2-Substituted 4-(thio)chromenone 6-O-sulfamates: Potent inhibitors of human steroid sulfatase

Article abstract of DOI:10.1021/jm020878w

Steroid sulfatase (STS) has emerged as a highly attractive target for the therapy of a number of disorders. Starting with the known inhibitor estrone sulfamate (1) as lead compound and with the finding that steroid sulfamates containing a nonaromatic A-ri

Full text of DOI:10.1021/jm020878w

4310
J . Med. Chem. 2002, 45, 4310-4320
2-Su bstitu ted 4-(Th io)ch r om en on e 6-O-Su lfa m a tes: P oten t In h ibitor s of Hu m a n
Ster oid Su lfa ta se
Peter Nussbaumer,* Philipp Lehr, and Andreas Billich
Novartis Research Institute Vienna, Brunnerstrasse 59, A-1235 Vienna, Austria
Received March 15, 2002
Steroid sulfatase (STS) has emerged as a highly attractive target for the therapy of a number
of disorders. Starting with the known inhibitor estrone sulfamate (1) as lead compound and
with the finding that steroid sulfamates containing a nonaromatic A-ring are inactive, chromen-
4-one sulfamates were designed, prepared, and tested for their ability to block human STS.
This new class of nonsteroidal inhibitors shows high potency when the sulfamate group and
the side chain are situated in diagonally opposite positions (i.e., 2,6- and 3,7-substitution
pattern). The highest activity is achieved with fully branched, bulky aliphatic side chains and
with thiochromen-4-one as the core element. 2-(1-Adamantyl)-4H-thiochromen-4-on-6-O-
sulfamate (6c) is the most potent STS inhibitor discovered so far, and it is about 170-fold
superior to 1. As with 1, all chromenone sulfamates are irreversible inhibitors of STS with a
biphasic time course of inactivation.
In tr od u ction
The enzyme steroid sulfatase (E.C. 3.1.6.2., STS)
catalyzes the hydrolysis of the sulfate esters of 3-hy-
droxy steroids, which are inactive transport or precursor
forms of the active 3-hydroxy steroids. Major substrates
are the 3-O-sulfates of estrone, dehydroepiandrosterone
(DHEA), pregnelonone, and cholesterol.1 STS is a 65
kDa membrane-bound protein, predominantly associ-
ated with the endoplasmatic reticulum,2 and present in
almost all mammalian tissues.3 In recent years, this
enzyme has received considerable attention due to its
potential involvement in the pathogenesis of a number
of diseases such as:
F igu r e 1. Structures of estrone sulfamate (1), the tricylic
coumarin COUMATE 667 (2), and the chromenone-based
(i) Br ea st Ca n cer . Estrone sulfate appears to be a
major source of active estrogens in mammary tumors,
sulfamates described here.
especially in women after menopause.4,5 While estrone
sulfate itself does not bind to the estrogen receptor, it
development of androgenetic alopecia (male pattern hair
is converted first by STS to unconjugated estrone and
loss)14 and of acne15 has been proposed.
then by 17â-hydroxysteroid dehydrogenase to estradiol,
(iii) Cogn itive Dysfu n ction . DHEA sulfate as a
which binds to the receptor with high affinity. STS
neurosteroid affects a number of neurotransmitter
levels are elevated in breast tumors,6 and this is a
systems, including those involving acetylcholine, gluta-
predictor of tumor recurrence.7 Inhibitors of STS block
mate, and GABA, resulting in increased neuronal excit-
estrone sulfate-dependent breast cancer cell prolifera-
ability.16 STS inhibition was shown to enhance learning
tion in vitro8,9 and in vivo.10,11 DHEA sulfate can also
and spatial memory in the rat.17
be converted in a metabolic pathway involving STS to
(iv) Im m u n e F u n ction s. STS has been proposed to
play a role in regulating T-helper cell functions by
an agonist of the estrogen receptor (androst-5-ene-3â,-
17â-diol)9,12 resulting in the stimulation of breast cancer
mobilizing DHEA in macrophages as an accessory signal
cell growth. In addition to breast cancer, STS activity
for T-cells.18 STS inhibition was shown to be protective
might also be involved in estrogen-dependent cancer of
in models of contact allergy and collagen-induced ar-
the endometrium and androgen-dependent prostate
thritis in rodents.19
carcinomas.5
Thus, inhibitors of STS have been considered as
potential new therapeutic agents for the treatment of a
range of different diseases, with breast cancer having
(ii) An d r ogen -Dep en d en t Sk in Disea ses. The skin
is capable of converting DHEA to active androgens,
namely, testosterone and dihydrotestosterone.13 DHEA
received the most attention so far (see ref 20 for review).
is formed in the skin from the abundant systemic
The most active STS inhibitors described so far are
precursor DHEA sulfate. An involvement of STS in the
sulfamic acid esters, such as estrone sulfamate (1,
Figure 1), which irreversibly inactivate the enzyme.21
* To whom correspondence should be addressed. Tel: ++43-1-86634-
However, estrone sulfamate is not suitable for therapy
395.
Fax.
++43-1-86634-354.
E-mail:
peter.nussbaumer@
pharma.novartis.com.
due to its strong estrogenic activity.22 Therefore, recent
10.1021/jm020878w CCC: $22.00 © 2002 American Chemical Society
Published on Web 08/13/2002

2-Substituted 4-(Thio)chromenone 6-O-Sulfamates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19 4311
Sch em e 1. Synthesis of STS Inhibitors 3-7a
Ta ble 1. Inhibition of Purified Human STS by
Chromenone-Based Sulfamatesa
position of
IC50
(nM)
compd X, Y OSO2NH2
R
substituent (R)
phenyl
1
56
3a
3b
3c
3d
3e
3f
3g
3h
3i
O
O
O
O
O
O
O
O
O
6
6
6
6
6
6
6
6
6
2
2
2
2
2
2
2
2
2
672
1620
1620
190
722
403
78
benzyl
2-phenylethenyl
2-phenylethyl
n-propyl
n-nonyl
1,1-dimethylnonyl
tert-butyl
2,2,3,3-tetramethyl-
cyclopropyl
cyclopentyl
cyclohexyl
cyclodocecyl
4-pentylbicyclo[2.2.2]-
oct-1-yl
22
45
3j
3k
3l
O
O
O
O
6
6
6
6
2
2
2
2
134
62
45
3m
11
3n
3o
4a
4b
5a
5b
6a
6b
6c
7a
7b
7c
7d
7e
7f
O
O
O
O
O
O
S
S
S
O
O
O
6
6
7
7
7
7
6
6
6
6
6
6
6
6
6
2
2
3
3
2
2
2
2
2
2
2
2
2
2
2
1-adamantyl
nor-adamantyl
phenyl
cyclohexyl
n-propyl
cyclohexyl
phenyl
tert-butyl
1-adamantyl
phenyl
cyclohexyl
1-adamantyl
cyclohexyl
1-adamantyl
cyclohexyl
5.6
11
437
50
>100 000
6330
62
5.6
0.34
2580
280
140
24 300
291
11 200
OH, H
OH, H
H, H
a
For a definition of substituent R, see Table 1.
a
For generic structures, see Scheme 1.
efforts have focused on the synthesis of nonestrogenic
sulfamate type STS inhibitors.9,10,23-25 Among these,
tricyclic coumarin sulfamates have been reported with
good inhibitory activity and lacking estrogenic potential,
e.g., COUMATE 667 (2, Figure 1).11,24 In a complemen-
tary approach, we aimed at exploring aryl sulfamates
with a bicyclic ring system, whereby the bicyclic core
structure serves as an estrone A,B-ring mimic and the
side chains fill the space of the steroidal C,D-rings. We
designed and synthesized novel nonsteroidal aryl sulfa-
mates featuring a 4-(thio)chromenone ring system (3-
6, Figure 1) as the core element, which resulted in the
discovery of highly potent STS inhibitors. Recently, we
reported the synthesis and biological properties of two
prototypes, i.e., 3f,h .9 In this paper, we describe the
structure-activity relationships (SAR) obtained for this
novel class of STS inhibitors.
gel followed by crystallization. The yield of isolated,
analytically pure sulfamates depended on how quickly
the pure product could be isolated (i.e., long purification
and manipulation times reduced the yield). This may
be due to reduced stability in solution as opposed to the
solid state, where the compounds are stable.
Product 3d was obtained by selective hydrogenation
of the exocyclic double bond in 3c. Catalytic hydrogena-
tion was also used for the synthesis of chromane
analogues 7 starting from chromenones 3a ,k ,n , respec-
tively. Depending on the reaction time, either the
chroman-4-one derivatives 7a -c or, after continued
hydrogenation, the corresponding 4-hydroxychromanes
(7d ,e; stereochemistry not elucidated) and chromane
(7f) could be obtained as the main products.
