N-Acyl arylsulfonamides as novel, reversible inhibitors of human steroid sulfatase

Article abstract of DOI:10.1016/j.bmcl.2004.11.069

Steroid sulfatase (STS) is an attractive target for a range of oestrogen- and androgen-dependent diseases. In search of novel chemotypes of STS inhibitors, we had previously identified nortropinyl-arylsulfonylureas 1; however, while these compounds were good inhibitors of purified STS (lowest K_i = 76 nM), they showed only weak inhibition of STS activity in cells (lowest IC₅₀ around 2 μM). Extended structure-activity relationship studies involving modification of the phenylacetyl side chain and replacement of the nortropine element by simpler scaffolds led to the discovery of N-acyl arylsulfonamides, more specifically N-(Boc-piperidine-4-carbonyl)- benzenesulfonamides, as STS inhibitors, some of which exhibit improved cellular potency (best IC₅₀ = 270 nM).

Full text of DOI:10.1016/j.bmcl.2004.11.069

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1235–1238
N-Acyl arylsulfonamides as novel, reversible inhibitors of
human steroid sulfatase
Philipp Lehr, Andreas Billich, Barbara Wolff and Peter Nussbaumer*
Novartis Institutes for BioMedical Research, Brunnerstrasse 59, A-1235 Vienna, Austria
Received 11 October 2004; revised 16 November 2004; accepted 25 November 2004
Available online 23 December 2004
Abstract—Steroid sulfatase (STS) is an attractive target for a range of oestrogen- and androgen-dependent diseases. In search of
novel chemotypes of STS inhibitors, we had previously identified nortropinyl–arylsulfonylureas 1; however, while these compounds
were good inhibitors of purified STS (lowest Ki = 76 nM), they showed only weak inhibition of STS activity in cells (lowest IC50
around 2 lM). Extended structure–activity relationship studies involving modification of the phenylacetyl side chain and replace-
ment of the nortropine element by simpler scaffolds led to the discovery of N-acyl arylsulfonamides, more specifically N-(Boc-piper-
idine-4-carbonyl)-benzenesulfonamides, as STS inhibitors, some of which exhibit improved cellular potency (best IC50 = 270 nM).
Ó 2004 Elsevier Ltd. All rights reserved.
Table 1. Inhibitory activity of test compounds against purified human
STS and against STS over-expressed in CHO cells (for structures see
Fig. 1)
1. Introduction
Steroid sulfatase (STS) catalyses the desulfation of ste-
roid hormone precursors, such as oestrone and dehydro-
epiandrosterone sulfate. The steroid sulfates are
delivered from the circulation to the target tissues, where
they are cleaved by STS as the first step in the local pro-
duction of oestrogens and androgens. Inhibition of STS
should result in reduced local levels of the active hor-
mones, and thus presents a potential new therapeutic
option in the treatment of oestrogen- and androgen-
dependent diseases, such as breast, endometrial and
prostate cancers, acne and androgenic alopecia.1–6 Clin-
ical validation of the concept is still missing, which
might reflect that the appropriate compound for clinical
testing has not been identified yet, although numerous
potent STS inhibitors have been described in the litera-
ture (for recent review see Ref. 6).
Compound
Purified STS
CHO–STS cells
IC50 (lM)
Ki (lM)
IC50 (lM)
EMATE
1a
1b
2
0.67a
0.056b
6.72
0.0841.89
0.03
2.40.91
0.076
––c
––
>100
>100
––
––
3a
3b
4a
4b
4c
14.5
16.1
––
3.6
8.6
>100
71
6.1
12.0
9.8
––
––
––
––
4d
4e
9.90
1.26
0.27
––
––
0.45
0.026
2.8
4f
0.012
4g
4h
4i
––
––
1.90
0.21
1.79
––
0.22
0.78
––
––
Recently, we reported on the discovery and preliminary
structure–activity relationship (SAR) of nortropinyl–
arylsulfonylureas 1 as reversible inhibitors of steroid sul-
fatase.7 Starting from 1a, compounds featuring high
inhibitory activity towards purified STS were identified,
with analogue 1b as the most active derivative (Table 1);
however, the compounds were only poor inhibitors of
STS in cells (best IC50 = 1.89 lM) (1b, Table 1). In this
4j
4k
––
––
10.2
a Taken from Ref. 11.
b Note that the IC50 value for the irreversible inhibitor EMATE
depends on incubation time; the value given is for the 1 h time point
of our standard assay (Ref. 9).
c Not done.
paper, we present extended SAR studies leading to the
discovery of N-acyl arylsulfonamide as novel scaffold
for STS inhibitors (4a–i). Among these compounds,
*
Corresponding author. Tel.: +43 1 86634 395; fax: +43 1 86634
354; e-mail: peter.nussbaumer@pharma.novartis.com
0960-894X/$ - see front matter Ó 2004Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.11.069

