2-(1-Adamantyl)-4-(thio)chromenone-6-carboxylic acids: Potent reversible inhibitors of human steroid sulfatase

Article abstract of DOI:10.1021/jm0407916

Steroid sulfatase (STS) is an attractive target for the potential therapy of a number of estrogen- and androgen-dependent disorders. Most potent STS inhibitors known so far act as irreversible enzyme blockers and feature an aryl sulfamate moiety; even min

Full text of DOI:10.1021/jm0407916

4268
J . Med. Chem. 2004, 47, 4268-4276
2-(1-Ad a m a n tyl)-4-(th io)ch r om en on e-6-ca r boxylic Acid s: P oten t Rever sible
In h ibitor s of Hu m a n Ster oid Su lfa ta se
Amarylla Horvath,* Peter Nussbaumer, Barbara Wolff, and Andreas Billich
Novartis Institute for Biomedical Research Vienna, Brunnerstrasse 59, A-1235 Vienna, Austria
Received February 23, 2004
Steroid sulfatase (STS) is an attractive target for the potential therapy of a number of estrogen-
and androgen-dependent disorders. Most potent STS inhibitors known so far act as irreversible
enzyme blockers and feature an aryl sulfamate moiety; even minor modifications at the
sulfamate group result in drastically decreased activity. On the basis of a recently reported
subclass of highly potent STS inhibitors, i.e., chromenone sulfamates, we now extended the
investigation of structure-activity relationships to hitherto unstudied sulfamate replacements.
Thereby, we discovered 2-(1-adamantyl)-4-(thio)chromenone-6-carboxylic acids (5d and 5j) as
potent, reversible inhibitors of STS. In a cell-free system using purified human STS, both new
inhibitors show similar Ki values (0.50 µM and 0.53 µM, respectively). However, the thio
analogue 5j is superior to 5d (IC50 ) 0.18 µM versus 9.4 µM) in a cellular assay system using
CHO cells overexpressing STS. Compound 5j is an example of a reversible STS inhibitor with
potent activity toward the target enzyme in a cellular test system. Moreover, 5d ,j are stable
and have no estrogenic potential.
In tr od u ction
sulfamate (EMATE, Figure 1),14 the sulfamate analogue
of the natural substrate estrone sulfate, has served as
the lead for all sulfamate-type inhibitors.10,15 One issue
associated with this type of compounds is their chemical
instability in solution (as reported for estrone sulfam-
ate),16-18 which might complicate or even impede de-
velopment and clinical use. Hydrolytic lability appears
to be an inherent problem for all aryl sulfamates, due
to the reactivity of the aryl sulfamate functionality.
All attempts to replace the sulfamate moiety in
EMATE by other functionalities reported in the litera-
ture19 have so far resulted in compounds with substan-
tially decreased or no STS inhibitory potency (examples
are summarized under general structures 2 and 3 in
Figure 1). We realized that in nearly all investigated
structures the steroidal skeleton (or a mimic thereof)
was connected to the headgroup functionality via a
heteroatom (O in structures 2a -f, S and N in structures
3a -k ). Therefore, we now explored sulfamate replace-
ments by introducing carbon-linked functional groups
(compounds 5a -j; Figure 1). For this study, the 2-ada-
mantyl-4-(thio)chromenone-6-yl scaffold, which we suc-
cessfully applied in a series of highly potent sulfamate-
type STS inhibitors (4a ,b; Figure 1),20 was used instead
of the estrone skeleton. This led to the discovery of
4-(thio)chromenone carboxylic acids (5d ,j) as potent
nonsteroidal STS inhibitors with reversible mode of
action. In this paper, we describe the discovery, syn-
thesis and the biological activity of this novel class of
inhibitors.
Steroid sulfatase (STS), the enzyme catalyzing the
hydrolysis of steroid-3-O-sulfates, has emerged as an
attractive drug target, since it is crucial for the local
production of active estrogens and androgens from their
systemic circulating sulfated precursors, namely estrone
sulfate and dehydroepiandrosterone (DHEA) sulfate, in
diseased tissues.1,2 Three areas where STS inhibitors
could be used in therapy have been identified: (i) Breast
cancer. There is evidence that the STS pathway is a
major source of estrogens in breast tissue,1-4 suggesting
STS inhibitors as potential agents for the treatment of
estrogen receptor-positive breast tumors. (ii) Androgen-
dependent skin diseases. DHEAS is a precursor for
active androgens in the skin; an important role of STS
in the pathogenesis of androgenetic alopecia and acne
has been proposed.5,6 (iii) Cognitive dysfunction. In the
brain STS may regulate the availability of DHEA as a
neurohormone; treatment of rats with inhibitors of STS
resulted in enhanced learning and spatial memory.7-9
So far, development of STS inhibitors is mainly in
preclinical phases, and the search for STS inhibitors
which are suitable as therapeutics is actively ongoing
(see ref 10 for a recent review).
To date, both reversible and irreversible STS inhibi-
tors are known. As an example of steroidal compounds
with a reversible mode of action, the 17R-4′-tert-butyl-
benzyl derivative of estradiol (1, Figure 1) with high
inhibitory activity has been reported.11 Recently, new
classes of reversible inhibitors were discovered, namely
nortropinyl-arylsulfonylureas12 and substituted phenyl
piperazines.13 However, the majority of potent STS
inhibitors known to date feature an aryl sulfamate
moiety and act in an irreversible manner. Estrone
Resu lts a n d Discu ssion
Ch em istr y. For the synthesis of the chromenone-
based test compounds, we started from the acyclic
precursor (6a -c) and constructed the bicyclic core
decorated with the 1-adamantyl residue at position 2
and three different substituents at position 6 (9a ) Me,
9b ) CH2COOMe, and 5c ) COOMe, Scheme 1). These
* To whom correspondence should be addressed. Phone: ++43-1-
86634-441, Fax. ++43-1-86634-354, E-mail: amarylla.horvath@
pharma.novartis.com.
10.1021/jm0407916 CCC: $27.50 © 2004 American Chemical Society
Published on Web 07/09/2004

Reversible Inhibitors of Human Steroid Sulfatase
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17 4269
F igu r e 1. Structures of estrone sulfamate (EMATE), 17-R-(tert-butylbenzyl)estradiol (1), previously described (refs 16a-h) O-linked
estrone derivatives 2a -f, S-, N-, C-linked estrone derivatives 3a -k , chromenone-based sulfamates 4a ,b (ref 17), and new test
compounds 5a -j.
