Inhibition of estrone sulfatase (ES) by alkyl and cycloalkyl ester derivatives of 4-[(aminosulfonyl)oxy] benzoic acid

Article abstract of DOI:10.1016/j.bmcl.2003.11.067

In our search for potent inhibitors of the enzyme estrone sulfatase (ES), we have undertaken the synthesis and biochemical evaluation of a range of esters of 4-[(aminosulfonyl)oxy] benzoic acid. The results of the study show that the synthesised compounds possess potent inhibitory activity, indeed the cyclooctyl derivative was found to be more potent than 667-COUMATE, which is currently undergoing clinical trials.

Full text of DOI:10.1016/j.bmcl.2003.11.067

Bioorganic & Medicinal Chemistry Letters 14 (2004) 605–609
Inhibition of estrone sulfatase (ES) by alkyl and cycloalkyl ester
derivatives of 4-[(aminosulfonyl)oxy] benzoic acid
Chirag K. Patel, Caroline P. Owen and Sabbir Ahmed*
Department of Pharmacy, School of Chemical and Pharmaceutical Sciences, Kingston University, Penrhyn Road,
Kingston upon Thames, Surrey KT1 2EE, UK
Received 11 September 2003;revised 29 October 2003;accepted 20 November 2003
Abstract—In our search for potent inhibitors of the enzyme estrone sulfatase (ES), we have undertaken the synthesis and bio-
chemical evaluation of a range of esters of 4-[(aminosulfonyl)oxy] benzoic acid. The results of the study show that the synthesised
compounds possess potent inhibitory activity, indeed the cyclooctyl derivative was found to be more potent than 667-COUMATE,
which is currently undergoing clinical trials.
#
2003 Elsevier Ltd. All rights reserved.
In the treatment of hormone dependent breast cancer,
extensive research has led to the synthesis of highly
selective and potent inhibitors of the cytochrome P-450
A number of aminosulfonate derivatives of both ster-
oidal and non-steroidal inhibitors has been investigated
(the sulfamate moiety is believed to be involved in the
irreversible inhibition of ES). An example of a potent
1
enzyme aromatase (AR). However, the enzyme estrone
2
sulfatase (ES) provides another source of estrogens and
is responsible for the catalysis of the conversion of the
stored (sulfated) form of the estrogens to the active
steroidal inhibitor is estrone-3-O-sulfamate (EMATE) —
a time and concentration dependent irreversible inhi-
bitor. However, this compound has been shown to pos-
sess potent estrogenic properties, and as a result, the
investigation into non-steroidal inhibitors has intensi-
fied. This search has resulted in the coumarin derived
compound, namely, 4-methylcoumarin-7-O-sulfamate
(
non-sulfated) form (Fig. 1). The same enzyme has also
been shown to catalyse the conversion of androgen sul-
fate (e.g., androstenediol sulfate) to the non-sulfated
androgen (Fig. 1).
Figure 1. Action of the enzyme ES on estrone sulfate and androstenediol sulfate.
*
0
Corresponding author. Tel.: +44-20-8547-2000;fax: +44-20-8547-7562;e-mail: s.ahmed@kingston.ac.uk
960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.11.067