Scheme 2 summarizes the syntheses of the 6- and
7-hydroxy(thio)chromenone precursors 8-11 applying
methods A-D. 2-Substituted 6-hydroxychromen-4-ones
8b-o were prepared from a common starting material,
i.e., 2,5-dihydroxyacetophenone (12), by following known
methods A or B.28 The hydroxy function at position 5
in 12 had to be protected for subsequent construction
of the heterocyclic ring system. This could be achieved
either by (i) using the same acyl residue as both building
block and hydroxy protecting group generating inter-
mediate 13 (method A) or (ii) by selective protection via
benzoylation (intermediate 15, method B), followed by
acylation to introduce the desired R substituent. In both
methods A and B, the double-acylated intermediates
underwent O f C migration of the acyl residues within
the hydroxyacetophenone moiety upon treatment with
sodium hydride. The protecting groups in position 5
were not affected under these conditions. The resulting
Resu lts a n d Discu ssion
Ch em istr y. Most of the test compounds 3-6 (for
characteristic physicochemical properties, see Table 3)
were prepared by introducing the sulfamate moiety in
the final step (Scheme 1; for definition of substituent
R, see Table 1). The sulfamoylation can be performed
by reacting the corresponding phenolic precursors 8-11
with amidochlorosulfonic acid26 (usually 2-3-fold ex-
cess) in dimethylformamide (DMF) or dimethylacet-
amide, either with or without addition of a base.27 We
routinely used sodium hydride (equimolar to excess
amidochlorosulfonic acid) in DMF. After aqueous work-
up, the pure sulfamates were isolated directly either by
crystallization (in most cases from 2-propanol) or by
chromatography of the crude product mixture on silica

4312 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19
Nussbaumer et al.
Sch em e 2. Methods A-D for the Synthesis of Key Intermediates 8, 9, and 11a
a
For a definition of substituent R, see Table 1.
crude (3-oxopropio)phenones 14 and 16 were then
cyclyzed to the O-protected 2-alkyl chromen-4-ones
without prior purification by treatment with formic acid
or hydrochloric acid in methanol. Deprotection was
achieved by basic hydrolysis (10% aqueous KOH in
dioxane) yielding 6-hydroxychromen-4-ones 8. For the
2-tert-butyl series, we developed a one pot reaction for
the cyclization and deprotection steps. Thus, cleavage
of the pivaloyl protecting group was accomplished by
just adding aqueous hydrochloric acid to the reaction
mixture.
boron tribromide in dichloromethane, generated the
substituted bicyclic heterocycles 11a -c.
STS In h ibition Assa y. Previous authors in the field
have used either placental microsomes21 or lysates of
cells transfected with an STS expression vector25 as the
source of enzyme activity. In contrast, we used highly
purified human STS obtained from a recombinant cell
line for our inhibition assays.9 Instead of radiolabeled
estrone sulfate or DHEA sulfate as used by other
authors, we used 4-methylumbelliferyl sulfate (4-MUS)
as a synthetic substrate for a convenient colorimetric
assay.9 We demonstrated for a series of compounds that
relative IC50 values measured with either estrone
sulfate or 4-MUS were identical (data not shown).
2-Substituted 7-hydroxychromen-4-ones 10a ,b were
synthesized analogously following method A but start-
ing from 2,4-dihydroxyacetophenone. Method C (Scheme
2) was used to prepare 3-substituted 7-hydroxychromen-
4-ones 9a ,b. Resorcinol (17) was acylated with acid
chlorides 18a ,b to produce intermediates 19a ,b, which
were cyclyzed to 9a ,b by treatment with triethyl ortho-
formate.29
SAR. Estrone sulfamate (1, Figure 1), the sulfamic
acid analogue of the natural substrate estrone sulfate,
was the most potent inhibitor of human STS known21
when we started our search for novel inhibitors. In our
assay system, the IC50 of estrone sulfamate is 56 ( 8
nM. STS accepts as substrates steroid sulfates not only
with an aromatic A-ring (estrone sulfate) but also with
a nonaromatic A-ring (e.g., DHEA sulfate and choles-
terol sulfate).1 When we synthesized and tested the
corresponding sulfamate derivatives of DHEA and
4-Mercaptoanisol (20) was the starting material for
the synthesis of thiochromen-4-ones 11a -c. Condensa-
tion of 20 with appropriately substituted ethyl 3-oxo-
propionates 21a -c in polyphosphoric acid, according to
a published procedure30 followed by ether cleavage using

2-Substituted 4-(Thio)chromenone 6-O-Sulfamates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19 4313
cholesterol as potential inhibitors in analogy to estrone
sulfamate, these compounds surprisingly turned out to
be completely inactive up to the highest test concentra-
tions of 30 µM. From these data, we concluded that an
aromatic A-ring is essential for STS inhibition and thus
concentrated our efforts on analogues with the sulfa-
mate group attached to a phenolic group. All sulfamate
type STS inhibitors described by other authors also fall
into this structural category.20
significantly superior to estrone sulfamate (1). Further-
more, the 1-adamantyl analogue 3n was the most potent
inhibitor of the chromenone series (IC50 ) 5.6 nM) with
a 10-fold improved activity relative to 1.
Then we asked whether the 3-R-7-OSO2NH2 substitu-
tion pattern on the chromenone core could also provide
compounds with good inhibitory activities against hu-
man STS. This was confirmed by two derivatives, i.e.,
4a (R ) phenyl) and 4b (R ) cyclohexyl). Comparable
activity was obtained for both compounds relative to
their 2,6-regioisomers 3a ,k (Table 1). Thus far, we only
investigated chromenone compounds with the side chain
R situated diagonally opposite to the sulfamate group.
This gives the closest fit when overlaying the steroidal
lead 1 with the substituted chromenone sulfamate
inhibitors. Here, the bicyclic core and the side chain
mimic the A,B- and the C,D-rings of estrone, respec-
tively. To test the validity of this model, we synthesized
and tested derivatives 5a ,b having a 2,7-substitution
pattern on the chromenone core. As expected, both
compounds were by at least 2 orders of magnitude less
active than their 2,6-regioisomers (compare 5a with 3f
and 5b with 3k , Table 1).
The inhibitory potency of chromenone sulfamates
could be further increased by formally replacing the
oxygen atom in the heterocycle by sulfur as demon-
strated by compounds 6a -c (Table 1). These thio-
chromenone analogues are 4- to 17-fold more potent
than their respective oxo-analogues (3a ,h ,n ). With an
IC50 value of 0.34 nM, compound 6c is the most potent
STS inhibitor discovered so far and is about 170-fold
more potent than the steroidal lead compound 1.
Finally, we investigated the influence of reducing the
keto function and the double bond in the chromenone
sulfamates on inhibitory activity. The racemic chroman-
4-one analogues (7a -c) were found to be slightly but
consistently less potent than the corresponding chromen-
4-one-based inhibitors (3a ,k ,n ). Additional reduction of
the keto function resulted in a further loss of activity
(derivatives 7d -f). Because the activities of compounds
7a -f were substantially lower as compared to their
parent chromenone derivatives, no efforts were made
to explore the influence of stereochemistry on inhibitory
potency.
Estrogenic activity of an STS inhibitor, as encoun-
tered in the case of 1,22 is prohibitive for its use in the
treatment of estrogen-dependent cancer. We evaluated
the potential estrogenicity of a selection of our chrome-
none-based STS inhibitors in two different systems and
obtained a SAR that does not correlate with the SAR
for STS inhibition. These results will be presented
elsewhere.
In this study, we describe a series of novel nonste-
roidal STS inhibitors featuring a 4-(thio)chromenone
ring system, characterized by the general formulas 3-6
in Scheme 1. The inhibitory potencies of the compounds
(IC50 values) against the pure enzyme were used for the
establishment of SAR (Table 1).
Structural and biological similarities between steroi-
dal estrogens and hydroxy-substituted flavones ()
2-phenylchromenones) are well-documented.31 There-
fore, it is not surprising that compound 3a (flavone
sulfamate) exhibits inhibitory activity against STS
(Table 1), albeit with 12-fold less potency than the
steroidal lead compound 1. We were concerned about
potential estrogenic activity of flavone derivatives and,
therefore, proceeded with 2-alkyl-substituted 4-chrome-
none 6-O-sulfamates. When the phenyl substituent
present in 3a was shifted away from the bicyclic core
by a C1 (3b) or a CdC spacer (3c), the inhibitory activity
decreased roughly by a factor of 2.5. However, potency
increased when the spacer in 3c was hydrogenated to
give an ethylene bridge between the phenyl ring and
the bicyclic core. Compound 3d was superior to all
previous analogues with a phenyl-containing side chain
(IC50 ) 190 nM). The results obtained with 3a -d
indicated that there is considerable space available for
side chain substituent R and that a substituent con-
nected to the chromenone core by an sp3 carbon might
be preferred over those connected by an sp2 carbon.
We then explored simple aliphatic chains, both un-
branched and branched, for the substituent R. Within
the unbranched series, a tendency for better activity by
longer side chains (3f vs 3e, Table 1) was observed.
