Novel inhibitors of the enzyme estrone sulfatase (ES)

Article abstract of DOI:10.1016/S0960-894X(01)00086-5

We report the initial results of our study into a series of simple 4-sulfamated phenyl alkyl ketones as potential inhibitors of the enzyme estrone sulfatase. The results of the study show that these compounds are potent inhibitors, possessing greater inhibitory activity than COUMATE, but weaker activity than EMATE. Furthermore, the compounds are observed to be irreversible inhibitors.

Full text of DOI:10.1016/S0960-894X(01)00086-5

Bioorganic & Medicinal Chemistry Letters 11 (2001) 841–844
Novel Inhibitors of the Enzyme Estrone Sulfatase (ES)
Sabbir Ahmed,* Karen James, Caroline P. Owen, Chirag K. Patel and Mijal Patel
School of Chemical and Pharmaceutical Sciences, Kingston University, Penrhyn Road,
Kingston upon Thames, Surrey, KT1 2EE, UK
Received 5 October 2000; accepted 2 February 2001
Abstract—We report the initial results of our study into a series of simple 4-sulfamated phenyl alkyl ketones as potential inhibitors
of the enzyme estrone sulfatase. The results of the study show that these compounds are potent inhibitors, possessing greater inhi-
bitory activity than COUMATE, but weaker activity than EMATE. Furthermore, the compounds are observed to be irreversible
inhibitors. # 2001 Elsevier Science Ltd. All rights reserved.
In the treatment of hormone dependent breast cancer,
extensive research has been undertaken to produce
compounds which are both potent and selective inhibi-
tors of the cytochrome P-450 enzyme aromatase
(AR).1,2 However, the use of AR inhibitors does not
result in the inhibition of all of the biosynthetic pro-
cesses which lead to estrogen formation. That is, the
enzyme estrone sulfatase (ES) converts the stored (sul-
fated) form of the estrogens to the active (non-sulfated)
form (Fig. 1), thereby allowing the stimulation of
tumours via a non-AR pathway (which, in general, is
not blocked by AR inhibitors).
joining the phenyl ring to the sulfamate group.5 We
have recently shown that the stabilisation of the phen-
oxide ion6 and that the incorporation of electron-with-
drawing groups within the phenyl ring can result in a
significant increase in the inhibitory activity of com-
pounds, thus alkyl sulfamates are poorer acids (in com-
parison to phenols) and are therefore weaker inhibitors
of ES.6
In an effort to: overcome the lack of detailed informa-
tion regarding the active site of ES; probe the nature of
the proposed pharmacophore; and rationalise the inhi-
bitory activity of the aminosulfonate based compounds,
we initiated a series of SAR determination studies.
From the results of our initial molecular modelling
A number of steroidal inhibitors3,4 have been investi-
gated as potent inhibitors of this enzyme, including
EMATE (a time- and concentration-dependent irrever-
sible steroidal inhibitor) and COUMATE (an irreversible
non-steroidal inhibitor) (Fig. 2). However, since
EMATE has been shown to possess potent estrogenic
properties, the investigation into non-steroidal inhibi-
tors has intensified.
In general, the potent inhibitors contain an amino-
sulfonate moiety which is believed to be involved in the
irreversible inhibition of ES. From the consideration of
the results obtained with the known sulfamate contain-
ing steroidal and non-steroidal inhibitors, a ‘definitive
model’ was proposed where it was suggested that the
most fundamental and basic requirements for inhibition
was the phenolic ring, and a bridging oxygen atom
Figure 1. Action of the enzyme ES on estrone sulfate.
*Corresponding author. Tel.:+44-20-8547-2000; fax: +44-20-8547-
7562; e-mail: ch_s534@kingston.ac.uk
Figure 2. Structures of EMATE and COUMATE.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00086-5