Sch em e 1a
a
Reagents and conditions: (a) adamantane-1-carbonyl chloride, DMAP, pyridine, rt, 18 h, 83-99%; (b) K2CO3, 2-propanol, reflux, 75
min; (c) HCOOH, 100 °C, 45 min, 52-84% over two steps; (d) 20% aqueous HCl, dioxane, 4 h, 100 °C, 74-90%; (e) SOCl2, dichloroethane,
cat. DMF, reflux, 4 h, quant.; (f) NH3, EtOH, 0 °C, 30 min., 37%.
central intermediates were then transformed into test
compounds 5a ,b,d -h by a variety of functional group
manipulations (Schemes 1-3). Adamantyl esters 7a -c
were prepared by acylation of the appropriately substi-
tuted 4-hydroxy-3-acetophenones 6a -c with adaman-
tane-1-carbonyl chloride in the presence of (dimethyl-
amino)pyridine in pyridine. Subsequent base-catalyzed
Baker-Venkataraman rearrangement21 provided the
1,3-diketones 8a -c, which were used without purifica-
tion in the next synthetic step. Cyclization with formic
acid was performed analogously to published proce-
dures22 and yielded the chromenones 5c and 9a ,b.
Treatment of 6-methyl chromenone 9a with N-bromo-
succinimide (NBS) and a catalytic amount of 2,2′-azobis-
(2-methylpropionitrile) (AIBN) in benzene afforded the
monobromide 10, which was converted into 5a using
Amberlite IRA 900 (carbonate form) exchange resin
(Scheme 2).23 Reaction of 9a with an excess of NBS
provided dibromide 11 in good yield, which was hydro-
lyzed with potassium acetate in acetic acid to the
aldehyde 5b. Compound 5b was also an intermediate
in the synthesis of the hydroxyacetyl derivative 5f.
Thus, methyl Grignard addition, followed by Swern
oxidation provided ketone 12, which after R-bromination

4270 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17
Horvath et al.
Sch em e 2a
a
Reagents and conditions: (a) 1.2 equiv of NBS, AIBN, benzene, reflux, 2 h, 72%; (b) 2.5 equiv of NBS, AIBN, benzene, reflux, 2 h,
84%; (c) Amberlite IRA 900, HCO3- form, toluene, 2 h, reflux, 67%; (d) CH3COOK, CH3COOH, 120°C, 12 h, 56%; (e) CH3MgI, THF, 0 °C,
1 h, 58%; (f) (COCl)2, DMSO, CH2Cl2, -68 °C, 1 h, then Et3N, 80%; (g) Br2, CH3COOH, 70 °C, 3 h, 68%; (h) (CH3)3N+CH2COO-, DMF,
CH2Cl2, EtOH; Na2CO3, H2O, rt, 24 h, 61%.
Sch em e 3a
a
-
Reagents and conditions: (a) NBS, AIBN, benzene, 75%; (b) Amberlite IRA 900 HCO3 form, toluene, reflux, 24 h, 59%; (c) DMSO,
(COCl)2, CH2Cl2, -68 °C, 1 h; then TEA, 59%; (d) 20% HCl, dioxane, 80 °C, 1.5 h, 91%.
Sch em e 4a
a
Reagents and conditions: (a) PPA, 90°C, 2 h, 34%; (b) Pd(OAc)2, Zn(CN)2, Ca(OH)2, PPh3, DMF, 100 °C, 1 h, 36%; (c) 20% HCl,
dioxane, 120 °C, 16 h, 50%.
using bromine in acetic acid gave compound 13. Treat-
ment with betaine and subsequent ester hydrolysis
yielded test compound 5f (Scheme 2). The carboxylic
acids 5d ,g were synthesized from the corresponding
esters 5c and 9b by hydrolysis using 20% aqueous HCl
in dioxane (Scheme 1). Conversion of the carboxylic acid
5c into the acid chloride by treatment with thionyl
chloride and subsequent reaction with ammonia in
ethanol gave amide 5e (Scheme 1). The oxo-acetic acid
group in 5h (Scheme 3) was introduced by first reacting
acetic acid methyl ester 9b with N-bromosuccinimide,
followed by hydrolysis with Amberlite IRA 900 carbon-
ate exchange resin to produce intermediate 14. Com-
pound 5h was then obtained by Swern oxidation and
subsequent acidic ester hydrolysis.
The thiochromenone-nitrile 5i (Scheme 4) was pre-
pared by condensation of commercially available 4-bromo-
benzenethiol (15) with 3-(adamantan-1-yl)-3-oxopro-
pionic acid ethyl ester (16) in polyphosphoric acid to give
the cyclic product 17,24 followed by palladium-mediated
cyanation with zinc cyanide in the presence of calcium
hydroxide. Acid-catalyzed hydrolysis of the nitrile 5i
gave the thiochromenone carboxylic acid 5j.
STS In h ibition Assa ys. As described earlier,20,25 we
used highly purified human STS obtained from a
recombinant cell line with 4-methylumbelliferyl sulfate

Reversible Inhibitors of Human Steroid Sulfatase
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17 4271
Ta ble 1. Effect of Inhibitors against Purified Human STS and
against STS Overexpressed in CHO Cells
(4-MUS) as a synthetic substrate for a convenient
colorimetric assay to determine IC50 or Ki values of
inhibitors.
Cellu la r STS Assa y. Cellular activities of both
reversible and irreversible inhibitors were evaluated in
CHO cells stably transfected with human STS (CHO-
STS) using the fluorogenic substrate 4-MUS, as de-
scribed.26
Sta bility Stu d ies in Aqu eou s Solu tion . To assess
potential degradation at physiological pH, test com-
pounds were dissolved in 0.1 M Tris-HCl buffer, pH
7.5/acetonitrile 1:1 (acetonitrile being included to en-
hance solubility), and solutions (100 µM) were incubated
at 37°C for up to 1 week. Aliquots were analyzed for
degradation by HPLC.
enzyme assay cellular assay
compound
X
R
Ki [µM]
IC50 [µM]
EMATE
4a
4b
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
-
O
S
-
0.67a
0.02b
0.026c
0.024c
∼30
a
OSO2NH2
OSO2NH2
CH2OH
CHO
0.19
0.21
a
O
O
O
O
O
O
O
O
S
32
>20
>50
>30
COOMe
COOH
>30
0.50
9.4
CONH2
COCH2OH
CH2COOH
C(O)COOH
CN
>50
3.2
>50
2.2
>50
0.53
>30
>30
>30
12.3
>30
S
COOH
0.18
a
These values were determined by Purohit et al.31 (for EMATE)
SAR. Several studies exploring the SAR around the
sulfamate moiety in prototype inhibitor EMATE and on
surrogates of the natural substrate estrone sulfate have
been published.19 In Figure 1, compounds 2 are ex-
amples for estrone derivatives with O-linked head-
groups and compounds 3a -j for S- and N-linked varia-
tions. The common result of these studies was that any
modification, even a minor one, such as alkylation at
the nitrogen atom of the sulfamate moiety (2a ),27 led to
a substantial decrease or complete loss of STS inhibitory
activity. The most active compound coming out of these
studies was analogue 2f featuring the methylthiophos-
phonate moiety as sulfate surrogate; but its activity (Ki
) 14.6 µM) did not approach that of the potent sulf-
amate-type inhibitors. However, it was shown that 2f
and the corresponding 3-O-phosponate of estrone (2d )
were able to inhibit STS activity in intact MCF-7 breast
cancer cells.19a Interestingly, all active compounds with
a modified headgroup were characterized as reversible
inhibitors of STS. The only derivative of EMATE, where
the modified headgroup is linked to the estrone skeleton
via a carbon atom and STS inhibitory activity has been
reported, is 3-desoxyestrone-3-methylenesulfonic acid
3k (Figure 1) with a high Ki value of 140 µM.19d
Searching for novel, potent, and stable STS inhibitors,
we used the potent compounds 4a ,b (Figure 1) as
starting points, based on our previous finding, that
4-(thio)chromenone 6-O-sulfamates with bulky alkyl
groups at position 2 are more potent than EMATE. We
prepared some analogues of the above-mentioned sulfate
surrogates (e.g., sulfonic acid, phosphonic acid, phos-
phate) using the 2-(1-adamantyl)-4-(thio)chromenone-
6-yl scaffold and basically confirmed the reported SAR
findings on estrone-based derivatives: the compounds
were inactive or only weakly active with IC50 values g
40 µM. The present report summarizes the results from
extended SAR studies focusing on chromenone ana-
logues with C-linked functionalities as headgroup (5a -
j, Figure 1). Their ability to inhibit human STS was
assessed in the enzyme assay using purified protein and
in the cellular assay using CHO-STS cells. The results
are summarized in Table 1 in comparison to prototype
inhibitor EMATE and chromenone sulfamates 4a ,b.