Moreover, bulkiness at the carbon atom attached to the
heterocycle was identified as a key factor for high STS
inhibitory potency of the compound class. This finding
was deduced by comparing the results obtained with
inhibitors 3f (R ) nonyl, IC50 ) 403 nM) and 3g (R )
1,1-dimethylnonyl, IC50 ) 78 nM), as well as by the low
IC50 value of 22 nM obtained with tert-butyl analogue
3h . Thus, 3h was the first chromenone-based inhibitor
superior to the steroidal lead 1. The importance of a
branched, bulky substituent R for high activity was
confirmed in a series of compounds with cycloalkyl side
chains (3i-o). As observed for the linear alkyl substit-
uents, we found that potency correlates with the size of
the ring cycle (compare 3j, R ) cyclopentyl; 3k , R )
cyclohexyl; and 3l, R ) cyclododecyl; Table 1). The
activity of the tetramethylcyclopropyl derivative 3i is
higher than expected for a small ring analogue, but it
probably results from the bulkiness introduced by the
four methyl substituents. Even higher activity was
achieved by introduction of bi- and tricycloalkyl sub-
stituents, which are bulky and are fully branched at the
attachment site to the chromenone core. With IC50
values of 11 nM, the inhibitors 3m ,o show potency
Kin etics of STS In a ctiva tion . We expected the
chromenone-based sulfamates to be irreversible inhibi-
tors of STS, similar to the lead compound estrone
sulfamate (1).21 We tested for enzyme inactivation by
incubating human placental microsomes (which contain
STS activity) with inhibitor, followed by separation of
unbound inhibitor by binding to dextran-coated char-
coal21 (note that purified Triton-solubilized STS cannot
be used in this assay since the detergent disturbs
binding of the inhibitor to the charcoal). Using this
method, we observed a time- and concentration-depend-
ent inactivation of STS activity in placental microsomes

4314 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19
Nussbaumer et al.
Ta ble 2. Kinetic Parameters of Selected Chromenone-Based
STS Inhibitors
Ki
(µM)
kinact
kinact/KI
IC50
(nM)
compd
(sec-1
)
(104/M/sec)
3f
3h
3j
3k
3l
3m
3n
3o
4b
5b
6c
7c
7d
7f
1.7
0.9
0.73
1.0
1.1
0.5
0.19
0.22
1.2
0.006
0.070
0.011
0.035
0.040
0.074
0.040
0.042
0.070
0.020
0.115
0.017
0.018
0.016
0.35
7.8
1.5
403
22
134
62
45
11
5.6
11
50
3.5
3.64
14.8
21.2
19.1
5.83
0.083
54.8
0.91
0.034
0.05
24
6330
0.21
1.86
53.7
32
0.34
140
24 300
11 200
F igu r e 2. Time- and concentration-dependent inhibition of
STS activity in placental microsomes by 3n . (A) Time course
of inactivation at different inhibitor concentrations (b, 5 µM;
3, 2.5 µM; [, 1.25 µM; O, 0.31 µM; ×, 0.16 µM). The initial
enzyme activity was set to one; the curves are generated by
fitting of the data to a double-exponential decay. (B) Double
reciprocal plot of the apparent first-order rate constants kapp
vs the inhibitor concentrations.
F igu r e 3. Correlation between kinact/KI and IC50 values for
chromenone-based STS inhibitors.
by our inhibitors, as exemplified in Figure 2A for
compound 3n . To explore whether inhibition of STS
activity by our compounds was truly irreversible, we
inactivated purified recombinant STS with inhibitors
3h ,n or 6c and then extensively dialyzed the protein or
passed it through a size exclusion column to separate
it from unbound inhibitor. We did not observe any
recovery of enzyme activity after these procedures,
indicating that the test compounds act as irreversible
inhibitors.
Similar to the case of estrone sulfamate,21 a biphasic
time course of inactivation was observed (Figure 2A).
The data can be fitted to a double-exponential decay.
Thus, the initial decrease in STS activity follows pseudo-
first-order kinetics. From a double-reciprocal plot of the
apparent first-order rates of inactivation (kapp) vs inhibi-
tor concentration (see Figure 2B), the inhibition con-
the reactivity of the sulfamate group in the enzyme-
inhibitor complex differs by factors of up to 20. This may
be explained by a combination of (i) electronic effects of
substituents on the electrophilic sulfur of the sulfamate
group and (ii) steric effects influencing the orientation
of the sulfamate moiety with respect to the nucleophilic
reaction partner on the enzyme (see ref 24a for a
postulated mechanism of STS inactivation by sulfa-
mates). Analysis of the data set obtained suggests that
steric effects may be the dominating factor. For instance,
inhibitors 3k (4-chromenone), 7c (4-chromanone), 7d (4-
hydroxychromane), and 7f (chromane) show very similar
kinact values, but the electronic properties of their
heterocyclic core elements differ substantially. Simi-
larly, we would have expected more than a factor of 2
in kinact between the regioisomers 3k (sulfamate func-
tionality in meta position to the carbonyl group of the
chromenone system) and 4b (sulfamate in para position
to the carbonyl), if electronic effects by the substituents
play a major role. The most potent inhibitor found here
(6c) features not only one of the lowest KI values but
also the fastest rate of inactivation.
stant (KI) and the rate constant of inactivation (kinact
)
can be calculated.32 Table 2 lists KI and kinact values for
a selection of chromenone-based STS inhibitors. The
ratio kinact/KI, as well as the IC50 value, are measures
for the overall efficiency of an irreversible inhibitor; in
fact, we observed a linear correlation (r2 ) 0.962)
between these two parameters (Figure 3).
Con clu sion s
The KI value, i.e., the dissociation constant of the
noncovalent enzyme-inhibitor complex, is in the range
of 0.2-2 µM for most compounds tested here. In
contrast, compound 5b (with substituents on the chrome-
none skeleton in 2,7-position) and compounds 7d ,f
(which lack the keto function) have much higher KI
values (∼24-54 µM), indicating that these configura-
tions do not favor inhibitor binding.
We discovered novel nonsteroidal aryl sulfamates
featuring a (thio)chromenone ring system as potent STS
inhibitors and established the SAR of this compound
class. The bicyclic ring system apparently serves as an
estrone A,B-ring mimic, and the side chains, which have
to be situated diagonally opposite relative to the sulfa-
mate group (i.e., 2,6- or 3,7-substitution pattern), occupy
the space of the steroidal C,D-rings. Aliphatic side
chains yield higher activity than aromatic moieties, and
fully branched, bulky groups are superior to linear
chains. Most preferred are bicyclic and tricyclic aliphatic
The kinact value describes the rate of covalent reaction
of the inhibitor with the enzyme. The values determined
here are in the range between 0.006 and 0.12 s-1. Thus,