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S. Ahmed et al. / Bioorg. Med. Chem. Lett. 11 (2001) 841–844
study and a consideration of potential mechanisms for
ES (Fig. 3), we concluded that logP may also be an
important factor in the inhibition process. That is, we
concluded that the role of the carbon backbone is to
favour the formation of the ROꢀ ion (thus the require-
ment for the phenolic moeity) and the expulsion of the
ROꢀ due to the high logP of carbon backbone within
ROꢀ. We also hypothesised that as a result of the high
logP requirement, the reaction catalysed by ES ‘appears’
to be an irreversible reaction.
phenyl ketones would possess the appropriate char-
acteristics. Here, we report the initial results of our
study where we have undertaken: the synthesis of deri-
vatives of 4-sulfamated phenyl ketones; the in vitro
biochemical evaluation of the synthesised compounds;
and the evaluation of the mode of action, i.e., reversible
or irreversible inhibition.
Chemistry
With respect to the requirement of pKa in the irrever-
sible inhibition of ES,6 we hypothesised that the sulfa-
mic acid produced is the inhibitory moeity and that the
carbon backbone aids the synthesis of sulfamic acid.
That is, we took the above mechanism and postulated
an inhibition mechanism involving the sulfamoyl moi-
ety—that is, an attack by the NH2 of sulfamic acid
produced as a result of the desulfatation reaction results
in the production of an imine type product (Fig. 4). We
have recently shown that the lack of production of sul-
famic acid results in the lack of irreversible inhibition—
alkyl sulfamates in partcular show this trend.
In the synthesis of the 4-aminosulfonated derivatives of
4-hydroxyphenyl ketones, modified literature proce-
dure5,7 (Scheme 1) was followed and was found to pro-
ceed well and in good yield without any major
problems. The synthesis of 4-nonanoylphenyl sulfamate
is given as an example—it should be noted that since 4-
hydroxynonanophenone is commercially available, it
was not synthesised via the initial Friedel–Crafts acyla-
tion reaction.
4-Nonanoylphenyl sulfamate (7)
In order to verify our hypothesis with respect to the
importance of logP, we undertook a design process so
as to incorporate the increasing logP whilst restricting the
pKa of the parent phenol. We concluded that sulfamated
Sodium hydride (NaH) (60% dispersion in mineral oil,
0.18 g, 4.5 mmol) was added to a stirred solution of 4-
hydroxynonophenone (1 g, 4.27 mmol) in dimethyl
formamide (DMF) (10 mL) under an atmosphere of
Figure 3. Mechanism of action of ES on estrone sulfate.
Figure 4. Mechanism for the irreversible inhibition of ES.

S. Ahmed et al. / Bioorg. Med. Chem. Lett. 11 (2001) 841–844
843
Scheme 1. Synthesis of the 4-sulfamate derivative of the substituted benzoic acid (a=acid chloride/AlCl3/DCM; b=NaH/H2NSO2Cl/toluene).
nitrogen gas at 0 ꢁC. After evolution of hydrogen had
ceased, aminosulfonyl chloride in toluene (10 mL,
ꢂ10 mmol) was added in one portion and the reaction
allowed to stir for 10 h. The reaction was then quenched
with saturated sodium bicarbonate (NaHCO3) solution
(50 mL), extracted into dichloromethane (DCM)
(2ꢃ50 mL), washed with water (3ꢃ30 mL) and dried
over anhydrous magnesium sulfate (MgSO4). The mix-
ture was filtered and the solvent removed under vacuum
to give a yellow oil which solidified on addition of
water. The crude product was purified using flash chro-
matography to give (7) (0.32g, 23.9%) as a white solid
(mp 102–104 ꢁC; Rf 0.57 ether/petroleum ether 40–60 ꢁC
(70:30)).
was removed and added to 5 mL scintillation cocktail
(TRITONX). The aliquots were counted for 3 min. All
samples were run in triplicate. Control samples with no
inhibitor were incubated simultaneously. Blank samples
were obtained by incubating with boiled microsomes.
Irreversible ES assay
The irreversible inhibition was determined using the
procedure described by Purohit et al. (1998)9 using
EMATE (10 mM), COUMATE (100 mM) and sulfa-
mated phenyl ketones (700 mM). Placental microsomes
(18 mg/mL, 55 mL) were incubated with each of the
inhibitors (25 mL in ethanol, removed with a stream of
nitrogen) in Tris–HCl buffer (50 mM, pH7.2, 945 mL) at
37 ꢁC for 10 min. A control tube with no inhibitor was
incubated simultaneously (100% tubes). An aliquot
(100 mL) in triplicate, was taken from each sample and
tested for ES activity using the procedure above, except
that 900 mL of Tris–HCl buffer was added to the assay
tubes. A second aliquot (100 mL) in triplicate, was sub-
jected to dialysis at 4 ꢁC for 16 h, with regular changes
of Tris–HCl buffer. The microsomes were then removed
from the dialysis tubing and tested for ES activity as
described above.
n(max) (Film) cmꢀ1: 3389.0, 3289.0 (NH2), 1682.3
(C¼O), 1377.9, 1181.8 (S¼O). dH (CDCl3): 7.99 (2H, d,
J=9 Hz, ArH), 7.42(2H, d, J=9 Hz, ArH), 5.17 (2H, s,
NH2), 2.94 (2H, t, J=7 Hz, COCH2CH2), 1.72(2H, m,
COCH2CH2CH2), 1.35 (10H, m, COCH2CH2[CH2]5-
CH3), 0.88 (3H, t, J=7 Hz, -CH3). dC (d6-Acetone):
154.6 (C¼O), 138.3, 136.1, 130.4, 122.8 (C-Ar) 38.8,
32.3, 29.7, 23.0 (CH2), 14.1 (CH3). MS m/z obtained
MH+ 314.1422, (C15H23NO4S)H+ requires 314.1426.
ES assay
The results of the biochemical evaluation of the synthe-
sised sulfamated phenyl ketones (as well as EMATE
and COUMATE within our assays for comparison) are
shown in Table 1 together with the relative potencies
against the latter two compounds. Consideration of the
results show that these compounds are potent inhibitors
of ES, with compound 7 being only 6.8 times weaker
than EMATE. In comparison to COUMATE, we
observe that a number of the synthesised compounds
are equipotent or are much stronger inhibitors than
COUMATE, indeed compounds 5, 6 and 7 are 2.4, 2.1
and 3.5 times more potent, respectively. These three
compounds would therefore appear to be some of the
most potent non-steroidal compounds known to date.
The synthesised compounds were further evaluated to
determine their mode of action. It was discovered that
In the biochemical evaluation, the standard literature
method was used.8 The total assay volume was 1 mL.
3H-estrone sulfate (25 mL, 50 mM/tube; 750,000 dpm)
and the inhibitors (various concentrations) dissolved in
ethanol were added to a 10 mL assay tube, and the eth-
anol removed with a stream of nitrogen. Tris–HCl buf-
fer (0.05 M, pH 7.2, 0.2 mL) was added to each tube.
Placental microsomes were then diluted with Tris–HCl
buffer (115 mg/mL). The microsomes and assay tubes
were pre-incubated for 5 min at 37 ꢁC in a shaking water
bath prior to the addition of the microsomes (0.8 mL) to
the tubes. After 20 min incubation (at 37 ꢁC), toluene
(4 mL) was added to quench the assay, and the tubes
placed on ice. The quenched samples were vortexed for
45 s and centrifuged (3000 rpm, 10 min). 1 mL of toluene

