4,4′-Benzophenone-O,O′-disulfamate: A potent inhibitor of steroid sulfatase

Article abstract of DOI:10.1016/S0960-894X(02)00381-5

We investigated whether the benzophenone moiety can be used as core element of steroid sulfatase (STS) inhibitors. While 4- and 3-benzophenone-O-sulfamates inhibit STS with IC₅₀ values between 5 and 7 μM irrespective of additional hydroxy and m

Full text of DOI:10.1016/S0960-894X(02)00381-5

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2093–2095
4,40-Benzophenone-O,O 0-disulfamate: A Potent Inhibitor
of Steroid Sulfatase
Peter Nussbaumer,* Melitta Bilban and Andreas Billich
NOVARTIS Research Institute, Brunnerstrasse 59, A-1235 Vienna, Austria
Received 26 March 2002; revised 14 May 2002; accepted 16 May 2002
Abstract—We investigated whether the benzophenone moiety can be used as core element of steroid sulfatase (STS) inhibitors.
While 4- and 3-benzophenone-O-sulfamates inhibit STS with IC50 values between 5 and 7 mM irrespective of additional hydroxy
and methoxy substituents at the second phenyl ring, benzophenone-O,O0-disulfamates show increased activity. With an IC50 value
of 190 nM the 4,40-derivative is the first small monocyclic STS inhibitor coming close to the potency of the steroidal standard
estrone sulfamate. # 2002 Elsevier Science Ltd. All rights reserved.
Steroid sulfatase (estrone sulfatase, E.C. 3.1.6.2., STS)
regulates the local production of estrogens from sys-
temic precursors, such as estrone sulfate and dehy-
droepiandrosterone sulfate in normal and malignant
breast tissues. There is increasing evidence that in breast
and endometrial tissues the steroid sulfatase pathway is
the major source of estrogens, which support the growth
of endocrine-dependent tumours. Inhibitors of STS are
therefore considered as potential new therapeutic agents
for the treatment of estrogen-dependent cancers.1
photophores, but its steric requirement limits wider
applicability.5
We asked whether for STS the benzophenone moiety
itself could be used as core element of a ligand and not
only as an appendix to known ligands. To estimate the
magnitude of the binding affinity of benzophenone-
based ligands we prepared the corresponding sulfamates
and tested them for their inhibitory potencies, analo-
gously to the successful search for a new STS substrate.6
Following this approach, we discovered benzophenone
disulfamates with high inhibitory potency against STS.
Both irreversible and reversible inhibitors of human
STS have been reported.2 All potent irreversible inhibi-
tors feature the sulfamate functionality, which is
responsible for the irreversible mode of action. While
with irreversible inhibitors blocking of enzyme activity
in the nanomolar range can be achieved, the few known
reversible inhibitors are less potent by several orders of
magnitude. The design of potent, reversible inhibitors is
hampered by the lack of the 3-D-structure of STS. In
contrast to the related arylsulfatases A and B,3,4 infor-
mation on structure and catalytic mechanism of STS is
not available. Photolabeling of the active site of an
enzyme is a way to get insight into the residues involved
in catalysis. We, therefore, envisaged the design and
synthesis of photolabeling ligands for STS. Incorpora-
tion of a benzophenone substituent in photolabeling
substrates has proven several advantages over other
Chemistry
The test compounds 1a–1l were synthesized by sulfa-
moylation of the corresponding hydroxybenzophenones
using sodium hydride followed by amidochlorosulfonic
acid7 (3-fold excess of both reagents) in dimethylforma-
mide. With 3,30- and 3,40-dihydroxybenzophenone as
starting material both, the mono- and the disulfamated
products were obtained (1g+1j and 1i+1k, respec-
tively). All test compounds were purified by silica gel
1
chromatography and characterized by H NMR, MS
(ESI), and, in the case of new compounds, also by
elemental analysis.
The non-commercially available phenolic precursors
were prepared as outlined in Scheme 1. Addition of in
situ generated methoxyphenyllithium salts 2 to TBDMS
protected hydroxybenzaldehydes 3 gave benzhydrol
*Corresponding author. Tel.: +43-1-86634-395; fax: +43-1-86634-
354; e-mail: peter.nussbaumer@pharma.novartis.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00381-5