2-Substituted 4-(Thio)chromenone 6-O-Sulfamates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19 4315
Ta ble 3. Physicochemical Properties of Chromenone-Based STS Inhibitors 3-7
methoda/
yieldb (%)
mp (°C)
1H NMR (DMSO-d6) δ
3b
3c
3d
3e
3f
157-160
8.08 (br.s, 2H), 7.85 (d, J ) 2.8 Hz, 1H), 7.72 (d, J ) 9 Hz, 1H), 7.63 (dd, J ) 2.8 + 9 Hz, 1H),
7.26-7.40 (m, 5H), 6.31 (s, 1H), 4.06 (s, 2H)
B/58
A/44
A/60
A /61
B/75
185-188
155-158
148
8.14 (br.s, 2H), 7.88 (d, J ) 3 Hz, 1H), 7.86 (d, J ) 9.2 Hz, 1H), 7.68-7.80 (m, 4H),
7.42-7.53 (m, 3H), 7.27 (d, J ) 16.2 Hz, 1H), 6.54 (s, 1H)
8.11 (br.s, 2H), 7.83 (d, J ) 2.9 Hz, 1H), 7.74 (d, J ) 9 Hz, 1H), 7.65 (dd, J ) 2.9 + 9 Hz, 1H),
7.21-7.32 (m, 5H), 6.26 (s, 1H), 3.03 (s, 4H)
8.10 (br.s, 2H), 7.86 (d, J ) 2.8 Hz, 1H), 7.75 (d, J ) 9 Hz, 1H), 7.65 (dd, J ) 2.8 + 9 Hz, 1H),
6.30 (s, 1H), 2.66 (t, J ) 7.4 Hz, 2H), 1.72 (sext, J ) 7.4 Hz, 2H), 0.96 (t, J ) 7.4 Hz, 3H)
8.10 (br.s, 2H), 7.86 (d, J ) 2.9 Hz, 1H), 7.76 (d, J ) 9 Hz, 1H), 7.64 (dd, J ) 2.9 +9 Hz, 1H),
6.30 (s, 1H), 2.68 (t, J ) 7.5 Hz, 2H), 1.68 (qui, J ) 7.5 Hz, 2H), 1.20-1.38 (m, 12H),
0.85 (t, J ) 7.5 Hz, 3H)
8.07 (br. s, 2H), 7.86 (d, J ) 2.9 Hz, 1H), 7.78 (d, J ) 9 Hz, 1H), 7.66 (dd, J ) 2.9 +9 Hz, 1H),
6.25 (s, 1H), 1.62-1.71 (m, 2H), 1.29 (s, 6H), 1.13-1.23 (m, 12H), 0.80 (t, J ) 7 Hz, 3H)
8.10 (br s, 2H), 7.86 (d, J ) 2.9 Hz, 1H), 7.79 (d, J ) 9 Hz, 1H), 7.66 (dd, J ) 2.9 + 9 Hz, 1H),
6.28 (s, 1H), 1.33 (s, 9H)
92
3g
3h
3i
122
B/69
A/79
B/64
A/74
A/64
B/64
B/63
B/56
B/74
180
148
8.08 (br s, 2H), 7.85 (d, J ) 2.8 Hz, 1H), 7.76 (d, J ) 9 Hz, 1H), 7.62 (dd, J ) 2.8 + 9 Hz, 1H),
6.24 (s, 1H), 1.70 (s, 1H), 1.26 (s, 6H), 1.20 (s, 6H)
3j
158-160
170-171
168-170
185
8.10 (br s, 2H), 7.85 (d, J ) 2.8 Hz, 1H), 7.75 (d, J ) 9 Hz, 1H), 7.64 (dd, J ) 2.8 + 9 Hz, 1H),
6.30 (s, 1H), 3.04-3.20 (m, 1H), 1.95-2.09 (m, 2H), 1.61-1.85 (m, 6H)
3k
3l
8.09 (br s, 2H), 7.85 (d, J ) 2.8 Hz, 1H), 7.76 (d, J ) 9 Hz, 1H), 7.65 (dd, J ) 2.8 + 9 Hz, 1H),
6.24 (s, 1H), 2.64 (tt, J ) 3.2 + 11.3 Hz, 1H), 1.08-2.03 (m, 10H)
8.09 (br s, 2H), 7.83 (d, J ) 2.8 Hz, 1H), 7.76 (d, J ) 9 Hz, 1H), 7.64 (dd, J ) 2.8 + 9 Hz, 1H),
6.37 (s, 1H), 2.83 (qui, J ) 6. 3 Hz, 1H), 1.23-1.85 (m, 22H)
3m
3n
3o
8.08 (br s, 2H), 7.84 (d, J ) 2.8 Hz, 1H), 7.76 (d, J ) 9 Hz, 1H), 7.65 (dd, J ) 2.8 + 9 Hz, 1H),
6.17 (s, 1H), 1.78-1.88 (m, 6H), 1.40-1.49 (m, 6H), 1.08-1.34 (m, 8H), 0.86 (t, J ) 7.4 Hz, 3H)
8.08 (br s, 2H), 7.85 (d, J ) 2.9 Hz, 1H), 7.78 (d, J ) 9 Hz, 1H), 7.65 (dd, J ) 2.9 + 9 Hz, 1H),
6.18 (s, 1H), 2.08 (br.s, 3H), 1.91-1.97 (m, 6H), 1.67-1.81 (m, 6H)
166-168
165-167
8.09 (br s, 2H), 7.86 (d, J ) 2.8 Hz, 1H), 7.74 (d, J ) 9 Hz, 1H), 7.65 (dd, J ) 2.8 + 9 Hz, 1H),
6.27 (s, 1H), 2.70 (t, J ) 6.6 Hz, 1H), 2.36 (br.s, 2H), 2.05-2.13 (m, 2H), 1.61-1.99 (m, 8H)
8.60 (s, 1H), 8.34 (br.s, 2H), 8.24 (d, J ) 8.8 Hz, 1H), 7.55-7.63 (m, 3H), 7.38-7.49 (m, 4H)
8.28 (br s, 2H), 8.20 (s, 1H), 8.14 (d, J ) 8.7 Hz, 1H), 7.52 (d, J ) 2.2 Hz, 1H),
7.37 (dd, J ) 2.2 + 8.7 Hz, 1H), 2.62-2.75 (m, 1H), 1.65-1.83 (m, 5H), 1.24-1.40 (m, 5H)
8.30 (br.s, 2H), 8.09 (d, J ) 8.7 Hz, 1H), 7.52 (d, J ) 2.2 Hz, 1H), 7.36 (dd, J ) 2.2 + 8.7 Hz, 1H),
6.28 (s, 1H), 2.65 (t, J ) 7.4 Hz, 2H), 1.72 (sext, J ) 7.4 Hz, 2H), 0.96 (t, J ) 7.4 Hz, 3H)
8.29 (br.s, 2H), 8.08 (d, J ) 8.7 Hz, 1H), 7.54 (d, J ) 2.2 Hz, 1H), 7.35 (dd, J ) 2.2 + 8.7 Hz, 1H),
6.22 (s, 1H), 2.64 (tt, J ) 3.3 + 11.3 Hz, 1H), 1.18-2.03 (m, 10H)
8.24 (d, J ) 2.7 Hz, 1H), 8.20 (br.s, 2H), 8.12 (d, J ) 8.8 Hz, 1H), 7.82-7.89 (m, 2H),
7.70 (dd, J ) 2.7 + 8.8 Hz, 1H), 7.56-7.64 (m, 3H)
8.17 (d, J ) 2.7 Hz, 1H), 8.16 (br.s, 2H), 8.04 (d, J ) 8.8 Hz, 1H), 7.64 (dd, J ) 2.7 + 8.8 Hz, 1H),
6.98 (s, 1H), 1.39 (s, 9H)
4a
4b
183
160-163
C/34
C/27
5a
5b
6a
6b
6c
7a
7b
7c
148-150
185-187
247-250
150-153
220
A/42
A/54
D/52
D/85
D/54
61
8.16 (d, J ) 2.7 Hz, 1H), 8.14 (br.s, 2H), 8.02 (d, J ) 8.8 Hz, 1H), 7.64 (dd, J ) 2.7 + 8.8 Hz, 1H),
6.93 (s, 1H), 2.10 (br.s, 3H), 1.96-2.02 (m, 6H), 1.71-1.78 (m, 6H)
178-180
136-138
192-194
CDCl3/CD3OD ) 5/1: 7.82 (d, J ) 3 Hz, 1H), 7.37-7.55 (m, 6H), 7.12 (d, J ) 9 Hz, 1H),
5.52 (dd, J ) 3.2 + 13 Hz, 1H), 3.12 (dd, J ) 13 + 17 Hz, 1H), 2.93 (dd, J ) 3.2 + 17 Hz, 1H)
CDCl3: 7.73 (d, J ) 3 Hz, 1H), 7.45 (dd, J ) 3 + 9 Hz, 1H), 7.01 (d, J ) 9 Hz, 1H),
5.49 (br.s, 2H), 4.10-4.21 (m, 1H), 2.58-2.70 (m, 2H), 1.63-2.00 (m, 7H), 1.03-1.35 (m, 6H)
7.96 (br.s, 2H), 7.58 (d, J ) 3 Hz, 1H), 7.43 (dd, J ) 3 + 9 Hz, 1H), 7.15 (d, J ) 9 Hz, 1H),
4.04 (dd, J ) 2.5 + 14 Hz, 1H), 2.83 (dd, J ) 14 + 16.7 Hz, 1H), 2.58 (dd, J ) 2.5 + 16.7 Hz, 1H),
2.01 (br.s, 3H), 1.55-1.78 (m, 12H)
20
49
7d
7e
147-149
Foam
133
7.82 (br.s, 2H), 7.30 (d, J ) 2.9 Hz, 1H), 6.99 (dd, J ) 2.9 + 8.8 Hz, 1H), 6.75 (d, J ) 8.8 Hz, 1H),
5.56 (d, J ) 6.3 Hz, 1H), 4.68-4.80 (m, 1H), 3.89-3. 98 (m, 1H), 2.06-2.17 (m, 1H),
1.49-1.92 (m, 7H), 1.00-1.34 (m, 5H)
73
31
8
7.82 (br.s, 2H), 7.30 (d, J ) 2.9 Hz, 1H), 6.99 (dd, J ) 2.9 + 8.8 Hz, 1H), 6.76 (d, J ) 8.8 Hz, 1H),
5.55 (d, J ) 6.3 Hz, 1H), 4.69-4.80 (m, 1H), 3.65 (d, J ) 11.1 Hz, 1H),
2.13 (dd, J ) 5.6 + 12. 4 Hz, 1H), 1.99 (br.s, 3H), 1.49-1.77 (m, 12H)
CDCl3: 6.97-7.04 (m, 2H), 6.74-6.81 (m, 1H), 4.98 (br.s, 2H), 3.72 (ddd, J ) 2.1 + 6.2 + 10 Hz, 1H),
2.68-2.89 (m, 2H), 1.02-2.05 (m, 13 H)
7f
a
b
Method used for the synthesis of the phenolic key intermediates 8-11. Yields (not optimized) of isolated, analytically pure products.
R groups such as the adamantyl residue in compounds
3n and 6c. Inhibitory potency is further increased by
replacing the chromenone with the thiochromenone core
element. 2-(1-Adamantyl)-4H-thiochromenone 6-O-sulf-
amate (6c) meets all of the defined criteria for high
activity and is the most potent inhibitor discovered so
far (about 170-fold superior to estrone sulfamate).
Like estrone sulfamate and related aryl sulfamates,
these compounds are irreversible inhibitors of STS with
a biphasic course of inactivation. When comparing
highly and weakly potent analogues, the differences in
KI values are much more pronounced than those in kinact
values. This indicates that the binding to the enzyme,
rather than the reactivity of the subsequent covalent
modification, is the dominating factor for the overall
efficiency of the chromenone-based inhibitors.
Exp er im en ta l Section
A. Ch em ica l Syn th esis. 1. Gen er a l Meth od s. 6-Hydroxy-
2-phenyl-4H-1-benzopyran-4-one (8a ) was purchased from
Aldrich. 6-Hydroxy-2-phenyl-4H-1-thiobenzopyran-4-one (11a )
was synthesized according to a published procedure,30 and
amidochlorosulfonic acid was prepared according to the method
of Appel and Berger.26

4316 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19
Nussbaumer et al.