844
S. Ahmed et al. / Bioorg. Med. Chem. Lett. 11 (2001) 841–844
Table 1. Inhibition data for compounds 1–11 and the relative potencies of some of the synthesised compounds in comparison to EMATE and
COUMATE
Compound
R
IC50 (mM)
Potency w.r.t
EMATE
Potency w.r.t
COUMATE
1
2
3
4
5
6
7
8
H
CH3
2
ꢄ10.1
54
0.0019
0.0016
0.0126
0.0239
0.1000
0.0893
0.1471
0.0385
0.0079
0.0019
0.01520.3636
1
0.0472
0.0397
0.3015
0.5742
2.4000
2.1429
3.5294
0.9231
0.1905
0.0456
302ꢄ6.7
39.8ꢄ1.4
20.9ꢄ0.38
5.0ꢄ0.36
5.6ꢄ0.19
3.4ꢄ0.13
13ꢄ0.05
63ꢄ1.83
263ꢄ5.5
33ꢄ1.1
C3H7
C4H9
C6H13
C7H15
C8H17
C9H19
Ph
CH¼CHPh
CH2Ph
—
9
10
11
EMATE
COUMATE
0.5ꢄ0.001
12ꢄ0.16
24
1
—
0.0417
the enzyme did not recover after the incubation with the
synthesised compounds, i.e., the compounds are irre-
versible inhibitors of ES.
insight into the structural features required for increased
inhibitory activity.
From the consideration of the results within Table 1, it
would appear that an optimum chain length appears to
be C6 to C8. Furthermore, the data supports our
hypothesis that the carbon backbone of the inhibitors
plays a crucial role in the mechanism of inhibition by
sulfamated compounds, which are then able to release
sulfamic acid as the inhibiting moiety involving the
attack of an aldehydic group by the NH2 group at the
active site (as proposed in Fig. 4).
References
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In conclusion, we have synthesised a range of sulfa-
mated phenyl ketones, which have proved to be some of
the most potent non-steroidal compounds known to
date, only the recently reported 667-COUMATE is
known to be of greater potency. The compounds are
therefore good lead compounds in the search for potent
non-steroidal inhibitors and have allowed us further