.
Ten microliters of inhibitor stock solutions in 50%
DMSO were added to the wells of a 96-well microtiter
plate containing 70 ll of 0.1 M Tris, pH 7.0. Ten micro-
liters of a 2.0 mM MUS stock solution in 0.1 M Tris–
HCl, pH 7.0, was added. The assay was initiated by add-
ing 10 lL STS (115 nM stock solution in 20 mM Tris–
HCl, pH 7.4, 0.1% Triton X-100). The concentration
of inhibitor ranged from 7 to 250 lM. The final concen-
tration of MUS was 200 lM. The reaction was followed
as described above. Eleven concentrations of inhibitor
bracketing the IC50 value were used for each compound.
The initial rates of enzyme activity in relative fluores-
cence units per second (RFU/s) were used to determine
the IC50. The ratio of the initial rate in the presence of
inhibitor (Vi) to that in the absence of inhibitor (Vo)
was calculated and plotted as a semi-log curve in Grafit,
from which the IC50 value was calculated based on the
following equation: Vi = Vo/[1 + ([I]/IC50)S] + B, where:
Vi is the initial rate of reaction at an inhibitor concentra-
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