Melting points were determined on a Reichert Thermovar
microscope and are not corrected. The temperature is given
in Celsius units. Thin-layer chromatography was performed
using silica gel F254 plates (Merck) detection by UV light or
potassium permanganate. Column chromatography was per-
formed using silica gel 60 (0.040-0.063 mm; Merck), pressure
3-5 bar. NMR spectra were recorded at 250 and 400 MHz,
respectively (Bruker WM 250 and Bruker Avance 400 spec-
trometer) with (CH3)4Si as internal standard. Chemical shifts
are given in δ units. Elemental analyses were performed by
Novartis Services AG and Solvias AG, repectively, both in
Basle, Switzerland.
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 3-6. Su lfa m oyla tion Rea ction . Syn th esis of Su l-
fam ic Acid 2-(1,1-Dim eth yleth yl)-4-oxo-4H-1-ben zopyr an -
6-yl Ester (3h ). Sodium hydride (95%, 0.6 g, 23.8 mmol) was
carefully added to a solution of 8h (4 g, 18.3 mmol) in dry DMF
(40 mL). After it was stirred for 30 min at room temperature,
the mixture was cooled (ice/water) and amidochlorosulfonic
acid26 (6.3 g, 55 mmol) was added in portions. The mixture
was stirred for an additional 3 h at room temperature and
subsequently concentrated in vacuo. The residue was parti-
tioned between water and ethyl acetate, and after the layers
were separated, the aqueous layer was extracted with ethyl
acetate. The combined organic layers were dried (MgSO4) and
evaporated to give the crude product, which was purified either
by crystallization from toluene or by chromatography on silica
gel (cyclohexane/ethyl acetate ) 2/1) to yield the sulfamate
3h (4.3 g, 79%) as colorless crystals; mp 180 °C. 13C NMR
(DMSO-d6): δ 177.1, 176.6, 154.3, 147.2, 129.2, 123.9, 120.7,
117.8, 106.1, 36.8, 27.9.
3. Gen er a l P r oced u r e for th e Syn th esis of Test Com -
p ou n d s 7a -f. Hyd r ogen a tion . Syn th esis of Su lfa m ic
Acid 2-Cycloh exyl-3,4-d ih yd r o-4-oxo-2H-1-ben zop yr a n -
6-yl Ester (7b), Su lfa m ic Acid 2-Cycloh exyl-3,4-d ih yd r o-
4-h yd r oxy-2H-1-ben zop yr a n -6-yl Ester (7d ), a n d Su lfa m -
ic Acid 2-Cycloh exyl-3,4-d ih yd r o-2H-1-ben zop yr a n -6-yl
Ester (7f). A solution of 3k (83 mg, 0.26 mmol) in ethyl acetate
was hydrogenated over palladium (8 mg, 10% on charcoal) at
atmospheric pressure and room temperature for 3 h. The
mixture was filtered over Celite, and the filtrate was evapo-
rated in vacuo. The residue was chromatographed on silica
gel (cyclohexane/ethyl acetate ) 2/1) to give 7b (17 mg, 20%)
as the first fraction, followed by 7d (61 mg, 73%) as the second
fraction. When the reaction time was extended to 14 h, a small
amount of 7f (8%) was isolated after chromatography as
colorless crystals, mp 133 °C, in addition to 7d as the main
product. Analogously to the synthesis of 7b,d , test compounds
7a ,c,e were prepared starting from 3a ,n , respectively. Com-
pound 3d was synthesized in 60% yield by the same procedure
starting from 3c, but the reaction time was limited to 40 min
in order to achieve a selective reduction of the exocyclic double
bond. Compound 3d was purified by chromatography followed
by crystallization from toluene; mp 155-158 °C.
4. Syn t h esis of St a r t in g Ma t er ia ls. 4.1. Met h od A.
Syn th esis of 2-(1,1-Dim eth yleth yl)-6-h yd r oxy-4H-1-ben -
zop yr a n -4-on e (8h ). 4.1.1. Syn th esis of 2,2-Dim eth ylp r o-
p a n oic Acid 2-Acetyl-1,4-p h en ylen e Ester (13, R ) t-Bu -
tyl). A solution of 12 (5 g, 32.8 mmol) in dry pyridine (35 mL)
was treated with pivaloyl chloride (10 g, 83 mmol) under
cooling with an ice-bath to keep the reaction temperature at
about 20 °C. After it was stirred for 18 h at room temperature,
the solvent was partially distilled off in vacuo. The residue
was poured onto ice and hydrochloric acid (32%, 30 mL) and
extracted with diethyl ether. The combined organic layers were
washed with aqueous sodium carbonate solution and water,
dried (MgSO4), and evaporated to give the crude title com-
pound (10.6 g, 100%), which was used in the next step without
further purification, as yellow crystals. 1H NMR (CDCl3): δ
7.45 (d, J ) 2.8 Hz, 1H), 7.23 (dd, J ) 2.8 + 8.7 Hz, 1H), 7.04
(d, J ) 8.7 Hz, 1H), 2.54 (s, 3H), 1.38 (s, 9H), 1.36 (s, 9H).
dissolved in dry DMF (30 mL) and added slowly at 0 °C under
argon to a suspension of sodium hydride (95% pure, 870 mg,
34.4 mmol) in dry DMF (30 mL). After the mixture was stirred
for an additional 2 h at 0-5 °C, acetic acid (2.5 mL) was added
cautiously to the mixture, and then, it was poured into water
(300 mL) and extracted with ethyl acetate (3 × 70 mL). The
combined organic layers were washed with saturated aqueous
sodium chloride solution, dried (MgSO4), and evaporated to
yield the crude title compound (11 g, ∼100%) as an orange
semicrystalline mass, which was used in the next step without
further purification.
4.1.3. 2,2-Dim eth ylpr opan oic Acid 2-(1,1-Dim eth yleth yl)-
4-oxo-4H-1-ben zop yr a n -6-yl Ester . A solution of crude 14
(R ) tert-butyl; 4.33 g, 13.6 mmol) in methanol (30 mL) was
treated with concentrated aqueous hydrochloric acid (15 mL).
Then, dioxane was added until a clear solution was achieved.
The mixture was stirred at ambient temperature for 3 h,
poured into water, and extracted with ethyl acetate. The
combined organic layers were washed cautiously with aqueous
sodium carbonate solution, dried over magnesium sulfate, and
evaporated in vacuo. The crude product was either directly
used in the next step or purified by chromatography on silica
gel to give the title compound as colorless crystals; mp 84-86
1
°C. H NMR (CDCl3): δ 7.82 (d, J ) 2.8 Hz, 1H), 7.48 (d, J )
9 Hz, 1H), 7.36 (dd, J ) 2.8 + 9 Hz, 1H), 6.28 (s, 1H), 1.37 (s,
9H), 1.36 (s, 9H).
4.1.4. Syn th esis of 2-(1,1-Dim eth yleth yl)-6-h yd r oxy-4H-
1-ben zop yr a n -4-on e (8h ). 2,2-Dimethylpropanoic acid 2-(1,1-
dimethylethyl)-4-oxo-4H-1-benzopyran-6-yl ester (2 g, 6.61
mmol) was dissolved in dioxane (60 mL) and treated with 2 N
aqueous sodium hydroxide solution (12 mL, 24 mmol). The
mixture was heated to 60 °C overnight, then poured into water,
and washed with ethyl acetate. The aqueous layer was
acidified with 15% aqueous hydrochloric acid followed by
extraction with ethyl acetate. Subsequent drying over mag-
nesium sulfate and evaporation yielded 8h (1.34 g, 93%) as
1
colorless crystals; mp 170 °C. H NMR (CDCl3): δ 8.10 (br s,
1H), 7.88 (d, J ) 3 Hz, 1H), 7.39 (d, J ) 9 Hz, 1H), 7.28 (dd,
J ) 3 + 9 Hz, 1H), 6.30 (s, 1H), 1.36 (s, 9H).
Alternatively, the ring closure of crude 14 to the correspond-
ing O-acylated hydroxy-4H-1-benzopyran-4-ones can be achieved
by heating in formic acid to 100 °C (see also method B), and
further transformation of these intermediates into compounds
8 is also possible by acidic ester cleavage using concentrated
aqueous hydrochloric acid in methanol and/or dioxane. For the
synthesis of compound 8h from 14h (R ) tert-butyl), a one
pot procedure was also developed.
4.1.5. Alter n a tive Syn th esis of 2-(1,1-Dim eth yleth yl)-
6-h yd r oxy-4H-1-ben zop yr a n -4-on e (8h ). Crude 14 (R ) tert-
butyl; 10.4 g, 30.2 mmol) was heated in formic acid (60 mL) to
100 °C for 1 h. Then, hydrochloric acid (32%, 10 mL) and water
(5 mL) were added and the mixture was stirred for an
additional hour at 100 °C. The cooled mixture was poured onto
ice/water (500 mL) and extracted with ethyl acetate. The
combined organic layers were washed cautiously with satu-
rated aqueous sodium bicarbonate solution (3 × 100 mL), dried
over magnesium sulfate, and concentrated in vacuo. The dark
green residue was chromatographed on silica gel (cyclohexane/
ethyl acetate ) 2/1) to give 8h (4.9 g, 74%).
Analogously, the following compounds were prepared.
