Bioorganic & Medicinal Chemistry Letters 10 (2000) 967±969
New Fluorogenic Substrate for the First Continuous Steroid
Sulfatase Assay
Melitta Bilban, Andreas Billich, Manfred Auer and Peter Nussbaumer*
NOVARTIS Research Institute, Brunnerstrasse 59, A-1235 Vienna, Austria
Received 18 June 1999; accepted 24 February 2000
AbstractÐThe screening for new inhibitors of steroid sulfatase requires an ecient test system. To overcome the shortcomings of
the available discontinuous ¯uorimetric assay, several coumarin-type compounds were investigated as potential new substrates. 3,4-
Benzocoumarin 7-O-sulfate was found to have appropriate substrate properties for the establishment of the ®rst direct continuous
assay of steroid sulfatase. # 2000 Elsevier Science Ltd. All rights reserved.
Steroid sulfatase (estrone sulfatase, E.C. 3.1.6.2.) is
involved in the local production of estrogens in normal
and malignant breast tissues by catalysing the desulfa-
tion of estrone sulfate to estrone. There is increasing
evidence that the steroid sulfatase pathway is the major
We envisaged designing coumarin derivatives exhibiting
properties that would allow both shortcomings to be
overcome, i.e., low binding anity of the sulfate and
insucient ¯uorescence of the phenolic cleavage pro-
duct at physiological pH. The sulfamate analogues of
both the natural and the assay substrate (Scheme 2),
estrone 3-O-sulfamate (3) and 4-methylcoumarin 7-O-
sulfamate (4), are irreversible inhibitors of steroid sul-
1
source of estrogens in breast and endometrial tumours.
Inhibitors of steroid sulfatase are therefore considered
as potential new therapeutic agents for the treatment of
estrogen-dependent cancers.
5
fatase. Their relative inhibitory potencies correlate with
the ranking of the KM values of the corresponding sul-
For the evaluation of potential inhibitors three types of
steroid sulfatase assays are available: a radiometric
fates as substrates (3: rIC =1, K =95 mM; 4:
50
M
rIC =30, K =275 mM; see also Table 1).
50
M
2
3
assay using the radiolabeled natural substrate H-
3
estrone sulfate, colorimetric assays using p-nitrophenyl
(
Based on these data we decided to ®rst prepare the sul-
6
pNPS) or p-nitrocatechol sulfate (pNCS) and a ¯uori-
famate derivatives 5±12 of our target molecules (Fig. 1)
3
metric assay using 4-methylumbelliferyl sulfate (1; 4-
MUS, Scheme 1). The usefulness of the non-radiometric
substrates is limited by their relatively high KM values
and to test their inhibitory potencies (Table 1) in order
to rapidly screen for the magnitude of the enzyme
binding anity. Only for selected compounds with high
inhibitory activities the synthesis of the corresponding
sulfates and the evaluation of their usefulness as sub-
strate was planned.
(
736, 680, and 275 mM for pNPS, pNCS, and 4-MUS,
respectively as compared to 95 mM for estrone sulfate;
A. Billich, unpublished). Furthermore, these are dis-
continuous assays because at the pH optimum of the
steroid sulfatase (7.5) only little change in absorbance or
In 1985 Koller and Wolfbeis reported on the preparation of
¯
uorescence intensity is observed. Alkalisation is required
several coumarin-based compounds with pK values ꢀ7
a
to generate the absorbing/¯uorescing phenolates of the
reaction products; e.g. 4-methylumbelliferone (2a; 4-MU,
Scheme 1) as the product of 4-MUS shows low ¯uores-
cence intensity at pH 7.5, but the phenolate (2b) shows a
strong signal at pH>10. Therefore, we argued that a
continuous assay would require a substrate that yields
and suggested the corresponding sulfates as substrates for
continuous assays of aryl sulfatases.7 The most promising
compound according to their results was 3-(2-benzothia-
,8
8
zolyl)coumarin-7-O-sulfate. Following our strategy, we
synthesised the corresponding sulfamate 5 and tested it
against steroid sulfatase. Compound 5 was found to be at
least 250 times less potent than the standard 3 and also
considerably less active than the 4-methylumbelliferyl ana-
logue 4 (Table 1). The most likely reason for this ®nding is
the fact that Koller and Wolfbeis used preparations of
arylsulfatases A and B in their investigations. These
a phenolic product with a lower pK , hence producing a
a
signal at physiological pH.4
*Corresponding author. Fax: +43-1-86634-354; e-mail: peter.
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00144-X