and Nussbaumer et al.20 (for 4a ,b) using the method of Kitz and
Wilson for irreversible inhibitors. This value was identified in
b
the cellular assay setups for both irreversible and reversible
inhibitors as described in Wolff et al.26 c These IC50 values were
determined in the CHO-STS assay for irreversible inhibitors,
using fixed cells.26
Also the carboxylic ester derivative 5c, which was an
intermediate in the synthesis of the corresponding acid
5d , was devoid of inhibitory activity. In contrast, car-
boxylic acid 5d showed marked inhibition in the enzyme
assay and was also active in the cellular system,
although the potency was not as pronounced as against
the purified enzyme. This difference might be due to
poor cell penetration properties of 5d , which is quite
frequently found for carboxylic acids. Amide derivative
5e again did not exhibit inhibitory activity.
In the next series of test compounds the chain length
was increased. Oxo alcohol 5f was moderately active
with a Ki value of 3.2 µM in the enzyme assay.
Surprisingly, carboxylic acid 5g, the homologue of 5d ,
was inactive, whereas the oxo derivative 5h again
showed inhibitory activity against the purified enzyme
in the low µM range. Although analogue 5h was inferior
to 5d in the enzyme assay roughly by a factor of 4, 5h
was almost as potent as 5d in the cellular assay.
Because of the interesting potency obtained with 5d ,
the corresponding thiochromenone analogue 5j was also
investigated. In the cell-free assay, 5j was as potent as
5d , but in the cellular test system compound 5j was
found to be substantially superior, displaying an IC50
value of 180 nM (compared to 9.4 µM for 5d ). The deter-
mining factor for this remarkable difference has not
been elucidated so far, but the increase in lipophilicity
caused by the exchange of the oxygen to sulfur might
result in enhanced cell penetration of the thio analogue.
The carboxylic acids 5d and 5j were analyzed in more
detail. The compounds were found to be reversible
inhibitors, as enzyme activity could be fully recovered
upon incubation of the enzyme-inhibitor complexes
with dextran-activated charcoal (data not shown). Ki-
netic measurements with STS and the substrate 4-MUS
in the presence of the carboxylic derivative 5d are
shown in Figure 2A, where data are plotted in an S/V
versus S diagram (Hanes plot). The linearity of the
secondary plot of Kapp/Vapp versus inhibitor concentra-
tion (I) (Figure 2B) indicates purely competitive inhibi-
tion, the Ki value being 0.50 µM. In analogy, Ki) 0.53
µM was measured for 5j. It is conceivable that the
chromenone moiety of these inhibitors binds in a fashion
We started our investigation with the primary alcohol
5a and increased stepwise the oxidation level of the
headgroup and the spacer length between the oxy
function and the chromenone scaffold. Alcohol 5a showed
only very weak inhibitory activity (Table 1), and alde-
hyde 5b was inactive at the highest test concentration.

4272 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17
Horvath et al.
indication for STS inhibitors, we asked whether a
chromenone carboxylic acid would also block the enzyme
in MCF-7 cells, a commonly used breast cancer cell line.
For compound 5d an IC50 value of 137 ( 15 nM was
determined. Thus, STS inhibitors of the carboxylic acid
type may also be useful to inhibit estrone sulfate-
dependent growth of breast cancer cells.
Estrogenicity is an issue associated with many STS
inhibitors which prevents their usefulness in estrogen-
dependent diseases, although more recently potent
nonestrogenic inhibitors have been identified.10,17,28
Derivatives 5d ,j did not show any affinity to the human
estrogen receptors R and â up to the highest test
concentration of 100 µM; for comparison, phenols 4a ,b,
as the hydrolytic products of the corresponding sulf-
amates, showed binding to the receptors with ED50
values in the range of 0.2 to 1.5 µM.17
Since the chromenone carboxylic acids represent a
new class of STS inhibitors, we also asked whether they
would exert any antiproliferative activity as a sign of
cellular toxicity. We used the human keratinocyte cell
line HaCaT as a sensitive assay system.32 There was
no effect of compounds 5d ,g on HaCaT cell proliferation
up to the highest test concentration of 30 µM.
Con clu sion s
By extending structure-activity relationship inves-
tigations to hitherto unstudied sulfamate replacements
on STS inhibitors featuring the 2-adamantyl-4-(thio)-
chromenone-6-yl scaffold, we discovered 2-(1-adaman-
tyl)-4-(thio)chromenone-6-carboxylic acids (5d and 5j)
as potent, reversible inhibitors of STS. All other modi-
fications, including variation of the oxidation state and
chain length, gave inactive or poorly active compounds.
The new inhibitors 5d and 5j show similar Ki values
(0.5 µM and 0.53 µM, respectively) against purified
recombinant human STS. In a cellular assay system
using CHO cells overexpressing STS, the thio analogue
5j is substantially superior to 5d (IC50 value ) 0.18 µM
versus 9.4 µM). Compound 5j is an example of a
reversible STS inhibitor showing potent activity toward
the target enzyme in a cellular test system.
F igu r e 2. Mode of action and Ki determination of inhibitor
5d . A: rate of substrate turnover (V) at various substrate
concentrations (S) in the presence of inhibitor 5d , plotted in a
Hanes diagram; B: secondary plot of Kapp/Vapp ratios against
inhibitor concentration (I).
similar to the active site of the enzyme as the A and B
ring of the steroid sulfates, while the carboxylic acid
moiety acts as a mimic of the sulfate group, both being
negatively charged at physiologic pH. Although not
formally proven, we assume that the weaker compounds
5a , 5f, and 5h also act as reversible, competitive
inhibitors.
When comparing the Ki values in Table 1, it has to
be taken into account that EMATE, 4a , and 4b are
irreversible inhibitors; thus, their efficacy is not only
determined by the binding to the active site (as indi-
cated by the Ki value), but also by the rate of covalent
modification of the enzyme (as indicated by the kinact
value).20 Therefore, the IC50 values for inhibition of STS
in the cellular assays by the sulfamates are lower by at
least 2 orders of magnitude than for the carboxylic acid
derivatives 5d and 5j.