(E)-6-Hyd r oxy-2-(2-p h en yleth en yl)-4H-1-ben zop yr a n -
4-on e (8c). Yield 93%, yellowish crystals, mp 219-222 °C. 1H
NMR (CDCl3): δ 8.93 (br.s, 1H), 7.52-7.64 (m, 4H), 7.35-7.44
(m, 4H), 7.24 (dd, J ) 3 + 9 Hz, 1H), 6.77 (d, J ) 16 Hz, 1H),
6.28 (s, 1H).
In ter m ed ia te: (E,E)-3-P h en ylp r op en oic Acid 2-(2-P h e-
n yleth en yl)-4-oxo-4H-1-ben zop yr a n -6-yl Ester . Yield 95%,
1
yellow crystals, mp 179-181 °C. H NMR (CDCl3): δ 7.99 (d,
J ) 2.8 Hz, 1H), 7.92 (d, J ) 16 Hz, 1H), 7.56-7.67 (m, 6H),
7.54 (dd, J ) 2.8 + 9 Hz, 1H), 7.38-7.48 (m, 6H), 6.81 (d, J )
16 Hz, 1H), 6.66 (d, J ) 16 Hz, 1H), 6.35 (s, 1H).
4.1.2. Syn th esis of 2,2-Dim eth ylp r op a n oic Acid 3-(4,4-
Dim eth yl-1,3-d ioxop en tyl)-4-h yd r oxyp h en yl Ester (14, R
) t-Bu tyl). Crude 13 (R ) tert-butyl; 10.5 g, 32.8 mmol) was
6-Hyd r oxy-2-p r op yl-4H-1-ben zop yr a n -4-on e (8e). Yield
86%, colorless crystals, mp 150 °C. 1H NMR (DMSO-d6): δ 9.97
(br.s, 1H), 7.47 (d, J ) 9 Hz, 1H), 7.27 (d, J ) 3 Hz, 1H), 7.19

2-Substituted 4-(Thio)chromenone 6-O-Sulfamates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19 4317
(dd, J ) 3 + 9 Hz, 1H), 6.15 (s, 1H), 2.60 (t, J ) 7.4 Hz, 2H),
1.69 (sext, J ) 7.4 Hz, 2H), 0.94 (t, J ) 7.4 Hz, 3H).
2-Cyclop en tyl-6-h yd r oxy-4H-1-ben zop yr a n -4-on e (8j).
Yield 93%, colorless crystals, mp 174 °C. 1H NMR (DMSO-
d6): δ 7.47 (d, J ) 9 Hz, 1H), 7.27 (d, J ) 3 Hz, 1H), 7.18 (dd,
J ) 3 + 9 Hz, 1H), 6.09 (s, 1H), 2.97-3.13 (m, 1H), 1.92-2.08
(m, 2H), 1.58-1.82 (m, 6H).
10% aqueous potassium hydroxide solution in dioxane as
described for the synthesis of 8h (method A) gave the title
compound in 93% yield as colorless crystals; mp 104 °C. 1H
NMR (DMSO-d6): δ 9.96 (s, 1H), 7.47 (d, J ) 9 Hz, 1H), 7.27
(d, J ) 3 Hz, 1H), 7.18 (dd, J ) 3 + 9 Hz, 1H), 6.15 (s, 1H),
2.61 (t, J ) 7.5 Hz, 2H), 1.65 (qui, J ) 7.5 Hz, 2H), 1.16-1.38
(m, 12H), 0.84 (t, J ) 7.5 Hz, 3H).
Analogously, the following compounds were prepared.
In ter m ed ia te: Cyclop en ta n eca r boxylic Acid 2-Cyclo-
p en tyl-4-oxo-4H-1-ben zop yr a n -6-yl Ester . Yield 56%, col-
orless crystals, mp 71 °C. 1H NMR (CDCl3): δ 7.83 (d, J ) 2.7
Hz, 1H), 7.45 (d, J ) 9 Hz, 1H), 7.36 (dd, J ) 2.7 + 9 Hz, 1H),
6.21 (s, 1H), 2.93-3.07 (m, 2H), 1.60-2.13 (m, 16H).
2-Cycloh exyl-6-h yd r oxy-4H-1-ben zop yr a n -4-on e (8k ).
2-Ben zyl-6-h yd r oxy-4H-1-ben zop yr a n -4-on e (8b). Yield
1
79%. H NMR (CDCl3): δ 9.10 (br.s, 1H), 7.50 (d, J ) 2.9 Hz,
1H), 7.20-7.35 (m, 6H), 7.18 (dd, J ) 2.9 + 9 Hz, 1H), 6.03 (s,
1H), 3.91 (s, 2H).
2-(1,1-Dim eth yln on yl)-6-h yd r oxy-4H-1-ben zop yr a n -4-
1
1
on e (8g). Yield 83%. H NMR (CDCl3): δ 7.94 (d, J ) 3 Hz,
Yield 94%, colorless crystals, mp 171 °C. H NMR (CDCl3): δ
1H), 7.40 (d, J ) 9 Hz, 1H), 7.29 (dd, J ) 3 + 9 Hz, 1H), 6.31
(s, 1H), 1.62-1.72 (m, 2H), 1.32 (s, 6 H), 1.20-1.29 (m, 12H),
0.84 (t, J ) 7 Hz, 3H).
8.23 (br. s, 1H), 7.92 (d, J ) 2.9 Hz, 1H), 7.37 (d, J ) 9 Hz,
1H), 7.26 (dd, J ) 2.9 + 9 Hz, 1H), 6.20 (s, 1H), 2.54 (tt, J )
3.3 + 11.5 Hz, 1H), 1.96-2.08 (m, 2H), 1.72-1.92 (m, 4H),
1.25-1.58 (m, 4H).
6-Hydr oxy-2-(2,2,3,3-tetr am eth ylcyclopr opyl)-4H-1-ben -
zop yr a n -4-on e (8i). Yield 69%, colorless crystals, 173 °C (from
In ter m ed ia te: Cycloh exa n eca r boxylic Acid 2-Cyclo-
h exyl-4-oxo-4H-1-ben zop yr a n -6-yl Ester . Yield 51%, color-
1
toluene). H NMR (DMSO-d6): δ 9.96 (s, 1H), 7.46 (d, J ) 9
1
Hz, 1H), 7.26 (d, J ) 3 Hz, 1H), 7.16 (dd, J ) 3 + 9 Hz, 1H),
6.09 (s, 1H), 1.63 (s, 1H), 1.23 (s, 6H), 1.17 (s, 6H).
2-Cyclod od ecyl-6-h yd r oxy-4H-1-ben zop yr a n -4-on e (8l).
Yield 78%, colorless crystals, mp 219 °C. 1H NMR (DMSO-
d6): δ 10.02 (br.s, 1H), 7.47 (d, J ) 9 Hz, 1H), 7.27 (d, J ) 3
Hz, 1H), 7.18 (dd, J ) 3 + 9 Hz, 1H), 6.20 (s, 1H), 2.76 (qui, J
) 6.2 Hz, 1H), 1.22-1.86 (m, 22H).
less crystals, mp 104-106 °C. H NMR (CDCl3): δ 7.83 (d, J
) 2.8 Hz, 1H), 7.45 (d, J ) 9 Hz, 1H), 7.35 (dd, J ) 2.8 + 9
Hz, 1H), 6.16 (s, 1H), 2.47-2.66 (m, 2H), 2.02-2.14 (m, 4H),
1.23-1.95 (m, 16H).
7-Hydr oxy-2-pr opyl-4H-1-ben zopyr an -4-on e (10a). Yield
89%, colorless crystals, mp 140-143 °C. 1H NMR (DMSO-d6):
δ 10.75 (br.s, 1H), 7.84 (d, J ) 8.7 Hz, 1H), 6.88 (dd, J ) 2.2
+ 8.7 Hz, 1H), 6.80 (d, J ) 2.2 Hz, 1H), 6.08 (s, 1H), 2.57 (t, J
) 7.5 Hz, 2H), 1.68 (sext, J ) 7.5 Hz, 2H), 0.94 (t, J ) 7.5 Hz,
3H).
2-Cycloh exyl-7-h yd r oxy-4H-1-ben zop yr a n -4-on e (10b).
Yield 84%, colorless crystals, mp 197 °C. 1H NMR (DMSO-
d6): δ 10.78 (br.s, 1H), 7.83 (d, J ) 8.7 Hz, 1H), 6.88 (dd, J )
2.2 + 8.7 Hz, 1H), 6.81 (d, J ) 2.2 Hz, 1H), 6.02 (s, 1H), 2.47-
2.60 (m, 1H), 1.20-1.98 (m, 10H).
In ter m ed ia te: 6-Ben zoyloxy-2-cyclod od ecyl-4H-1-ben -
zop yr a n -4-on e. Yield 42%, colorless crystals, mp 105-107 °C.
1H NMR (CDCl3): δ 8.17-8.26 (m 2H), 7.97-8.02 (m, 1H),
7.62-7.72 (m, 1H), 7.47-7.58 (m, 5H), 6.20 (s, 1H), 2.78 (qui,
J ) 6.5 Hz, 1H), 1.32-1.90 (m, 22H).