1236
P. Lehr et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1235–1238
O
O
O
O
H
N
O
H
N
N
O
N
S
S
O
O
O
O
Cl
R
1a R = 4-Cl
1b R = 3,5-diCF3
2
O
O
R
O
N
O
N
H
N
O
H
N
O
N
S
S
O
O
O
O
R
Cl
4a R = 4-Cl
4b R = 2-Cl
4c R = 3-Cl
3a R = benzyl
3b R = t-butyl
4d R = 2,6-diCl
O
4e R = 3,5-diCl
Cl
O
N
4f Ph-R =
H
N
O
S
S
Br
O
O
Cl
4g R = 4-OMe
4j R = H
4k R = 3,5-diCl
4h R = 2,5-diOMe
R
4i R = 3,5-diCF3
Figure 1. Structural formulae of STS inhibitors.
derivatives with improved cellular activity were identi-
fied (Fig. 1).
All test compounds were characterized by 1H NMR,
(HR)MS, TLC and HPLC, and the structures fully com-
plied with the analytical data.
2. Chemistry
3. Steroid sulfatase assays
Sulfonylurea-type inhibitors 2 and 3 were synthesized by
reacting 4-chlorophenylsulfonyl isocyanate with the cor-
responding secondary amines (Scheme 1). The required
nortropine building block was prepared by O-acylation
with pivaloyl chloride using an N-Boc-protection/depro-
tection protocol. Synthesis of acylsulfonamides 4a–i
(Scheme 2) started from substituted arylsulfonyl chlo-
rides, which were readily converted into the correspond-
ing sulfonamides using ion exchange resin Dowex 50W
X4to sequester excess of ammonia. Without further
purification, crude sulfonamides were N-acylated in high
yields with N-Boc-piperidinecarboxylic acid applying
propylphosphonic anhydride (PPA) as coupling reagent.
Benzyl analogues 4j,k were prepared analogously.
Compounds were tested in an enzymatic assay using hu-
man STS (purified to homogeneity from recombinant
CHO cells) as described.8 In brief, the STS-catalyzed
cleavage of 4-methylumbelliferyl sulfate (4-MUS) was
monitored at 37 °C in a fluorimetric assay (1 h incuba-
tions) in the absence or presence of inhibitor; initial
velocity, v, was calculated from the linear phase of the
reaction as FU (fluorescence units) per min. Inhibition
constants (Ki values) were calculated from kinetic data
obtained at different inhibitor concentrations using
[S]/v versus [S] diagrams (Hanes plots). IC50 values were
measured using the method described previously.9 The
concentrations of 4-MUS were varied between 0.05
O
O
O
O
O
R
O
O
O
O
R
N
N
N
+
+
N
H
N
N
H
SO2NCO
O O
O O
S
S
N
H
N
O
a)
a)
Cl
H
Cl
Cl
3
2
Scheme 1. Synthesis of arylsulfonylureas. Reaction conditions: (a) toluene or pyridine, rt, 4–16 h, 60–85% yield.

