Nortropinyl-arylsulfonylureas as novel, reversible inhibitors of human steroid sulfatase

Article abstract of DOI:10.1016/j.bmcl.2003.08.019

Steroid sulfatase (STS) has emerged as an attractive target for a range of estrogen- and androgen-dependent diseases. Searching for novel chemotypes as STS inhibitors, we identified nortropinyl-arylsulfonylurea 3 as a hit from high-throughput screening. A

Full text of DOI:10.1016/j.bmcl.2003.08.019

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3673–3677
Nortropinyl-Arylsulfonylureas as Novel,
Reversible Inhibitors of Human Steroid Sulfatase
Peter Nussbaumer,a,* Dieter Geyl,b Amarylla Horvath,a
Philipp Lehr,a Barbara Wolffa and Andreas Billicha
aNovartis Research Institute Vienna, Brunnerstrasse 59, A-1235 Vienna, Austria
bNovartis Institutes for Biomedical Research, Basel, Switzerland
Received 12 June 2003; revised 1 August 2003; accepted 11 August 2003
Abstract—Steroid sulfatase (STS) has emerged as an attractive target for a range of estrogen- and androgen-dependent diseases.
Searching for novel chemotypes as STS inhibitors, we identified nortropinyl-arylsulfonylurea 3 as a hit from high-throughput
screening. A series of analogues was prepared in order to explore the essential structural elements for STS inhibition, and first
structure–activity relationships were established. Mechanistic investigations revealed that the compounds are reversible, competitive
inhibitors of STS.
# 2003 Elsevier Ltd. All rights reserved.
Steroid sulfatase (STS) acts as the first enzyme in meta-
bolic pathways converting sulfated steroid hormone
precursors (e.g., estrone sulfate and dehydroepian-
drosterone sulfate) to the active hormones which bind
to estrogen or androgen receptors. Therefore, STS has
been recognized as a potential drug target for hormone-
dependent diseases.1À4 STS inhibitors have been sug-
gested for the therapy of breast, endometrial and pros-
tate cancer,1,2 and for androgen-dependent skin
diseases, such as acne.5 To date, the therapeutic concept
of STS inhibition is still at a preclinical stage, with only
one compound (667COUMATE6) being announced to
enter clinical trials in the near future.
the reactivity of the sulfamate functionality, and might
complicate or even impede development and clinical
use. While one class of steroidal reversible STS inhibi-
tors lacking the sulfamate group (see compound 2 in
Fig. 1 as an example) has been described by Poirier and
co-workers,9,10 its activity does not approach that of the
most potent sulfamate-type inhibitors.
In this situation, discovery of new, preferentially non-
steroidal inhibitor classes would be welcome. As a result
of a high-throughput screening for STS inhibitors, we
obtained compound 3 (Fig. 1), initially synthesized by
Fulvio Gadient in 1967 as potential hypoglycemic agent
(for closely related analogues, see ref 11), as a hit. In this
paper we describe the inhibitory profile of 3 and some
initial structure–activity relationships (SARs) of a novel
class of STS inhibitors.
The most potent STS inhibitors known to date feature
an aryl sulfamate moiety and act in an irreversible
manner. Estrone sulfamate (1, Fig. 1), discovered by
Potter and co-workers in 1994,7 has served as the lead
for all sulfamate-type inhibitors.3,4 In general, there are
two issues associated with this compound class: poten-
tial estrogenicity and chemical instability. Several recent
studies have addressed the estrogenicity issue, resulting
in the identification of potent, non-estrogenic inhibitors
(for review, see ref 4). However, the chemical instability
in solution (as described for estrone sulfamate8) appears
to be an inherent problem for all aryl sulfamates, due to
Chemistry
The synthesis of the nortropinyl-arylsulfonylureas 6 was
accomplished by reacting the central intermediate 412
with various arylsulfonyl isocyanates 5 (Scheme 1) in
toluene or pyridine. 5a (R=4-Cl), 5b (R=4-F), 5d
(R=H), and 5e (R=4-Me) were commercially avail-
able, while 5c (R=4-Br), 5f (R=4-CF3), and 5g
(R=3,5-diCF3) were prepared in situ from the corre-
sponding sulfonyl chlorides by treatment with NaOCN
and pyridine in acetonitrile according to a published
*Corresponding author. Tel.: +43-1-86634-395; fax: +43-1-86634-
354; e-mail: peter.nussbaumer@pharma.novartis.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.08.019