606
C. K. Patel et al. / Bioorg. Med. Chem. Lett. 14 (2004) 605–609
(COUMATE) and its derivatives, such as 667- and 669-
COUMATE (Fig. 2).
whilst the mode of action was determined using a
1
1
method involving dialysis of bound/unbound inhibitor.
We have previously shown that the requirement of the
phenyl group within both the steroidal and, in parti-
cular, the non-steroidal inhibitors, is necessary for the
cleavage of the S–O–Ar bond resulting in the formation
In the calculation of the logP (Table 1) of the amino-
sulfonated compounds, we discovered that very little
was known about the contribution of the sulfamate
group towards the overall logP of the molecule. In an
effort to simplify our logP calculations and therefore
remove any potential problems with the calculated
values, we used the parent non-sulfamated compounds
as a guide to the determination of the potential opti-
3ꢀ5
of the phenoxide ion
and sulfamic acid (the latter is
therefore the inhibiting moiety which results in the for-
mation of an irreversibly bound imine species at the
6
,7
active site of ES ). The requirement of logP and the
nature of the role of this physicochemical property in
TM
mum logP (using Projectleader ). The determination
of the pK (Table 1) of the starting phenols involved a
6
the inhibition of ES has also been rationalised by us
and from the consideration of the potential mechanism
a
1
2
spectroscopic technique that considered the change in
UV absorption by the phenolic group under: acidic (pH
2);pH9, and;basic (pH 11) conditions.
7
for the de-sulfatation reaction catalysed by ES, we
proposed that the role of logP was to aid the exit of the
carbon backbone from the active site.
Experimentally determined IC50 and pKa values are
summarised in Table 1 together with the calculated logP
of the non-sulfamated starting phenol. From an initial
consideration of the inhibitory activity, we observe that
the compounds synthesised possess potent inhibitory
activity, for example, heptyl 4-aminosulfonyl benzoate
(14) is the most potent straight chain containing com-
pound possessing greater inhibitory activity than COU-
MATE but lower inhibitory activity than EMATE and
667-COUMATE. Cyclooctyl 4-aminosulfonyl benzoate
(28) is found to be the most potent cyclic ester based
compound and is more potent than 667-COUMATE,
which is currently undergoing clinical trials. The
Here, we report the initial results of a study in an
attempt to consider the contribution of size (and there-
fore steric hindrance) to the overall inhibitory activity of
ES inhibitors. We have therefore undertaken: the
synthesis of a number of straight chain and cyclic esters
of 4-sulfamated benzoic acid;biochemical evaluation
(
including the mode of action, i.e., reversible or irrever-
sible);the determination of physicochemical properties
such as logP, pK , as well as the size of the overall
(
a
molecule), and the rationalisation of the inhibitory
activity involving the superimposing of the inhibitors
onto the estrone sulfate backbone of the transition-state
of the reaction catalysed by ES.
In the synthesis of the 4-aminosulfonated derivatives of
7
benzoic acid, modified literature procedure (Scheme 1)
was followed and was found to proceed well and in
good yield without any major problems. The syntheses
8
of methyl 4-hydroxybenzoate (1) and methyl 4-[(ami-
9
nosulfonyl)oxy]benzoate (2) are described as examples.
The synthesised compounds were then evaluated for ES
inhibition using standard literature method to deter-
1
0
mine the initial screening inhibition and IC50 values,
Scheme 1. Synthesis of the 4-O-sulfamate derivative of the alkyl and
cycloalkyl esters of benzoic acid [a=ROH/Á/toluene;b=NaH/
H NSO Cl/DMF or DMA] (R=Me to Dec or cyclobutyl to cyclooctyl).
2 2
Figure 2. Structures of EMATE, COUMATE and two tricyclic deri-
vatives of COUMATE.
Figure 3. Plot of IC50 versus logP of the synthesised straight chain
containing compounds.

C. K. Patel et al. / Bioorg. Med. Chem. Lett. 14 (2004) 605–609
607
synthesised compounds were found to block ES in an
irreversible manner similar to EMATE, COUMATE
and 667-COUMATE.
within the series of compounds based on the straight
chain esters, an ‘optimum’ logP value is observed
between 3.5 and 4.3. It is interesting to note that the
calculated logP of the carbon backbone of a number of
the known potent inhibitors of ES is also close to the
optimum observed within the current study, for exam-
ple, EMATE and 669-COUMATE are calculated to
possess logP values of 3.8 and 3.4, respectively.
A more detailed consideration of pK and IC data
a
50
from the current study shows that within the series of
compounds synthesised in this study, the pK of all of
a
the benzoic acid based compounds fall within a rela-
tively narrow range, that is, the values range from
approximately 8.0–8.9 and that there is no correlation
In an effort to rationalise the inhibitory activity of the
straight chain containing esters and the cyclic moiety
containing esters, we superimposed compounds 14 and
28 (the most potent compound from each range) onto
the transition-state for the reaction catalysed by ES
(Fig. 4) — the derivation and use of the transition-state
between IC50 and pK within this range of compounds.
a
Consideration of the logP (for the parent phenolic
compound) and IC50 data shows that a good correlation
exists between these two parameters (Fig. 3), as such,
a
Table 1. Showing the calculated logP and the pK of the non-sulfamated derivatives, the inhibitory activity and the mean IC50 values (n=3) of the
synthesised compound
Compd
R
logP
pK
a
Initial screening
% inhibition)
IC50/mM
(
a
2
4
6
8
CH
3
1.49
1.84
2.30
2.70
3.10
3.49
3.89
4.29
4.68
5.08
2.68
3.08
3.47
3.87
3.80
1.60
2.65
8.28ꢁ0.07
8.22ꢁ0.09
8.03ꢁ0.11
8.07ꢁ0.09
8.47ꢁ0.13
8.52ꢁ0.15
8.27ꢁ0.10
8.09ꢁ0.13
8.39ꢁ0.09
8.09ꢁ0.13
8.73ꢁ0.02
8.81ꢁ0.10
8.88ꢁ0.08
8.09ꢁ0.01
8.37ꢁ0.05
8.00ꢁ0.14
8.27ꢁ0.05
25.4
25.6
31.6ꢁ1.23
31.6ꢁ1.95
13.2ꢁ0.4
10.5ꢁ0.28
5.9ꢁ0.44
3.8ꢁ0.16
3.4ꢁ0.25
5.0ꢁ0.26
4.8ꢁ0.17
22.4ꢁ0.48
9.3ꢁ0.07
1.7ꢁ0.07
0.5ꢁ0.028
0.17ꢁ0.007
0.5ꢁ0.001
13.8ꢁ0.07
0.21ꢁ0.01
a
C
C
C
2
3
4
H
H
H
5
7
9
a
42.6
48.3
a
a
1
1
1
1
1
2
2
2
2
2
0
2
4
6
8
0
2
4
6
8
C
5
H
11
H
13
H
15
H
17
H
19
64.4
72.6
a
C
6
C
7
C
8
C
9
a
69.5
63.0
a
a
76.2
36.5
a
C
CycloC
10
H
21
b
5
H
9
62
84
b
CycloC
CycloC
CycloC
—
6
H
11
H
13
H
15
b
7
8
87
b
82
68.6
47.6
d
a
EMATE
COUMATE
6
c
—
—
67-COUMATE
56
a
b
c
[
[
[
[
I] of 10 mM.
I] of 20 mM.
I] of 12 mM.
I] of 0.25 mM.
d
Figure 4. Superimpositioning of compounds 14 (in green) and 28 (in red) onto the transition-state of the reaction catalysed by ES (the hydrogen
bonding groups are shown as lines and the estrone sulfate backbone in grey).