Sta bility of Ch r om en on e Ca r boxylic Acid s a n d
Cor r esp on d in g Su lfa m a tes. One issue associated
with sulfamate-type STS inhibitors is their limited
stability in solution.16-18 Therefore, the stability of
carboxylic acid analogues 5d ,j and, as references, of
their corresponding sulfamates 4a ,b in buffered aqueous
solution at pH 7.5 was compared. Chromenone carbox-
ylic acid derivatives 5d ,j did not show any measurable
degradation during 1 week at 37 °C. In contrast,
sulfamates 4a and 4b both decomposed to the corre-
sponding phenols with half-lives of 36 and 20 h,
respectively.
Exp er im en ta l Section
A. Ch em ica l Syn th esis. 1. Gen er a l Meth od s. 1-(2-
Hydroxy-5-methylphenyl)ethanone (6a ) and 4-bromobenzene-
thiol (15) were purchased from Aldrich. 3-Acetyl-4-hydroxy-
benzoic acid methyl ester (6c) and 3-acetyl-4-hydroxybenzene-
acetic acid methyl ester (6b) were synthesized according to a
published procedure.22 Melting points were determined on a
Reichert Thermovar microscope and are uncorrected. All
reactions were monitored by thin-layer chromatography per-
formed by use of silica gel F254 plates (Merck), detected by
UV light or potassium permanganate. Column chromatogra-
phy was carried out on silica gel 60 (40-63 µm, Merck) using
pressures up to 5 bar with the indicated solvent system. The
NMR spectra were recorded at 250 MHz (Bruker WM 250)
and at 400 MHz (Bruker Avance 400 spectrometer) with
tetramethylsilane as internal standard. Chemical shifts are
given in δ units. All reactions were carried out under an
atmosphere of argon unless otherwise noted. Tetrahydro-
furan (THF) was obtained anhydrous by distillation over
LiAlH4; all other solvents were dried by storing over 3- or 4-Å
molecular sieves. Elemental analyses were performed by
Novartis Services AG and Solvias AG, respectively, both in
Basle, Switzerland. High-resolution mass spectra (HMRS)
were recorded on a Finnigan MAT900.
F u r th er Biologica l Eva lu a tion of Ch r om en on e
Ca r boxylic Acid s. Since breast cancer is an important

Reversible Inhibitors of Human Steroid Sulfatase
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17 4273
2. Gen er a l P r oced u r e for th e Syn th esis of Test Com -
p ou n d s 5c-e a n d 5g. (a ) Ad a m a n ta n e-1-ca r boxylic Acid
2-Acetyl-4-(m eth oxyca r bon yl)p h en yl Ester (7c). To a
solution of 3-acetyl-4-hydroxybenzoic acid methyl ester (6c,
1.86 g 10.22 mmol) and 4-(dimethylamino)pyridine (250 mg,
2.04 mmol) in anhydrous pyridine (18 mL) was added ada-
mantane 1-carbonyl chloride (2.44 g, 12.26 mmol) at room
temperature. After being stirred for 18 h, the mixture was
diluted with toluene (10 mL) and evaporated in vacuo. The
residue was partitioned between ethyl acetate (15 mL) and
water (5 mL), the organic layer was washed with saturated
aqueous sodium bicarbonate solution, water, and brine, and
dried over MgSO4. After evaporation of the solvent, the crude
product was purified by column chromatography (toluene/ethyl
acetate ) 4/1) to afford 7c (3.5 g, 99%) as colorless crystals;
precipitate was collected and washed with a water/dioxane
mixture (10:1) to give 5d (74%) as colorless crystals; mp 250-
251 °C. 1H NMR (CDCl3): δ 8.83 (d, J ) 2.1 Hz, 1H), 8.27 (dd,
J ) 2.1 + 8.8 Hz, 1H), 7.44 (d, J ) 8.8 HZ, 1H), 6.17 (s, 1H),
2.03-2.16 (m, 3H), 1.87-2.01 (m, 6H), 1.63-1.82 (m, 6H);
HRMS m/z 325.1444 [(M + 1) calcd for C21H20O4 325.1440];
Anal. (C21H20O4) C, H.
2-Adam an tan -1-yl-4-oxo-4H-ch r om en e-6-acetic acid (5g)
was prepared analogously, as described for 5d . Yield 85%,
colorless crystals; mp184-186 °C. 1H NMR (CDCl3): δ 8.09
(d, J ) 2.2 Hz, 1H), 7.61 (dd, J ) 2.2 + 8.6 Hz, 1H), 7.43 (d,
J ) Hz, 1H), 6.24 (s, 1H), 3.76 (s, 2H), 2.04-2.18 (m, 3H),
1.90-2.05 (m, 6H), 1.66-1.88 (m, 6H); HRMS m/z 3339.1598
[(M + 1) calcd for C21H22O4 339.1596]; Anal. (C21H22O4) C, H.
2-Ad a m a n t a n -1-yl-4-oxo-4H -ch r om e n e -6-ca r b oxa m -
id e (5e). A mixture of 5d (1 g, 3.1 mmol) and thionyl chloride
(0.47 g, 4 mmol) in dry 1,2- dichloroethane (4 mL) and DMF
(0.2 mL) was heated to reflux for 4 h. The solvent was
evaporated to dryness, and to the residue was added saturated
ammonia solution in ethanol (15 mL) at 0 °C. After being
stirred for 2 h at room temperature, the solvent was removed
under reduced pressure and the residue was purified by
column chromatography (toluene/ethyl acetate ) 1/2) to afford
5e (370 mg, 37%) as colorless crystals; mp 285-287 °C. 1H
NMR (DMSO-d6): δ 8.55 (d, J ) 2.2 Hz, 1H), 8.24 (dd, J ) 2.2
+ 8.8 Hz, 1H), 7.71 (d, J ) 8.8 Hz, 1H), 7.51 (s, br, 2H), 6.18
(s, 1H), 2.02-2.14 (m, 3H), 1.82-2.01 (m, 6H), 1.67-1.82 (m,
6 H); HRMS m/z 324.1603 [(M + 1) calcd for C20H21NO3
324.1600]; Anal. (C20H21NO3) C, H, N.
1
mp 97-101 °C. H NMR (CDCl3): δ 8.36 (d, J ) 2.1 Hz, 1H),
8.10 (dd, J ) 2.1 + 8.5 Hz, 1H), 7.05 (d, J ) 8.5 Hz, 1H), 3.88
(s, 3H), 2.52 (s, 3H), 1.97-2.02 (m, 6H), 1.86-1.87 (m, 3H),
1.65-1.71 (m, 6H).
