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

Article abstract of DOI:10.1016/S0960-894X(02)00383-9

In our search for potent inhibitors of the enzyme estrone sulfatase (ES), we have undertaken the synthesis and biochemical evaluation of a range of straight chain alkyl esters of 4-[(aminosulfonyl)oxy] benzoic acid. The results of the study show that the synthesised compounds possess greater inhibitory activity when compared to COUMATE, although they were all found to possess lower inhibitory activity with respect to EMATE. Furthermore, the data suggest a strong correlation between logP and IC₅₀ and therefore adds further support to our previous report where we suggested a link between inhibitory activity and hydrophobicity.

Full text of DOI:10.1016/S0960-894X(02)00383-9

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2391–2394
Inhibition of Estrone Sulfatase (ES) by Derivatives of
4
-[(Aminosulfonyl)oxy] Benzoic Acid
a,
b
a
Sabbir Ahmed, * Karen James and Caroline P. Owen
aSchool of Chemical and Pharmaceutical Sciences, Kingston University, Penrhyn Road, Kingston upon Thames, Surrey KT1 2EE, UK
bInstitute of Cancer Research, Sutton, Surrey, UK
Received 15 April 2002;revised 21 May 2002;accepted 23 May 2002
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 straight chain alkyl esters of 4-[(aminosulfonyl)oxy] benzoic acid. The results of the study show
that the synthesised compounds possess greater inhibitory activity when compared to COUMATE, although they were all found to
possess lower inhibitory activity with respect to EMATE. Furthermore, the data suggest a strong correlation between logP and IC50
and therefore adds further support to our previous report where we suggested a link between inhibitory activity and hydro-
phobicity. # 2002 Elsevier Science Ltd. All rights reserved.
6
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
stabilisation of the phenoxide ion and that the incor-
poration of electron-withdrawing groups within the
phenyl ring can further increase the inhibitory activity.
However, the requirement of logP and the nature of the
role of this physicochemical property in the inhibition
of ES has yet to be fully rationalised. From the con-
sideration of the potential mechanism for the de-sulfa-
tation reaction catalysed by ES (Fig. 3), we proposed
that the role of logP was to aid the exit of the carbon
backbone from the active site—which is presumably
polar in nature so as to mimic (and therefore stabilise)
the sulfate group.
1
,2
(
AR). However, the enzyme estrone sulfatase (ES)
provides another source of estrogens and is responsible
for the conversion of the stored (sulfated) form of the
estrogens to the active (non-sulfated) form (Fig. 1).
A number of steroidal inhibitors3 have been investi-
gated as potent inhibitors of this enzyme, including
estrone-3-O-sulfamate (EMATE)—a time and con-
centration dependent irreversible inhibitor. However,
this compound has been shown to possess potent estro-
genic properties, and as a result, the investigation into
non-steroidal inhibitors has intensified. This search has
resulted in the coumarin derived compound, namely,
ꢀ5
Here, we report the initial results of a study in an
attempt to consider the contribution of logP in the
overall inhibition of ES. We have therefore undertaken:
the synthesis of a number of straight chain esters of
4-sulfamated benzoic acid;biochemical evaluation
(including the mode of action, that is, reversible or
irreversible), and;the determination of the logP and p Ka
values of the esters of 4-hydroxybenzoic acid in an effort
4
-methylcoumarin-7-O-sulfamate (COUMATE) and
other derivatives, such as, 667- and 669-COUMATE
Fig. 2). In general, the aminosulfonate moiety is
(
believed to be involved in the irreversible inhibition of
ES.
We have recently shown that the requirement of the
phenyl group within both the steroidal and, in parti-
cular, the non-steroidal inhibitors, is necessary for the
*
8
Corresponding author. Tel.:+44-20-8547-2000x2433;fax:+44-20-
547-7562;e-mail: ch_s534@kingston.ac.uk
Figure 1. Action of the enzyme ES on estrone sulfate.
0
960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00383-9