608
C. K. Patel et al. / Bioorg. Med. Chem. Lett. 14 (2004) 605–609
(TS) in the rationalisation of the inhibitory activity of a
range of inhibitors of ES has been previously reported
by us and as such will not be discussed here in detail.
3. Ahmed, S.;James, K.;Owen, C. P.;Patel, C. K.;Patel,
M. Bioorg. Med. Chem. Lett. 2001, 11, 899.
4. Ahmed, S.;James, K.;Patel, C. K. Biochem. Biophys. Res.
Commun. 2000, 272, 583.
1
3
In summary, the construction of the probable TS
involved in the construction of the residues which have
been proposed to exist at the active site within the
5
6
7
. Owen, C. P.;James, K.;Sampson, L.;Ahmed, S.
Pharm. Pharmacol. 2003, 55, 85.
. Ahmed, S.;James, K.;Owen, C. P.;Patel, C. K.;Patel,
M. Bioorg. Med. Chem. Lett. 2001, 11, 2525.
J.
1
4
CaChe molecular modelling software on an IBM PC
compatible microcomputer. The completed structures
were then refined performing a pre-optimisation calcu-
lation, followed by a geometry optimisation. The oxy-
gen atom of the formylglycine residue was then attached
to the sulfonate group of estrone sulfate via a weak
bond whilst another hydrogen atom from a histadine
residue was attached to the phenolate oxygen atom of
the substrate. The saddle point for the reaction was then
calculated and the resulting TS structure was further
refined by performing a minimise gradient calculation.
The molecule’s vibrational transitions were calculated in
order to ‘verify’ the transition-state.
. Ahmed, S.;James, K.;Owen, C. P. J. Steroid Biochem.
Mol. Biol. 2002, 82, 425.
8. Methyl-4-hydroxybenzoate (1): Concd H SO (3 mL) was
2
4
added to a suspension of 4-hydroxy benzoic acid (3 g,
21.74 mmol) in methanol (20 mL) and the solution
refluxed for 1 h. After cooling to room temperature,
NaOH (ꢂ15 mL) was added to neutralise the solution.
The resulting mixture was allowed to stand for 15 min,
before being poured into a cool beaker, and made up to
500 mL with water. The white precipitate was filtered,
ꢃ
and dried (80 C), to give 1 (3.3 g, 99.9%) as a white
ꢃ
crystalline solid [mp 112–115 C; R 0.47 diethyl ether/
f
ꢃ
ꢀ1
:
petroleum ether 40–60 C (50/50)]. n(max.) (film) cm
263.0 (OH). 1688.2 (C¼O). d (CDCl ): 7.95 (2H, d,
J=8 Hz, ArH), 6.89 (2H, d, J=8 Hz, ArH), 6.06 (1H, s,
OH), 3.90 (3H, s, CH ). d (CDCl
): 167.2 (C¼O), 160.0,
31.8, 122.3, 115.1 (CAr), 52.0 (CH ). GCMS t 9.176
3
H
3
From the modelling study, in particular, the super-
impositioning of the most potent cycloalkyl and alkyl
inhibitors onto the steroid backbone within the TS, we
can clearly observe that the larger straight chain con-
taining compounds [such as 14 (shown in green)] are
able to undergo steric interaction with the area of the
active site which would normally undergo hydrogen
bonding with the estrone sulfate C(17)=O group. From
the superimpositioning of 28 [Fig. 4 (shown in red)], we
observe that the cyclooctyl moiety is far removed from
the hydrogen bonding group and therefore does not
undergo steric interactions with the protein backbone of
the active site. The enzyme–inhibitor complex for the
cyclic derivatives therefore appear to be more stable
than for the straight chain containing compounds,
resulting in the latter compounds possessing less potent
inhibitory activity.
3
c
3
1
3
R
+
m/z 152 (M ).
9
. Methyl 4-[(aminosulfonyl)oxy]benzoate (2): Sodium
hydride (NaH) (60% dispersion in mineral oil, 0.16 g, 4
mmol) was added to a stirred solution of 1 (0.5 g, 3.29
mmol) in dimethyl formamide (DMF) (20 mL) under an
ꢃ