(b) 2-Ad a m a n ta n -1-yl-4-oxo-4H-ch r om en e-6-ca r boxylic
Acid Meth yl Ester (5c). A suspension of 7c (10.67 g, 29.94
mmol) and potassium carbonate (12.41 g, 90 mmol) in 2-pro-
panol (120 mL) was refluxed for 75 min. The reaction mixture
was partitioned between ethyl acetate (400 mL) and water (100
mL), and the organic layer was separated, washed with water
and brine, dried over MgSO4, and evaporated in vacuo. The
crude product 3-[(3-adamantan-1-yl)-3-oxopropionyl]-4-hydroxy-
benzoic acid methyl ester 8c (8.09 g, 76%) was used in the
next step without further purification.
The crude product 8c (8.09 g, 22.7 mmol) was heated in
formic acid (60 mL) to 100 °C for 30 min. The solvent was
removed under reduced pressure, and the product was recrys-
tallized from acetone to afford pure 5c (5.33 g, 69%) as colorless
crystals; mp 211-212 °C. 1H NMR (CDCl3): δ 8.88 (d, J ) 2.1
Hz, 1H), 8.29 (dd, J ) 2.1 + 8.8 Hz, 1H), 7.49 (d, J ) 8.8 Hz,
1H), 6.28 (s, 1H), 3.93 (s, 3H), 2.07-2.016 (m, 3H), 1.92-2.02
(m, 6H), 1.68-1.87 (m, 3H); HRMS m/z 339.1600 [(M + 1) calcd
for C21H22O4 339.1596]; Anal. (C21H22O4) C, H.
3. Syn th esis of th e Test Com p ou n d 5a . (a ) 2-Ad a m a n -
ta n -1-yl-6-br om om eth ylch r om en -4-on e (10). A solution of
9a (2 g, 6.79 mmol), N-bromosuccinimide (1.33 g, 7.47 mmol),
and a catalytic amount of 2,2′-azobis(2-methylpropionitrile) in
benzene (100 mL) was heated to reflux for 2 h. The cooled
mixture was poured into saturated aqueous sodium bicarbon-
ate solution (20 mL) and extracted with ethyl acetate (50 mL).
The organic layer was washed with water and brine and dried
over MgSO4, and the solvent was evaporated under reduced
pressure. Purification by column chromatography (toluene/
ethyl acetate ) 10/1) afforded 10 (1.87 g 72%) as colorless solid.
1H NMR (CDCl3): δ 8.17 (d, J ) 2.3 Hz, 1H),7.69 (dd, J ) 2.3
+ 8.7 Hz, 1H), 7.45 (d, J ) 8.7 Hz, 1H), 6.2 (s, 1H), 4.56 (s,
2H), 2.06-2.20 (m, 3H), 1.90-2.04 (m, 6H), 1.68-1.9 (m, 6H).
(b ) 2-Ad a m a n t a n -1-yl-6-(h yd r oxym et h yl)ch r om en -4-
on e (5a ). A mixture of 10 (270 mg, 0.72 mmol) and Amberlite
IRA 900 carbonate form (820 mg, 2.87 mmol) in toluene (1.3
mL) was heated to 90 °C for 2 h. The reaction mixture was
cooled to room temperature, filtered, and washed with toluene/
methanol and evaporated to dryness. The residue was purified
by column chromatography (cyclohexane/ethyl acetate ) 1/1)
to give 5a as colorless crystals; mp 188-190 °C. 1H NMR
(CDCl3): δ 8.11 (d, J ) 2.2 Hz, 1H), 7.69 (dd, J ) 2.2 + 8.6
Hz, 1H), 7.42 (d, J ) 8.6 Hz), 6.17 (s, 1H), 4.77 (d, J ) 5.6 Hz,
2H), 2.66 (t, J ) 5.8 Hz, 1H), 2.08 - 2.23 (m, 3H), 1.89-2.08
(m, 6H), 1.65-2.08 (m, 6H); HRMS m/z 311.1648 [(M + 1) calcd
for C20H22O3 311.1647]; Anal. (C20H22O3) C, H.
4. Syn th esis of th e Test Com p ou n d s 5b a n d 5f. (a )
2-Adam an tan -1-yl-6-(dibr om om eth yl)ch r om en -4-on e (11).
According to the procedure described for 10, prepared from
9a with 2.5 equiv of N-bromosuccinimide to give colorless solid
(28% yield). 1H NMR (CDCl3): δ 8.21 (d, J ) 2.5, 1H), 8.0 (dd,
J ) 2.5 + 8.8 Hz, 1H), 7.52 (m, J ) 8.8 Hz, 1H), 6.72 (s, 1H),
6.21 (s, 1H), 2.08-2.2 (m, 3H), 1.92-2.04 (m, 6H), 1.68-1.88
(m, 6H).
(b) 2-Ad a m a n ta n -1-yl-4-oxo-4H-ch r om en e-6-ca r ba ld e-
h yd e (5b). A mixture of 11 (200 mg, 0.44 mmol) and potassium
acetate (260 mg, 2.65 mmol) in acetic acid (10 mL) was heated
to 120 °C for 12 h. To the hot reaction mixture was added 2 N
HCl (1.6 mL), followed by further heating for 30 min. The
cooled reaction mixture was evaporated to dryness, and the
residue was partitioned between ethyl acetate (15 mL) and
saturated aqueous sodium bicarbonate solution (5 mL). The
organic layer was washed with water and brine, dried over
MgSO4, and evaporated. The crude product was purified by
According to the procedure described for 7c, the following
compounds were prepared.
Ad a m a n ta n e-1-ca r boxylic a cid 2-a cetyl-4-m eth ylp h en -
yl Ester (7a ). Starting from 1-(2-hydroxy-5-methylphenyl)-
ethanone (6a , 6 g, 39.95 mmol). Yield (11.17 g, 83.7%), colorless
crystals; mp 94-96 °C. 1H NMR (CDCl3): δ 7.55 (d, J ) 2 Hz,
1H), 7.29 (ddd, J ) 2 + 0.6 + 8.2 Hz, 1H), 6.89 (d, J ) 8.2 Hz,
1 H), 2.53 (s, 3H), 2.37 (s, 3H) 2.08 (s, br, 9H), 1.77 (s, br, 6H).
Ad a m a n ta n e-1-ca r boxylic a cid 2-a cetyl-4-m eth oxyca r -
bon ylm eth ylp h en yl Ester (7b). Starting from (3-acetyl-4-
hydroxyphenyl)acetic acid methyl ester (6b, 3 g, 14.4 mmol).
Yield (5.28 g, 99%), colorless crystals; mp 103-107 °C. 1H NMR
(CDCl3): δ 7.66 (d, J ) 2.2 Hz, 1H), 7.43 (dd, J ) 2.3 + 8.3
Hz, 1H), 6.98 (d, J ) 8.3 Hz, 1H), 3.70 (s, 3H), 3.65 (s, 2H),
2.53 (s, 3H), 2.04-2.015 (m, 9H), 1.69-1.82 (m, 6H).
According to the procedure described for 5c, the following
compounds were prepared.