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P. Nussbaumer et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3673–3677
in analogy to a literature procedure and used without
purification.14 Coupling of the commercially available
acid 9 with the secondary amine 4 using n-propylpho-
sphonic acid anhydride (PPA) provided the carba-ana-
logue 10, and the unbridged piperidine derivatives 12
were prepared by treatment of amines 11a,b with sulfo-
nyl isocyanate 5a. Intermediate 11b was obtained by
acylation of the cyanohydrin of N-benzyl-4-piperidone
followed by debenzylation using catalytic hydrogena-
tion. All test compounds were characterized by 1H
NMR, MS, HRMS, TLC, and HPLC, and the struc-
tures fully complied with the analytical data.
Figure 1. Structural formulae of STS inhibitors.
Steroid sulfatase assays
Compounds were tested in an enzymatic assay using
human STS (purified to homogeneity from recombinant
CHO cells) as described.15 In brief, the STS-catalysed
cleavage of 4-methylumbelliferyl sulfate (4-MUS) was
monitored in a discontinuous fluorimetric assay in the
absence or presence of inhibitor; initial velocity v was cal-
culated from the linear phase of the reaction as FU (fluo-
rescence units) per min. Inhibition constants (Ki values)
were calculated from kinetic data obtained at different
inhibitor concentrations using [S]/v versus [S] diagrams
method.13 In this case, the isolation and purification of
the products was more laborious (repeated chromato-
graphy), resulting in low to moderate yields (8–50%),
but no efforts have been invested so far to optimise the
procedure. In contrast, usually good yields (60–85%)
were obtained when the purchased starting materials 5
were used. Benzoylurea analogue 8 was synthesised by
reacting amine 4 with benzoyl isocyanate 7, which was
prepared from 4-chlorobenzamide and oxalyl chloride
Scheme 1. Synthesis of nortropinyl-arylsulfonylureas. Reaction conditions: (a) toluene or pyridine, rt, 4–16 h, 6–85% yield; (b) Et3N/PPA/cat.
DMAP, DMF, rt, 16 h, 30% yield.

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3675
(Hanes plots). In some instances, IC50 values were
measured using the method described previously.16
Cellular activity of 3 was measured in CHO cells over-
expressing STS.17 In this assay, 3 was about 128 times
less potent than the irreversible standard compound 1.
To assess the effect of test compounds on the activity of
STS in intact cells, an assay using recombinant CHO
cells and 4-MUS as substrate was used as previously
described,17 using the assay variant for reversible inhib-
itors.
Some STS inhibitors, in particular 1,19 have been shown
to be estrogenic, which might limit their potential clin-
ical usefulness. Compound 3 did not stimulate the
growth of the estrogen-dependent breast cancer cell line
MCF-7 (assayed as described in ref 15) up to the highest
test concentration of 10 mM, and thus is devoid of
estrogenicity, at least in this in vitro cell culture system.
Characterisation of the hit compound (3)
High-throughput screening (HTS) of our substance col-
lections for STS inhibitors using the assay with purified
enzyme yielded compound 3 (Fig. 1) as a hit. Kinetic
analysis (Fig. 2) revealed that the compound is a pure
competitive inhibitor of STS with an inhibition constant
(Ki) of 890 nM. Pre-incubation of STS with 3 did not
enhance inhibition, indicating that it does not act as a
time-dependently inactivating inhibitor. Furthermore,
enzyme activity was fully recovered when the enzyme-
inhibitor complex was treated with dextran-activated
charcoal (data not shown), demonstrating that 3 acts as
a reversible inhibitor of STS.
Structure–activity relationships
After having established the mode of action of 3 we
explored which structural features were essential for the
inhibitory activity. When the tractability and synthetic
accessibility of the hit compound were analyzed, the
hydrazine moiety was recognized as a hurdle for rapid
SAR exploration. To reduce the synthetic complexity,
analogue 6a lacking the exocyclic nitrogen atom was
prepared and tested. Since 6a was only 2.7-fold less
active than 3 (based on the comparison of the Ki values
against purified STS, Table 1), we decided to prepare a
collection of further derivatives without the original
hydrazine structural element while keeping the phenyl-
acetate residue constant. This led to 6a–g as the first
series of derivatives; their STS inhibitory activities are
listed in Table 1. Among the halogen substituents
investigated, chlorine (6a) and bromine (6c) gave com-
parable results, whereas the fluorine derivative 6b was
The Ki for estrone sulfamate (1) has been determined to
be 670 nM.18 Thus, the binding affinities of 1 and 3
towards STS are of the same order of magnitude.
However, since 1 irreversibly inactivates STS, its IC50
was found to be approximately 10-fold lower than the
Ki value of 1 and 3 after a 1h incubation period under
our assay conditions.
Figure 2. Mode of action and Ki determination of inhibitor 3: (a) rate of substrate turnover (v) at various concentrations of 3; (b) same data plotted
in a Hanes diagram; (c) plot of Kapp/Vapp ratios against inhibitor concentration [I]; the linear correlation indicates pure competitive inhibition of the
enzyme, the Ki value is obtained from the intercept with the abscissa.