atmosphere of nitrogen gas at 0 C. After evolution of
hydrogen had ceased (after 30 min), aminosulfonyl chlo-
ride 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 bicar-
3
bonate (NaHCO ) solution (50 mL), extracted into di-
chloromethane (DCM) (2ꢄ50 mL), washed with water
(
(
3ꢄ30 mL) and dried over anhydrous magnesium sulfate
MgSO ). The mixture was filtered and the solvent
4
removed under vacuum to give a yellow oil which was
purified using flash chromatography to give 2 (0.24 g,
ꢃ
3
1.6%) as a pure white solid [mp 118–121 C; R 0.24
f
In conclusion, the results of the present study show that
whilst hydrophobicity is an important factor in deter-
mining the overall inhibitory activity of the sulfamate
based inhibitors of ES, the overall size of the molecule is
also important and that the combination of pK , logP
a
and overall inhibitor length can result in the production
of highly potent inhibitors such as 28.
ꢃ
diethyl ether/petroleum ether 40–60 C (50/50)]. n
(
max)
ꢀ1
(film) cm : 3376.1, 3274.0 (NH ), 1704.3 (C¼O), 1376.7,
2
1156.9 (S¼O). d
7.41 (2H, d, J=9 Hz, ArH), 5.10 (2H, s, NH
s, H C). d (CDCl
): 165.2 (C¼O), 154.0, 149.8, 131.6,
and 121.9 (CAr), 52.4 (CH
H
(CDCl
3
): 8.08 (2H, d, J=9 Hz, ArH),
), 3.93 (3H,
2
3
c
3
+
3
). MS m/z found: M
8 9 5
31.0198, (C H NO S) requires 231.0201.
+
2
3
1
0. ES assay: The total assay volume was 1 mL. H-estrone
sulfate (25 mL, 20 mM/tube;300,000 dpm) and the inhibi-
tors (50 mM/tube) dissolved in ethanol were added to a 10
mL assay tube, and the ethanol removed with a stream of
nitrogen. Tris–HCl buffer (0.05 M, pH 7.2, 0.2 mL) was
added to each tube. Placental microsomes were then dilu-
ted with Tris–HCl buffer (115 mg/mL). The microsomes
Acknowledgements
The high resolution mass spectra were undertaken by
the EPSRC National Mass Spectrometry centre at the
University of Wales College Swansea and the elemental
analysis were undertaken at the School of Pharmacy,
University College London.
ꢃ
and assay tubes were pre-incubated for 5 min at 37 C in a
shaking water bath prior to the addition of the micro-
somes (0.8 mL) to the tubes. After 20 min incubation (at
ꢃ
3
7 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 was removed and added to 5 mL scintilla-
tion 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 micro-
somes. It should be noted that EMATE and COUMATE
were synthesised within our laboratories using published
References and notes
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1998, 41, 1068.
8

C. K. Patel et al. / Bioorg. Med. Chem. Lett. 14 (2004) 605–609
609
procedures and used within our assays as the standard
compounds.
1. Irreversible ES assay: The irreversible inhibition was
determined using EMATE (10 mM), COUMATE (100
mM), 12 (700 mM) and 14 (700 mM). Placental microsomes
of Tris–HCl buffer was added to the assay tubes. A sec-
ond aliquot (100 mL) in triplicate, was subjected to dia-
lysis 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.
ꢃ
1
(
18 mg/mL, 55 mL) were incubated with each of the inhi-
bitors (25 mL in ethanol, removed with a stream of nitro-
gen) in Tris–HCl buffer (50 mM, pH 7.2, 945 mL) at 37 C
12. Harwood, L. M.;Moody C. J. In Experimental Organic
Chemistry;Blackwell, 1989;p 716.
ꢃ
for 10 min. A control tube with no inhibitor was incu-
bated 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
13. Ahmed, S.;James, K.;Owen, C. P.;Patel, C. K.;Patel,
M. Bioorg. Med. Chem. Lett. 2001, 11, 3001.
14. CaChe is a trademark of Oxford Molecular Ltd, Oxford
Science Park, Oxford, UK.