6-Hyd r oxy-2-(4-p en tylbicyclo[2.2.2]oct-1-yl)-4H-1-ben -
zop yr a n -4-on e (8m ). Yield 88%, colorless crystals, 180-182
1
°C. H NMR (DMSO-d6): δ 9.96 (br.s, 1H), 7.47 (d, J ) 9 Hz,
1H), 7.25 (d, J ) 3 Hz, 1H), 7.18 (dd, J ) 3 + 9 Hz, 1H), 6.03
(s, 1H), 1.75-1.86 (m, 6H), 1.37-1.50 (m, 6H), 1.04-1.35 (m,
8H), 0.86 (t, J ) 7 Hz, 3H).
In ter m ed ia te: Cycloh exa n eca r boxylic Acid 2-Cyclo-
h exyl-4-oxo-4H-1-ben zop yr a n -7-yl Ester . Yield 54%, color-
less crystals, mp 110 °C. H NMR (CDCl3): δ 8.19 (d, J ) 8.7
Hz, 1H), 7.24 (d, J ) 2.2 Hz, 1H), 7.08 (dd, J ) 2.2 + 8.7 Hz,
1H), 6.15 (s, 1H), 2.60 (tt, J ) 3.7 + 11 Hz, 1H), 2.51 (tt, J )
3.3 + 11 Hz, 1H), 1.20-2.12 (m, 20H).
1
In ter m ed ia te: 6-Ben zoyloxy-2-(4-p en tylbicyclo[2.2.2]-
oct-1-yl)-4H-1-ben zop yr a n -4-on e. Yield 49%, colorless crys-
1
tals, mp 124-126 °C. H NMR (CDCl3): δ 8.17-8.25 (m 2H),
7.96-8.00 (m, 1H), 7.61-7.72 (m, 1H), 7.47-7.58 (m, 5H), 6.20
(s, 1H), 1.82-1.95 (m, 6H), 1.43-1.56 (m, 6H), 1.08-1.37 (m,
8H), 0.89 (t, J ) 7 Hz, 3H).
4.2. Meth od B. Syn th esis of 6-Hyd r oxy-2-n on yl-4H-1-
ben zop yr a n -4-on e (8f). 4.2.1. Syn th esis of 5-Ben zoyloxy-
2-d eca n oyloxya cetop h en on e. Decanoyl chloride (3.8 g, 20
mmol) was added to a solution of 159 (5.1 g, 20 mmol) in dry
pyridine (50 mL). The solution was stirred for an additional 3
h at room temperature and then poured into 15% aqueous
hydrochloric acid (300 mL). Extraction with ethyl acetate,
followed by washing with aqueous sodium carbonate solution,
drying over magnesium sulfate, and evaporation yielded the
crude product (8.2 g, 100%), which was used in the next step
without further purification, as yellowish crystals. 1H NMR
(CDCl3): δ 8.18-8.23 (m, 2H), 7.62-7.72 (m, 1H), 7.67 (d, J
) 2.8 Hz, 1H), 7.48-7.58 (m, 2H), 7.41 (dd, J ) 2.8 + 8.7 Hz,
1H), 7.18 (d, J ) 8.7 Hz, 1H), 2.64 (t, J ) 7.5 Hz, 2H), 2.56 (s,
3H), 1.78 (qui, J ) 7.5 Hz, 2H), 1.22-1.42 (m, 12H), 0.89 (t, J
) 7.5 Hz, 3H).
4.2.2. 1-(5-Ben zoyloxy-2-h yd r oxyp h en yl)-1,3-d od eca n e-
d ion e (16, R ) Non yl). Following the procedure described
for the synthesis of 14, the title compound was obtained as
crude product from 5-benzoyloxy-2-decanoyloxyacetophenone
and used in the next step without further purification.
4.2.3. 6-Ben zoyloxy-2-n on yl-4H -1-b en zop yr a n -4-on e.
Crude 16 (R ) nonyl, 8.2 g, 20 mmol) was heated in formic
acid (50 mL) to 100 °C for 45 min. After it was cooled, the
solvent was distilled off in vacuo and the residue was chro-
matographed on silica gel (cyclohexane/ethyl acetate ) 8/1)
to give the protected chromenone (3.95 g, 50%) as colorless
crystals; mp 80 °C. 1H NMR (CDCl3): δ 8.18-8.23 (m, 2H),
8.00 (dd, J ) 1 + 2.3 Hz, 1H), 7.62-7.72 (m, 1H), 7.48-7.58
(m, 4H), 6.20 (s, 1H), 2.64 (t, J ) 7.5 Hz, 2H), 1.75 (qui, J )
7.5 Hz, 2H), 1.22-1.42 (m, 12H), 0.88 (t, J ) 7.5 Hz, 3H).
4.2.4. 6-Hyd r oxy-2-n on yl-4H-1-ben zop yr a n -4-on e (8f).
Treatment of 6-benzoyloxy-2-nonyl-4H-1-benzopyran-4-one with
6-Hydr oxy-2-(tr icyclo[3.3.1.13,7]dec-1-yl)-4H-1-ben zopy-
r a n -4-on e (8n ). Yield 95%, colorless crystals, mp 232 °C
1
(literature33 232-235 °C). H NMR (DMSO-d6): δ 9.98 (br.s,
1H), 7.49 (d, J ) 9 Hz, 1H), 7.26 (d, J ) 3 Hz, 1H), 7.19 (dd,
J ) 3 + 9 Hz, 1H), 6.04 (s, 1H), 2.02-2.12 (m, 3H), 1.88-1.96
(m, 6H), 1.66-1.78 (m, 6H).
In ter m ed ia te: 6-Ben zoyloxy-2-(tr icyclo[3.3.1.13,7]d ec-
1-yl)-4H-1-ben zop yr a n -4-on e. Yield 47%, colorless crystals,
1
mp 185 °C. H NMR (DMSO-d6): δ 8.15-8.23 (m 2H), 7.84-
7.88 (m, 1H), 7.72-7.83 (m, 3H), 7.58-7.68 (m, 2H), 6.18 (s,
1H), 2.04-2.13 (m, 3H), 1.95-2.01 (m, 6H), 1.70-1.80 (m, 6H).
2-[H exa h yd r o-2,5-m et h a n op en t a len -3a (1H )-yl]-6-h y-
d r oxy-4H-1-ben zop yr a n -4-on e (8o). Yield 95%, colorless
crystals, mp 190 °C. 1H NMR (DMSO-d6): δ 9.98 (br.s, 1H),
7.46 (d, J ) 9 Hz, 1H), 7.27 (d, J ) 3 Hz, 1H), 7.18 (dd, J ) 3
+ 9 Hz, 1H), 6.13 (s, 1H), 2.66 (t, J ) 6.6 Hz, 1H), 2.34 (br.s,
2H), 2.02-2.12 (m, 2H), 1.80-1.92 (m, 4H), 1.62-1.74 (m, 4H).
In ter m ed ia te: 6-Ben zoyloxy-2-[Hexa h yd r o-2,5-m eth a -
n op en ta len -3a (1H)-yl]-4H-1-ben zop yr a n -4-on e. Yield 71%,
1
colorless crystals, mp 200 °C. H NMR (CDCl3): δ 8.18-8.26
(m 2H), 8.00-8.03 (m, 1H), 7.48-7.71 (m, 5H), 6.27 (s, 1H),
2.76 (t, J ) 6.6 Hz, 1H), 2.42 (br.s, 2H), 2.10-2.18 (m, 2H),
1.84-1.98 (m, 4H), 1.64-1.78 (m, 4H).
4.3. Meth od C. Syn th esis of 3-Cycloh exyl-7-h yd r oxy-
4H-1-ben zop yr a n -4-on e (9b). 4.3.1. Syn th esis of 2-Cyclo-
h exyl-1-(2,4-d ih yd r oxyp h en yl)eth a n on e (19b). A suspen-
sion of 17 (1.54 g, 14.1 mmol) in dry dichloroethane was cooled
to 0-5 °C and treated with aluminum chloride (1.87 g, 14.1
mmol). Cyclohexaneacetyl chloride (2.3 g, 14.1 mmol) was
added slowly, and then, the mixture was stirred for 2 h at room

4318 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 19
Nussbaumer et al.
temperature and for 2 h at 70 °C. The cooled mixture was
poured into 1 N aqueous hydrochloric acid and extracted with
ethyl acetate. The combined organic layers were dried over
magnesium sulfate and evaporated. The residue was chro-
matographed on silica gel (cyclohexane/ethyl acetate ) 4/1)
to afford 19b (1.31 g, 40%) as colorless crystals; mp 98-100
°C. 1H NMR (CDCl3): δ 12.8 (s, 1H), 7.63-7.69 (m, 1H), 6.35-
6.40 (m, 2H), 2.74 (d, J ) 6.9 Hz, 2H), 1.62-1.98 (m, 7H),
0.94-1.37 (m, 4H).