2-Ad a m a n ta n -1-yl-6-m eth ylch r om en -4-on e (9a ). Start-
ing from 7a (5.2 g, 16.6 mmol). Yield (4.1 g, 84% over two
steps), colorless crystals; mp 150 °C. 1H NMR (CDCl3): δ 7.95
(d, J ) 2 Hz, 1H), 7.45 (dd, J ) 2 + 8.5 Hz, 1H), 7.34 (d, J )
8.5 Hz, 1H), 6.18 (s, 1H), 2.43 (s, 3H), 2.06-2.18 (m, 3H), 1.91-
2.03 (m, 6H), 1.70-1.86 (m, 6H); HRMS m/z 295.1698 [(M +
1) calcd for C20H22O2 295.1694].
(2-Ad a m a n ta n -1-yl-4-oxo-4H-ch r om en -6-yl) a cetic Acid
Meth yl Ester (9b). Starting from 7b (5.75 g, 14.2 mmol). Yield
(3.55 g, 71%), colorless crystals; mp 93-97 °C. 1H NMR
(CDCl3): δ 8.05 (d, J ) 2.29 Hz, 1H), 7.59 (dd, J ) 2.3 + 8.6
Hz, 1H), 7.43 (d, J ) 8.6 Hz, 1H), 6.19 (s, 1H), 3.74 (s, 2H),
3.70 (s, 3H), 2.08-2.17 (m, 3H), 1.94-1.99 (m, 6H), 1.73-1.84
(m, 6H); HRMS m/z 353.1754 [(M + 1) calcd for C22H24O4
353.1753].
(c) Ad a m a n t a n -1-yl-4-oxo-4H -ch r om en e-6-ca r b oxylic
Acid (5d ). A suspension of 5c (3 g, 8.86 mmol) was heated to
100 °C in a mixture of dioxane (100 mL) and 20% aqueous
HCl for 4 h. The mixture was stored at 5 °C for 18 h, and the

4274 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17
Horvath et al.
chromatography on silica gel toluene/ethyl acetate ) 6/4) to
give 5b (171 mg, 56%) as colorless crystals; mp 196-198 °C.
1H NMR (CDCl3): δ 10.09 (s, 1H), 8.66 (d, J ) 2.1 Hz, 1H),
8.20 (dd, J ) 2.1 + 9 Hz, 1H), 7.58 (d, J ) 9 Hz, 1H), 6.25 (s,
1H), 2.10-2.26 (m, 3 H), 1.97-2.09 (m, 6H), 1.69-2.09 (m,
6H); Anal. (C20H20O3) C, H.
NMR (CDCl3): δ 8.22 (d, J ) 2.4 Hz, 1H), 7.97 (dd, J ) 2.4 +
8.8 Hz, 1H), 7.5 (d, J ) 8 Hz, 1H), 6.20 (s, 1H), 5.45 (s, 1H),
3.80 (s, 3H), 2.06-2.23 (m, 3H), 1.93-2.00 (m, 6H), 1.70-1.88
(m, 6H).
(b) (2-Adam an tan -1-yl-4-oxo-4H-ch r om en -6-yl)h ydr oxy-
a cetic Acid Meth yl Ester (14). Amberlite IRA 900, carbonate
form (3.2 g, 11.2 mmol) was added to a solution of 2-adaman-
tan-1-yl-4-oxo-4H-chromen-6-yl)bromoacetic acid methyl ester
(1.36 g, 3.1 mmol) in dry toluene (60 mL), and the mixture
was stirred for 48 h at room temperature. The ion-exchange
resin was filtered off, the residue was washed with toluene,
and the solvent was concentrated in vacuo. The pure compound
14 (726 mg, 59%) was obtained after column chromatography
on silica gel (ethyl acetate) as a white solid. 1H NMR (CDCl3):
δ 8.23 (d, J ) 2.26 Hz, 1H), 7.74 (dd, J ) 2.2 + 8.7 Hz, 1H),
7.47 (d, J ) 8.7 Hz, 1H), 6.20 (s, 1H), 5.29 (d, J ) 5.08 Hz,
1H), 3.75 (s, 3H), 3.66 (d, J ) 5.1 Hz, OH), 2.06-2.24 (m, 3H),
1.92-2.07 (m, 6H), 1.69-1.88 (m, 6H); HRMS m/z 369.1704
[(M + 1) calcd for C22H24O5 369.1702].
(c) Syn th esis of 2-Ad a m a n ta n -1-yl-6-(1-(h yd r oxyeth -
yl))ch r om en -4-on e. To a stirred solution of 5b (700 mg, 2.27
mmol) in THF (15 mL) was added methylmagnesium iodide
(1 mL, 3 mmol of a 3 M solution in diethyl ether) at 0 °C. After
the reaction mixture was stirred at room temperature for 1 h,
the mixture was quenched with saturated ammonium chloride
solution (2 mL) and ethyl acetate (15 mL). The organic layer
was washed with water and brine, dried over MgSO4, and
evaporated under reduced pressure. The crude product was
purified by column chromatography (toluene /ethyl acetate )
4/1) to afford the title compound (431 mg, 59%) as colorless
crystals; mp 142-147 °C. 1H NMR (CDCl3): δ 8.08 (d, J )
2.28 Hz, 1H), 7.29 (dd, J ) 2.2 + 8.7 Hz, 1H), 7.41 (d, J ) 8.8
Hz, 1H), 6.16 (s, 1H), 5.0 (qa. J ) 6.4 Hz, 1H), 2.02-2.17 (m,
3H), 1.90-2.04 (m, 6H), 1.75-1.87 (m, 6H), 1.52 and 1.49 (2s,
3H).
(c) (2-Ad a m a n ta n -1-yl-4-oxo-4H-ch r om en -6-yl)oxoa ce-
tic Acid Meth yl Ester . According to the procedure described
for 12, prepared from 14. Yield 72% yield, colorless crystals;
1
mp 183-185 °C. H NMR (CDCl3): δ 8.8 (d, J ) 2.2 Hz, 1H),
(d ) Syn th esis of 6-Acetyl-2-a d a m a n ta n -1-ylch r om en -
4-on e (12). To a stirred solution of oxalyl chloride (409 mg,
3.25 mmol) in anhydrous dichloromethane (15 mL) at -68 °C
dimethyl sulfoxide (533 mg, 6.82 mmol) dissolved in dichloro-
methane (3 mL) was added over a period of 10 min. The
reaction mixture was stirred for 15 min followed by addition
of 2-adamantan-1-yl-6-(1-(hydroxyethyl))chromen-4-one (420
mg, 1.28 mmol) in 2 mL of dichloromethane within 15 min.
After being stirred for 1 h at -68 °C the reaction was allowed
to reach -30 °C, stirred for 15 min and then re-cooled to -68
°C. Triethylamine (471 mg, 4.6 mmol) was added and the
mixture was allowed to reach room temperature. The clear
yellow solution was partitioned between saturated aqueous
sodium bicarbonate (5 mL) and ethyl acetate (20 mL), the
organic layer was washed with brine, dried over MgSO4,
filtered and evaporated. Purification by chromatography on
silica gel (toluene/ethyl acetate ) 20/1) afforded 12 (270 mg,
8.33 (dd, J ) 2.2 + 8.8 Hz, 1H), 7.57 (d, J ) 8.8 Hz, 1H), 6.24
(s, 1H), 4.02 (s, 3H), 2.04-2.09 (m, 3H), 2.03-1.94 (m, 6H),
1.7-1.8 (m, 6 H).