P. Lehr et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1235–1238
1237
O
O
O
O
O
O
N
O
S
O
S
NH2
N
Cl
b)
a)
O O
+
O
O
R
R
S
N
H
R
HO
4a-e,g-i
O
O
Br
O
N
Cl
S
a, b)
O
Cl
Br
O O
S
S
Cl
N
H
O
Cl
S
Cl
4f
Scheme 2. Synthesis of acylsulfonamides 4a–i. Reaction conditions: (a) NH4OH/ethyl acetate, rt, 16 h, then Dowex 50WX4; (b) PPA/DIEA, cat.
DMAP, DMF, rt, 16 h, 85–96% overall yield.
and 2 mM in the case of measurement of steady-state
kinetics, and were fixed at 0.75 mM in the case of IC50
determinations.
Based on the SAR finding that the potency of sulfonyl-
urea-based STS inhibitors 1 was strongly dependent on
the aryl substitution pattern, we synthesized and tested a
collection of derivatives of 4a with different aryl substit-
uents. The results with selected analogues 4b–i are
shown in Table 1. When the para-chloro substituent in
4a is moved to the ortho and meta position (4b,c) po-
tency is substantially reduced. Considering that the
ortho-chloro analogue 4b does not show inhibitory
activity up to 100 lM, it is surprising that the 2,6-di-
chloro derivative 4d with an IC50 value of 6.1 lM is as
potent as 4a. Substantial improvement in potency was
achieved with the 3,5-dichloro compound 4e, which
shows an IC50 value against purified STS in the submi-
cromolar range. In the cellular assay system 4e displays
an IC50 value of 1.26 lM, which is only about 3-fold
higher than the inhibitory potency in the cell-free STS
assay. Good agreement between the CHO and purified
enzyme data were also observed for the other chlorophe-
nyl acylsulfonamides tested, that is, 4a and 4d. The high-
est potency in this series was obtained with the fully
halogenated analogue 4f, which features a 4-bromo-
2,5-dichloro-3-thienyl moiety as the aryl element. The
IC50 and Ki values of 4f against purified STS are 26
and 12 nM, respectively. Thus, compound 4f binds
much better to the target and reaches a comparable
IC50 relative to the standard irreversible inhibitor
EMATE. This is remarkable because acylsulfonamides
4 were proven to be reversible inhibitors of STS (data
not shown). In STS-overexpressing CHO cells, 4f is also
the most potent inhibitor out of this series with an IC50
value of 270 nM, exceeding the cellular activity of all
representatives of the original sulfonylurea lead class 1.7
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 (0.5 mM) was used as pre-
viously described,10 using the assay variant for reversible
inhibitors (using live cells and 4h incubations).
4. Discussion of biological results
Starting from lead compound 1a, we had been able to
enhance the potency of this new STS inhibitor class by
appropriate aryl substitution at the arylsulfonylurea
moiety.7 The present study deals with modification of
the bridged nortropine and the attached phenylacetyl
side chain, particularly motivated by the search for sim-
pler central scaffolds.
Initial attempts in this direction, partly disclosed in the
previous publication,7 had been unsuccessful. Thus, cut-
ting out the bridge in the nortropine element to generate
the corresponding piperidine analogue resulted in loss of
activity. Furthermore, the new derivative 2 featuring the
pivaloyl instead of the phenylacetyl ester in 1a was
found to be inactive (Table 1). When investigating addi-
tional analogues, we discovered that moderate inhibi-
tory activity can be achieved by a concomitant change
to a simpler template (i.e., piperazine in 3b) and to the
t-butoxycarbonyl residue as side chain. Surprisingly,
the corresponding benzyloxycarbonyl piperazine 3a with
a side chain similar to that of 1a was inactive. Further
variation of the central scaffold yielded compound 4a
in which formally one nitrogen atom of the piperazine
scaffold in 3b was replaced by carbon. Although this
compound shows 10-fold less inhibitory potency relative
to the lead 1a against purified STS, the cellular activities
of both compounds are in the same range (Table 1). This
result encouraged us to further investigate the potential
of acylsulfonamides of type 4a as STS inhibitors.
The results obtained with additional analogues 4g–i
(Table 1) demonstrate that STS inhibition can also be
achieved with other aryl substituents in acylsulfon-
amides 4. As seen in the previous sulfonylurea series with
1b,7 the 3,5-diCF3 substitution pattern yields a com-
pound with high inhibitory potency. Acylsulfonamide
analogue 4i is not as active as 1b against purified STS
but is superior to 1b with regard to cellular activity. In