2094
P. Nussbaumer et al. /Bioorg. Med. Chem. Lett. 12 (2002) 2093–2095
intermediates 4. Oxidation with manganese dioxide gen-
erated the benzophenone core element and subsequent
deprotection using HF yielded hydroxybenzophenones
5a–c. Dihydroxybenzophenones 5e and 5f were
obtained from 5b and 5c, respectively, by treatment with
Lewis acid (aluminum chloride for 5e, boron tribromide
for 5f). Similarly, 3,40-dimethoxybenzophenone8 (5d)
was transformed into 3-hydroxy-40-methoxybenzo-
phenone (5g) by treatment with boron tribromide and
to 3,40dihydroxybenzophenone (5h) using hydrobromic
acid in acetic acid.
core moiety of STS inhibitors by the report that already
simple tetralone and indalone sulfamates are able to
block STS activity.1 During the course of our studies,
4-acylphenyl-O-sulfamates including 4-benzophenone-
O-sulfamate (1a) itself were reported as inhibitors of
STS.11
Estrone sulfamate (EMATE, Fig. 1), a well character-
ized potent STS inhibitor, was used as reference com-
pound in our assay (Table 1). The first compounds we
investigated were 4- and 3-benzophenone-O-sulfamate
(1a,13 1b) without any additional substituents. Both
regioisomers inhibited STS and were about 100-fold less
active than estrone sulfamate. This agrees well with the
reported activity of 1a (126-fold less active than
EMATE).11 The comparable potency of the 3- and the
4-isomer might be explained by the possibility to over-
lay the sulfamate functionalities and the phenyl rings of
the two molecules. All potent STS inhibitors have a
stretched shape and published SAR studies circum-
stantiate that elongation of the (poly)cyclic core ele-
ments by side chains results in improved potency.
Therefore, we explored the influence of alkoxy and
hydroxy substituents in benzophenone-O-sulfamates,
which could serve as potential attachment sites for side
chains to enhance binding to the enzyme. Surprisingly,
all these derivatives (selected compounds 1c to 1i shown
in Table 1) exhibit very similar potency with IC50 values
in the low micromolar range (about 100 times less
potent than EMATE) indicating that electronic and
steric effects by these substituents are tolerated at all
positions of the second phenyl group. In the course of
the synthesis of 1g we also isolated the 3,30-disulfamate
1j. This by-product showed improved activity relative to
all benzophenone monosulfamates tested before. This
prompted us to synthesise and test the benzophenone
disulfamates 1k and 1l. Here a clear preference for the
Biological Results and Discussion
All known potent STS inhibitors are bi- or polycyclic
sulfamates with one exception: Li et al. reported on a
series of N-acyl tyramine sulfamates,9 with the best
representative showing only about 20-fold less activity
than the common standard estrone sulfamate.10 Eva-
luation of the structure–activity relationship (SAR) of
STS inhibitors suggests that a stretched shape of the
molecule is beneficial in all substance classes. However,
we were encouraged in investigating benzophenones as
Table 1. Inhibitory potencies (IC50) of benzophenone sulfamates 1a–l
against recombinant human steroid sulfatase in comparison to
EMATE
a
Compd
Position of
–OSO2NH2
STS, IC50
(mM)
R
EMATE
0.056
5.1
5.7
5.2
4.8
4.6
7.1
6.9
6.9
5.0
3.2
0.78
0.19
1a
1b
1c
1d
1e
1f
1g
1h
1i
4
3
4
4
4
3
3
3
3
3
3
4
H
H
3-OMe
2-OMe
2-OH
3-OMe
3-OH
4-OMe
4-OH
Scheme 1. Synthesis of non-commercially available hydroxybenzo-
phenone precursors 5.
1j
1k
1l
3-OSO2NH2
4-OSO2NH2
4-OSO2NH2
aAll compounds were tested using a fluorimetric assay.12 The substrate
(0.5 mM of 4-methylumbelliferyl sulfate) was incubated with STS
(1.5 nM) in the presence of graded concentrations of inhibitors at pH
7.5 and 37ꢀ C for 60 min. Then, 0.2 M NaOH was added and fluores-
cence intensity (lex=355 nm, lem=460 nm) was measured. The inhi-
bitory activities of the test compounds (IC50) were calculated using
non-linear regression (software Grafit).
Figure 1. General structure of benzophenone-O-sulfamates 1a–l (for
definition of substituent R see Table 1) and the structure of the ster-
oidal STS inhibitor estrone sulfamate (EMATE).