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Table 1. Inhibitory activities of test compounds against purified
human STS and against STS over-expressed in CHO cells
The compounds of this study were also tested for STS
inhibition in intact CHO cells over-expressing the
enzyme (Table 1). The cellular potencies of the new
compounds were relatively weak when compared to the
irreversibly acting standard 1. In general, the cellular
results correlated with the cell-free data with regard to
the ranking of the compounds. However, the improved
cell-free potency of derivatives 6f and, particularly, 6g
did not translate into significantly increased cellular
activity relative to the screening hit 3, indicating a cell
penetration problem for 6f,g. As the cell-free and cel-
lular data for compounds 3 and its ‘simplified’ analo-
gue 6a correlate very nicely, it can be ruled out that
the discrepant results obtained with 3 in comparison
to 6f,g are due to the structural modification in the
core region, that is elimination of the exocyclic nitro-
gen atom. Both analogues 6f,g feature the tri-
fluoromethyl group as substituent. One can speculate
that physicochemical properties of this moiety, that is
high lipophilicity, render the compounds less cell
permeable. In any case, insufficient cellular potency
was recognized as weakness of these first representa-
tives of the novel inhibitor class.
Compd
Purified STS
IC50 (mM)
CHO-STS cells
Ki (mM)
IC50 (mM)
1
3
0.67a
0.89
2.4
—
1.85
—
0.056b
—
—
50
—
0.03
3.84
6.72
39.2
6.15
>30
37.1
7.47
1.89
10.4
4.32
>30
24.5
c
6a
6b
6c
6d
6e
6f
6g
8
10
12a
12b
>100
—
16.6
—
0.084
—
6.2
0.53
0.076
5.8
—
—
>100
13
—
aTaken from ref 17 (there calculated by the method of Kitz and Wil-
son for an irreversible inhibitor).
bNote that the IC50 value for 1, an irreversible inhibitor, depends on
incubation time; the value given is for the 1h time point of our stan-
dard assay.15
cNot done.
substantially less effective. Analogue 6d, lacking an
additional substituent at the sulfonylated phenyl ring,
did not show any inhibition of STS up to the highest
test concentrations (30 or 100 mM). The formal intro-
duction of a para-methyl group (6e) restored some
inhibitory activity, but 6e was about 10-fold less potent
than the chloro and bromo analogues 6a and 6c. This
indicated that not only the size but also electronic effects
of the substituents were important for potent inhibitory
activity of this compound class. This finding was further
supported by the trifluoromethyl derivatives 6f and 6g.
While 6f was already more active than 6a against pur-
ified STS, compound 6g with two trifluoromethyl
groups at positions 3 and 5 of the sulfonylated phenyl
ring was the best inhibitor out of this series. Based on
the Ki values as a measure for binding of the compound
to the enzyme, 6g (76 nM) was found to be superior to
the irreversible inhibitor 1 (670 nM) by almost one
order of magnitude. Using purified STS, the IC50 values
of both compounds were determined to be in a similar
range (84 nM for 6g and 56 nM for 1, respectively).
In summary, we have discovered a novel class of rever-
sible, competitive inhibitors of STS and established first
insight into its SAR. Work is in progress to improve
cellular activity of the compounds.
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