(d ) 2-Ad a m a n ta n -1-yl-4-oxo-4H-ch r om en e-6-yl)oxoa ce-
tic a cid (5h ). Yield 90%, colorless crystals; mp 245-246 °C.
1H NMR (DMSO-d6): δ 8.54 (d, J ) 2 Hz, 1H), 8.30 (dd, J )
2 + 8.8 Hz, 1H), 7.85 (d, J ) 8.8 Hz, 1H), 6.23 (s, 1H), 2.02-
2.14 (m, 3H), 1.87-2.02 (m, 6H), 1.65-1.82 (m, 6H); 13CNMR
(DMSO-d6): δ 187.1, 177.1, 176.3, 165.69, 159.7, 134.1, 129.1,
128.4, 123.2, 120.3, 107.1, 40.9, 40.5, 40.2, 39.9, 39.5, 39.2, 39.1,
38.9, 38.25, 36.2, 27.7; HRMS m/z 339.1600 [(M + 1) calcd for
C
21H22O4 339.1596]; Anal. (C21H22O4) C, H.
6. Syn th esis of th e Test Com p ou n d s 5i a n d 5j. (a ) 2-
Ad a m a n ta n -1-yl-6-br om oth ioch r om en -4-on e (17) was pre-
pared as described in the literature,24 starting from 4-bromo-
benzenethiol (15, 1.89 g, 10 mmol) and ethyl 3-(1-adamantyl)-
3-oxopropionate (16, 2.5 g, 10 mmol) in polyphosphoric acid
(10 mL). Triturating of the crude product with hot EtOH gave
17 (1.25 g, 34%) as colorless crystals; mp 214-216 °C. 1H NMR
(DMSO-d6): δ 8.37 (dd, J ) 1.1 + 1,8 Hz, 1H), 7.91 (m, 3H),
6.94 (s, 1H), 2.05-2.14 (m, 3H), 1.96-2.03 (m, 6H), 1.69-1.78
(m, 6H).
1
66%) as colorless crystals; mp 183-185 °C. H NMR (CDCl3):
δ 8.72 (d, J ) 2.2 Hz, 1H), 8.29 (dd, J ) 2.2 + 8.8 Hz, 1H),
7.52 (d, J ) 8.8 Hz, 1H), 6.23 (s, 1H), 2.69 (s, 3H), 2.12-2.26
(m, 3 H), 1.97-2.02 (m, 6H), 1.55-1.85 (m, 6H).
(e) 2-Ad a m a n ta n -1-yl-6-(2-br om oa cetyl)ch r om en -4-on e
(13). A suspension of ketone 12 (700 mg, 2.17 mmol) in acetic
acid (15 mL) was heated to 70 °C followed by addition of
bromine (347 mg, 2.17 mmol). After the mixture was stirred
at 70 °C for 3 h, the solvent was evaporated and the residue
was purified by column chromatography (toluene/ ethyl acetate
) 10/1) to give 13 (591 mg, 68%). 1H NMR (CDCl3): δ 8.73 (d,
J ) 2.2 Hz, 1H), 8.31 (dd, J ) 2.2 + 8.8 Hz, 1H), 7.56 (d, J )
8.7 Hz, 1H), 6.24 (s, 1H), 4.54 (s, 2H), 2.05-2.18 (m, 3 H),
1.96-2.04 (m, 6H), 1.61-1.88 (m, 6H).
(f) 2-Ad a m a n ta n -1-yl-6-(2-h yd r oxya cetyl)ch r om en -4-
on e (5f). To a mixture of 13 (150 mg, 0.37 mmol) in anhydrous
DMF (2 mL) and dichloromethane (2 mL) was added a solution
of betaine (44 mg, 0.37 mmol) in anhydrous ethanol (1 mL).
After being stirred for 22 h, the solvents were evaporated to
dryness, the residue was taken up in dioxane (2 mL) and water
(2.5 mL), and potassium carbonate (200 mg, 1.86 mmol) was
added. The mixture was stirred for 30 min and partitioned
between water and ethyl acetate. The organic layer was
washed with water and brine, dried over MgSO4, and evapo-
rated. Purification by column chromatography (toluene/ethyl
acetate ) 2/1) provided 5f (77 mg, 61%) as colorless crystals;
mp 208-212 °C. 1H NMR (DMSO-d6): δ 8.51 (d, J ) 2 Hz,
1H), 8.27 (dd, J ) 2 + 8.4 Hz, 1H), 7.77 (d, J ) 8.5 Hz, 1H),
6.20 (s, 1H), 5.21 (s, br, 1H), 4.85 (s, 2H), 2.03-2.20 (m, 3H),
1.88-1.99 (m, 6 H), 1.71-1.77 (m, 6H); HRMS m/z 339.1600
[(M + 1) calcd for C21H22O4 339.1596]; Anal. (C21H20O4) C, H.
(b) 2-Ad a m a n ta n -1-yl-4-oxo-4H-th ioch r om en e-6-ca r bo-
n itr ile (5i). A mixture of 17 (565 mg, 1.5 mmol), palladium(II)
acetate (53 mg, 0.235 mmol), triphenylphosphine (123 mg, 0.47
mmol), and calcium hydroxide (115 mg, 1.55 mmol) in DMF
(5 mL) was heated to 100 °C for 90 min. After being cooled to
room temperature, the mixture was poured onto phosphate
buffer (pH 7) and extracted with ethyl acetate (3×). The
combined organic layers were dried over MgSO4, and evapo-
rated in vacuo. The crude product was purified by column
chromatography (cyclohexane/ethyl acetate ) 10/1) to give the
title compound (176 mg, 36%) as colorless crystals; mp 261
1
°C. H NMR (DMSO-d6): δ 8.59 (t, J ) 1.2 Hz, 1H), 8.12 (d, J
) 1.2 Hz, 3H), 6.96 (s, 1H), 2.04-2.15 (m, 3H), 1.93-2.04 (m,
6H), 1.67-1.80 (m, 6H). 8.91 (d, J ) 1.7 Hz, 1H); HRMS m/z
322.1263 [(M + 1) calcd for C20H19NOS 322.1266]; Anal.
(C20H19NOS) C, H, N.