P. Nussbaumer et al. /Bioorg. Med. Chem. Lett. 12 (2002) 2093–2095
2095
4-isomer was observed. Already the 3,40-disulfamate 1k
showed additionally increased potency and the 4,40-
disulfamate 1l was the best compound out of the series
being only 3.4-fold less active than EMATE. This find-
ing proved for the first time that highly efficient STS
inhibitory activity can be also achieved with a very
small aryl sulfamate lacking both a steroidal B-ring
mimicry and a side chain mimicking the steroidal D-ring
and beyond (see for instance elongated 17-estradiol
analogues14). It still has to be elucidated how the second
sulfamate moiety contributes to the substantial increase
in activity.
2. Poirier, D.; Ciobanu, L. C.; Maltais, R. Exp. Opin. Ther.
Pat. 1999, 9, 1083.
3. Lukatela, G.; Krauss, N.; Theis, K.; Selmer, T.; Giesel-
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3654.
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Harrop, S. J.; Hopwood, J. J.; Guss, J. M. Structure 1997, 5,
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5. Dorman, G.; Prestwich, G. D. Biochemistry 1994, 33, 5661.
6. Bilban, M.; Billich, A.; Auer, M.; Nussbaumer, P. Bioorg.
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7. Appel, R.; Berger, G. Chem. Ber. 1958, 91, 1339.
8. Blicke, F. F.; Weinkauff, O. J. J. Org. Chem. 1932, 54,
1446.
In summary, we have discovered 4,40-benzophenone-
O,O0-disulfamate (1l) as potent inhibitor of human STS.
Moreover, the results from this study indicate that the
benzophenone moiety might be suitable as a core struc-
ture for the design of photolabeling ligands for STS.
9. Li, P. K.; Milano, S.; Kluth, L.; Rhodes, M. E. J. Steroid
Biochem. Mol. Biol. 1996, 59, 41.
10. Purohit, A.; Williams, G. J.; Howarth, N. M.; Potter,
B. V. L.; Reed, M. J. Biochemistry 1995, 34, 11508.
11. Ahmed, S.; James, K.; Owen, C. P.; Patel, C. K.; Patel, M.
Bioorg. Med. Chem. Lett. 2001, 11, 841.
12. Roy, A. B. Methods Enzymol. 1987, 143, 207.
13. 1a was a known compound but for other use (Lo, Y. S.;
Walsh, D. A.; Uwaydah, I. M. US Patent 5025031 A, 1991;
Chem. Abstr. 1991, 115, 255820) and was not tested as steroid
sulfatase inhibitor until very recently.11
References and Notes
1. Woo, L. W. L.; Howarth, N. M.; Purohit, A.; Jejaz,
H. A. M.; Reed, M. J.; Potter, B. V. L. J. Med. Chem. 1998,
41, 1068.
14. Ciobanu, L. C.; Boivin, R. P.; Luu-The, V.; Labrie, F.;
Poirier, D. J. Med. Chem. 1999, 42, 2280.