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P. Lehr et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1235–1238
general, acylsulfonamide-based STS inhibitors 4 show
improved cellular activity over sulfonylureas 1 when
comparing activity towards purified and cellular STS.
So far, we have not identified a rationale for the ob-
tained SAR with regard to the aryl substitution, as both
electron-withdrawing and electron-donating groups
can yield potent inhibitors and a clear correlation with
o-, m-, and p-substitution is missing. However, in gen-
eral, double (or multiple) halogen substitution at posi-
tions 3 and 5 is preferred and consistently provides high
potency. We also investigated the potential of benz-
ylsulfonamides (4j,k) as STS inhibitors. Surprisingly,
the (aryl)-unsubstituted compound 4j was found to be
superior to its dichloro derivative 4k. As this subclass
did not show any advantage over the arylsulfonamide
analogues it was not followed further.
sulfonamides 4 as STS inhibitors with improved cellular
potency. These compounds are currently further profiled
in order to assess their potential use for a clinical proof-
of-concept trial.
References and notes
1. Poirier, D.; Ciobanu, L. C.; Maltais, R. Expert Opin. Ther.
Pat. 1999, 9, 1083.
2. Nussbaumer, P.; Billich, A. Expert Opin. Ther. Pat. 2003,
13, 605.
3. Winum, J. Y.; Scozzafava, A.; Montero, J. L.; Supuran, C.
T. Expert Opin. Ther. Pat. 2004, 14, 1273.
4. Purohit, A.; Woo, L. W.; Chander, S. K.; Newman, S. P.;
Ireson, C.; Ho, Y.; Grasso, A.; Leese, M. P.; Potter, B. V.;
Reed, M. J. J. Steroid Biochem. Mol. Biol. 2003, 86,
423.
For selected compounds (3b, 4a,f,i), measurement of
steady-state kinetics was performed and inhibition con-
stants (Ki) were determined. In all cases, the compounds
showed pure competitive inhibition, similar to our previ-
ous finding for the nortropinyl–arylsulfonylureas 1a and
1b.7 Selected test compounds were investigated for
chemical stability and oestrogenicity, issues associated
with some STS inhibitor classes, in particular the irre-
versibly acting arylsulfamates.6 As expected, the results
indicate that acylsulfonamides 4 are chemically stable
in bulk and do not exhibit oestrogenic activity in vitro
(data not shown).
5. Scozzafava, A.; Owa, T.; Mastrolorenzo, A.; Supuran, C.
T. Curr. Med. Chem. 2003, 10, 925.
6. Nussbaumer, P.; Billich, A. Med. Res. Rev. 2004, 24,
529.
7. Nussbaumer, P.; Geyl, D.; Horvath, A.; Lehr, P.; Wolff,
B.; Billich, A. Bioorg. Med. Chem. Lett. 2003, 13, 3673.
8. Billich, A.; Bilban, M.; Meisner, N. C.; Nussbaumer, P.;
Neubauer, A.; Ja¨ger, S.; Auer, M. Assay Drug Dev.
Technol. 2004, 2, 21.
9. Nussbaumer, P.; Lehr, P.; Billich, A. J. Med. Chem. 2002,
45, 4310.
10. Wolff, B.; Billich, A.; Brunowsky, W.; Herzig, G.; Lindley,
I.; Nussbaumer, P.; Pursch, E.; Rabeck, C.; Winkler, G.
Anal. Biochem. 2003, 318, 276.
In summary, by modification of the nortropine scaffold
and the phenylacetyl side chain we obtained N-acyl aryl-
11. Purohit, A.; Williams, G. J.; Howarth, N. M.; Potter, B.
V. L.; Reed, M. J. Biochemistry 1995, 34, 11508.