(c) 2-Ad a m a n t a n -1-yl-4-oxo-4H -t h ioch r om en e-6-ca r -
boxylic Acid (5j). A suspension of 5i (100 mg, 0. 31 mmol) in
a mixture of dioxane (10 mL) and 20% aqueous HCl was heated
to 100 °C for 16 h. After being cooled to room temperature,
the reaction mixture was diluted with water (2 mL) and
extracted with ethyl acetate (15 mL). The organic layer was
separated, and the aqueous layer was extracted with ethyl
acetate (2×). The combined organic layers were dried over
MgSO4 and evaporated. Purification by column chromatogra-
phy (dichloromethane/methanol ) 6/1) provided 5j (53 mg,
5. Syn th esis of th e Test Com p ou n d 5h . (a ) 2-(Ad a m a n -
ta n -1-yl-4-oxo-4H-ch r om en -6-yl)br om oa cetic a cid m eth yl
ester was prepared analogously to the procedure as described
for 10. Yield 75%, light yellow crystals; mp 132-134 °C. 1H
1
50%) as colorless crystals; mp 275-280 °C. H NMR (DMSO-
d6): δ 8.9 (d, J ) 1.7 Hz, 1H), 8.28 (dd, J ) 1.2 + 8.2 Hz, 2H),
7.86 (d, J ) 8.3 Hz, 1H),6.96 (s, 1H), 2.03-2.16 (m, 3H), 2.04-

Reversible Inhibitors of Human Steroid Sulfatase
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 17 4275
2.15 (m, 3H), 1.86-2.04 (m, 6H), 1.60-1.80 (m, 6H); HRMS
m/z 341.1215 [(M + 1) calcd for C20H19NOS 341.1211]; Anal.
(C20H20O3S) C, H.
4. Ha Ca T Ker a tin ocyte P r olifer a tion Assa y. HaCaT
cells32 were cultivated in DMEM (Gibco) containing 5% FCS.
For the proliferation assay, cells were detached by trypsiniza-
tion, suspended in fresh medium, and seeded into 96-well
microtiter plates at 4000 cells/0.2 mL/ well. After 24 h, the
medium was replaced with fresh medium containing graded
concentrations of test compounds. After 4 days of incubation,
the extent of cellular proliferation was measured by colori-
metric assay using sulforhodamine.33
B. Biology. 1. Assa y of P u r ified Hu m a n STS. STS was
produced from a clone of recombinant Chinese hamster ovary
(CHO) cells that stably express the human enzyme as de-
scribed previously.26 The purity of the final enzyme prepara-
tion was estimated to > 95%. Sulfatase activity was assessed
using the method by Eto et al.29 with modifications as
described.20 Briefly, 0.5 mM 4-MUS (Sigma) was used as
substrate at pH 7.5, 37 °C, and the formation of the reaction
product 4-methylumbelliferone was measured fluorimetrically
after alkalization.
5. Bin d in g t o E st r ogen R ecep t or s (E R ) r a n d â.
Fluorescence polarization-based ligand displacement assays
with ER-R and -â were performed as described,17 using
commercial assay kits obtained from PanVera (Madison, WI).
For inhibition studies, compounds were included at graded
concentrations from stock solutions in ethanol; final ethanol
content did not exceed 2%. IC50 values were calculated using
nonlinear regression (GraFit, Erithacus Software Ltd.). Values
reported are the mean of triplicate determinations, which
typically lie in the range of (20%.
6. Sta bility Stu d ies in Solu tion . Test compounds were
dissolved at a concentration of 10 mM in acetonitrile and
diluted 100-fold (final concentration: 100 µM) into 0.1 M Tris-
HCl buffer, pH 7.5/acetonitrile 1:1. Samples contained in
tightly closed vials were stored in the dark at 37 °C. Im-
mediately after preparation of the solutions and at various
time points up to 7 days, aliquots were analyzed by HPLC.
The extent of degradation was determined and, in the case of
sulfamates, first-order rate constants of hydrolysis and cor-
responding half-lives were calculated by linear regresssion
using the software Grafit (Elsevier). HPLC analyses for 4a ,b
were performed using a HP1090 chromatographic system
equipped with a Kratos UV detector set at 230 nm; a Merck
LiChrosorb column (RP-18, 7 µm, 250 × 4 mm) was eluted
with acetonitrile/10 mM ammonium sulfate (55/45, v/v), pH
6.0, at a flow rate of 1.5 mL/min at ambient temperature. For
5d ,j, an Agilent 1100 system was used equipped with an
XTerra Waters column (RP-8, 3.5 µm, 4.6 × 50 mm) using 95-
5% phosphate buffer (pH 5.6) /acetonitrile/1%aqueous TFA
(gradient) as eluent. Samples of the incubations were diluted
with the corresponding eluent and then injected directly into
the HPLC system.
In the case of compound 5d and 5j, Ki values were
determined by measuring substrate turnover (V) at various
substrate concentrations (S) in the presence of various inhibi-
tor concentrations (I), as detailed in Figure 2. Data were
plotted in an S/V vs S diagram (Hanes plot), and replotted in
a Km/Vmax vs I diagram. Ki values were calculated from the
intercept of the linear fit with the abscissa using GraFit. For
compounds 5a , 5f, and 5h , the Ki values were calculated from
the IC50 values, using the relation IC50 ) Ki(1 + S/Km) for
competitive inhibitors,30 with Km) 270 µM and S) 500 µM.
The reversibility of STS inhibition by 5d and 5j was
determined using the enzyme activity in microsomes from
human placenta as described.20 The assay involves incubation
of the microsomes with the inhibitor (for various times up to
6 h) followed by addition of dextran-coated charcoal to bind
free and noncovalently bound inhibitor and determination of
residual STS activity.
2. CHO-STS Assa y. The cellular assay for both irrevers-
ible (4a ,b) and reversible inhibitors (5a -j) was carried out as
described,26 using fixed and live cells, respectively. Briefly, cells
were seeded into 96-well plates (Nunc, Roskilde, Denmark)
at 30 000 cells/well in RPMI 1640 medium, supplemented with
10% FCS and 100 U/mL penicillin/100 µg/mL streptomycin,
and allowed to adhere overnight. They were then treated with
graded concentrations of inhibitors for 24 h or left untreated.
For the assay using fixed cells, the cells were fixed with 4%
paraformaldehyde for 10 min at room temperature and washed
four times with PBS. They were then incubated with 100 µL/
well 0.5 mM 4-MUS in 0.1 M Tris-HCl, pH 7.5 for 30 min. 50
µL/well of 1 M Tris-HCl, pH 10.4 (stop solution) were added
to stop the reaction. The reaction solutions were transferred
to white plates (Microfluor, Dynex, Chantilly, VA).
For the assay with live cells, cells were seeded into 96-well
plates and incubated with test compounds as above, except
that phenol red-free RPMI 1640 containing 10% FCS was used.
After 24 h, the medium was replaced by fresh medium (200
µL/well) containing the same substance concentrations plus
0.5 mM 4-MUS. The cells were then incubated at 37°C for an
additional 4 h. At this point, 100 µL supernatant were
transferred to white plates containing 50 µL stop solution.
The plates were read in a Fluoroskan II fluorescence
microtiter plate reader (Titertek, Salzburg, Austria) at excita-
tion and emission wavelengths of 355 and 460 nm, respec-
tively. Reagent blanks were subtracted from all values. The
fluorescence units (FU) were divided by the optical density
readings for cellular protein obtained after fixation and
staining of cells with sulforhodamine B (OD550), to correct for
variations in cell number. IC50 values were defined as the
concentrations of test substances required for 50% inhibition
of STS activity. All experiments were carried out in triplicate
for each sample and concentration.
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