4.3.2. 3-Cycloh exyl-7-h yd r oxy-4H-1-ben zop yr a n -4-on e
(9b). A mixture of 19b (1.30 g, 5.59 mmol), triethyl orthofor-
mate (1.24 g, 8.38 mmol), morpholine (2 g, 23 mmol), and DMF
(8 mL) was heated to 150 °C for 2 h. Then, the solvent was
distilled off and the residue was partitioned between water
and ethyl acetate. The organic layer was separated, dried over
magnesium sulfate, and concentrated in vacuo. The residue
was chromatographed on silica gel (cyclohexane/ethyl acetate
) 3/1) to yield 9b (169 mg, 12%). 1H NMR (DMSO-d6): δ 10.70
(br.s, 1H), 7.99 (s, 1H), 7.88 (d, J ) 8.8 Hz, 1H), 6.89 (dd, J )
2.3 + 8.8 Hz, 1H), 6.79 (d, J ) 2.3 Hz, 1H), 2.52-2.73 (m, 1H),
1.60-1.80 (m, 5H), 1.05-1.45 (m, 5H).
the cells was solubilized using Triton X-100. The protein then
was carried through a sequence of anion exchange and affinity
chromatography (DEAE Sephacel, ConA Sepharose, Blue
Sepharose); further purification was achieved on PBE94
chromatofocusing and Sephadex G-100 gel filtration columns.
The purity of the final enzyme preparation was estimated to
>95%. The identity of the protein was confirmed by its reaction
with anti-STS polyclonal and monoclonal antibodies in enzyme-
linked immunosorbent assay (ELISA) and on Western Blots
(kindly provided by Dr. J .-I. Kawano, Miyazaki, J apan)35 and
by N-terminal sequencing.
2. Assa y of P u r ified Hu m a n STS. Sulfatase activity was
assessed using a method originally described by Eto et al.36
with modifications. The assay was conducted in white 96 well
plates (Packard). Enzyme (0.6 units in 50 µL buffer; one unit
is defined as the amount of enzyme producing 1 nmol of
4-methylumbelliferone) was added to 100 µL of 0.75 mM
4-MUS (Sigma) solution in buffer (0.1 M Tris-HCl, pH 7.5,
0.1% Triton). After it was incubated for 60 min at 37 °C, 100
µL of 0.2 M NaOH was added and fluorescence was measured
(λex ) 355 nm, λem ) 448 nm) using a Titertek reader. For
inhibition studies, compounds were included at graded con-
centrations from stock solutions in ethanol; the final ethanol
content did not exceed 2%. IC50 values were calculated using
nonlinear regression (GraFit, Erithacus Software Ltd.). Values
reported here are the mean of triplicate determinations, which
typically lie in the range of (20%.
3. Micr osom es fr om Hu m a n P la cen ta . Placenta obtained
fresh after delivery was stripped of membranes and frozen at
-70 °C for storage. After it was thawed, the tissue was cut
into pieces and rinsed in 20 mM Tris-HCl, pH 7.4, 0.25 M
sucrose, and 0.02% azide. The pieces were blotted on filter
paper and then homogenized using an Ultraturax in 2 volumes
of 50 mM Tris-HCl, pH 7.8. After the homogenate was
centrifuged at 10 000g for 1 h, 4 °C, the supernatant was
collected and the pellet was frozen again at -70 °C. The pellet
was thawed and extracted again, and the whole procedure was
repeated a third time. The supernatants were pooled and
centrifuged at 100 000g, 4 °C, for 1 h. The microsomal pellet
was suspended in 50 mM Tris-HCl, pH 7.8, and the protein
concentration of the suspension was adjusted to about 10 mg/
mL by dilution with the buffer.
4. Deter m in a tion of KI a n d kin a ct. A 25 µL amount of
microsomal suspension was added to 75 µL of inhibitor dilution
in 0.1 M Tris-HCl, pH 7.5. The reaction was allowed to proceed
for various periods (1-20 min) and was then stopped by adding
a suspension of dextran-coated charcoal (5%/0.5%) in buffer.
The samples were then centrifuged at 2000g for 1 min. A 100
µL amount of supernatant was transferred to white 96 well
plates (Packard) followed by 75 µL of substrate solution (1.5
mM 4-MUS in 0.1 M Tris-HCl, pH 7.5). Plates were incubated
at 37 °C for 20 min. Then, the assay was stopped by addition
of 50 µL of 0.2 M NaOH. Fluorescence intensity was measured
in a Titertek instrument with λex ) 355 nm and λem ) 460
nm. The fluorescence data were normalized to the intensity
of the uninhibited reaction and plotted against the reaction
time. Data were fitted to a double-exponential decay using the
software Grafit, and the apparent rate constant for the first
phase (kapp) was obtained. As shown by Kitz and Wilson,32 the
relation kapp ) kinact/(1 + KI/[I]) describes the kinetics of
irreversible enzyme inhibition, where [I] is the inhibitor
concentration, KI is the inhibition constant, and kinact is the
rate constant of inactivation. KI and kinact were obtained from
a double-reciprocal plot of kapp vs [I]. Finally, the ratio kinact/KI
was calculated as a measure for the overall efficiency of
inactivation.
Analogously, 19a was prepared starting from 17 and phe-
nylacetyl chloride (18a ) in 70% yield and then converted into
9a , which was obtained in 47% yield as colorless crystals; mp
202-205 °C (literature34 212-214 °C).
4.4. Meth od D. Syn th esis of 6-Hyd r oxy-2-(tr icyclo-
[3.3.1.13,7]dec-1-yl)-4H-1-ben zoth iopyr an -4-on e (11c). 4.4.1.
Syn th esis of 6-Meth oxy-2-(tr icyclo[3.3.1.13,7]d ec-1-yl)-
4H-1-ben zoth iop yr a n -4-on e. 4-Methoxybenzenethiol (20; 1.4
g, 10 mmol) was added to polyphosphoric acid (12 mL)
preheated to 90 °C under mechanical stirring. At this tem-
perature, ethyl 3-(1-adamantyl)-3-oxopropionate (21c; 2.5 g,
10 mmol) was added very slowly to the mixture and stirring
was continued for 30 min after the addition. The cooled
mixture was vigorously stirred with ice/water and extracted
with ethyl acetate. The combined organic layers were washed
with brine, dried over magnesium sulfate, and evaporated in
vacuo. The crude product was purified by silica gel chroma-
tography (cyclohexane/ethyl acetate ) 6/1) to give the title
compound as colorless crystals (2.09 g, 64%); mp 140 °C. 1H
NMR (CDCl3): δ 7.93 (d, J ) 2.9 Hz, 1H), 7.53 (d, J ) 8.8 Hz,
1H), 7.22 (dd, J ) 2.9 + 8.8 Hz, 1H), 7.00 (s, 1H), 3.92 (s, 3H),
2.08-2.18 (m, 3H), 1.98-2.06 (m, 6H), 1.68-1.87 (m, 6H).
4.4.2. Syn th esis of 6-Hyd r oxy-2-(tr icyclo[3.3.1.13,7]d ec-
1-yl)-4H-1-ben zoth iopyr an -4-on e (11c). 6-Methoxy-2-(tricyclo-
[3.3.1.13,7]dec-1-yl)-4H-1-benzothiopyran-4-one (1.1 g, 3.5 mmol)
was dissolved in dry dichloromethane (30 mL) and treated with
boron tribromide (14 mL of 1 M solution in dichloromethane,
14 mmol) under ice-cooling. The mixture was stirred for 1 h
at room temperature and then poured onto ice/water. After
vigorous stirring, the organic layer was separated, washed
cautiously with aqueous sodium bicarbonate solution, dried
over magnesium sulfate, and concentrated in vacuo. The
residue was purified by chromatography on silica gel (cyclo-
hexane/ethyl acetate ) 4/1) to obtain 11c (603 mg, 55%) as
colorless crystals; mp 257 °C. 1H NMR (DMSO-d6): δ 10.20
(br.s, 1H), 7.72 (d, J ) 8.8 Hz, 1H), 7.65 (d, J ) 2.8 Hz, 1H),
7.21 (dd, J ) 2.8 + 8.8 Hz, 1H), 6.83 (s, 1H), 2.03-2.14 (m,
3H), 1.90-2.00 (m, 6H), 1.64-1.80 (m, 6H).
Analogously, the following compounds was prepared.
2-(1,1-Dim eth yleth yl)-6-h ydr oxy-4H-1-ben zoth iopyr an -
4-on e (11b). Yield 76%, colorless crystals, mp 188-190 °C
from 2-propanol. 1H NMR (DMSO-d6): δ 10.20 (br.s, 1H), 7.72
(d, J ) 8.8 Hz, 1H), 7.66 (d, J ) 2.8 Hz, 1H), 7.21 (dd, J ) 2.8
+ 8.8 Hz, 1H), 6.87 (s, 1H), 1.37 (s, 9H).
In ter m ed ia te: 2-(1,1-Dim eth yleth yl)-6-m eth oxy-4H-1-
ben zoth iop yr a n -4-on e. Yield 20%, viscous oil. 1H NMR
(CDCl3): δ 7.94 (d, J ) 2.9 Hz, 1H), 7.53 (d, J ) 8.8 Hz, 1H),
7.22 (dd, J ) 2.9 + 8.8 Hz, 1H), 7.03 (s, 1H), 3.93 (s, 3H), 1.43
(s, 9H).
Ack n ow led gm en t. We thank Dr. Anthony Winiski
for a critical reading of the manuscript.
B. Biology. 1. Exp r ession a n d P u r ifica tion of Hu m a n
STS. STS was produced from a clone of recombinant Chinese
hamster ovary cells that stably express the human enzyme.9
Briefly, the enzyme contained in the microsomal fraction of
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