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S. Ahmed et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2391–2394
to determine the effect of these two physicochemical
properties on the inhibitory activity of the ester based
compounds.
(Table 2). The determination of the pK of the starting
a
1
2
phenols involved a spectroscopic technique that con-
sidered the change in UV absorption by the phenolic
group under: acidic (pH 2);pH9, and;basic (pH 11)
In the synthesis of the 4-aminosulfonated derivatives of
5
conditions. The results of the pK determination are
a
shown in Table 2 and are found to be within narrow
range (between 8.0 and 8.5).
,7
benzoic acid, modified literature procedure (Scheme
) was followed and was found to proceed well and in
good yield without any major problems. The syntheses
1
8
of methyl 4-hydroxybenzoate and methyl 4-[(amino-
9
The results of the determination of the logP, IC50 and
pK values are shown in Tables 1 and 2, respectively.
sulfonyl)oxy]benzoate are described as examples. The
synthesised compounds were then evaluated for ES inhi-
bition using standard literature method so as to deter-
a
The compounds were also evaluated for their mode of
action—it was discovered that all the synthesised com-
pounds were irreversible inhibitors of ES. A more
detailed consideration of logP and IC50 data from the
current study shows that within a given series of com-
1
0
mine the initial screening inhibition and IC50 values,
whilst the mode of action was determined using a method
1
1
involving dialysis of bound/unbound inhibitor.
pounds (where pK is within a narrow range), the logP
a
In the calculation of the logP (Table 1) of the amino-
sulfonated compounds, we discovered that very little is
known about the contribution of the sulfamate group
of the inhibitors is an important factor in determining
the inhibitory activity (Fig. 4). Within the series of
compounds considered, an ‘optimum’ logP value is
observed at about 3.8 (for the parent phenolic com-
pound). 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 example, the
carbon backbones of two of the most potent inhibitors
known to date [EMATE and 669-COUMATE (Fig. 2)]
are calculated to possess logP values of 3.8 and 3.4,
respectively.
towards the overall logP of the molecule. In an effort to
TM
simplify our logP calculations (using Projectleader
)
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 optimum logP.
Although the pK values exist for a small number of the
a
phenols, we concluded that a more consistent approach
would be the determination of the pK values of the
parent phenols considered within our present study
a
In conclusion, the results of the present study show that
logP is an important factor in determining the inhibi-
tory activity of the sulfamate containing inhibitors of
ES. Results of our previous studies have also shown
that pK (which is related to the overall stability of the
a
ꢀ
OR ion) is also a factor to be considered. Within the
present study, we have maintained the pK at optimum
a
value through the use of esters of 4-hydroxybenzoic acid
as the starting compound. As a result, we have synthe-
sised some highly potent compounds which have served
as good lead compounds. As such, this is the first report
to show that these two factors can be combined in the
design of highly potent inhibitors of ES. Furthermore,
we have synthesised, through the consideration of the
physicochemical factors, some of the most potent non-
steroidal compounds known to date (the most potent,
14, being only 6.8 times weaker than EMATE, and 3.5
times more potent than COUMATE), only the tricyclic
Figure 2. Structures of EMATE and two coumarin-based inhibitors.
Figure 3. Mechanism of action of ES on estrone sulfate.

S. Ahmed et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2391–2394
2393
derivatives of COUMATE (namely, 667- to 6612-
COUMATE) are stronger inhibitors of ES.
Acknowledgements
The high resolution mass spectra were undertaken by
the EPSRC National Mass Spectrometry centre at the
University of Wales College Swansea.
References andNotes
1
2
3
. Feutrie, M. L.;Bonneterre, J. Bull. Cancer 1999, 86, 821.
. Brodie, A. M. H.;Njar, V. C. O. Steroids 2000, 65, 171.
. Howarth, N. M.;Purohit, A.;Reed, M. J.;Potter, B. L. V.
Scheme 1. Synthesis of the 4-sulfamate derivative of the substituted
benzoic acid [a=ROH/Á/toluene;b=NaH/H NSO Cl/toluene]
R=Me to Dec).
Steroids 1997, 62, 346.
. Woo, L. W. L.;Howarth, N. M.;Purohit, A.;Hejaz,
H. A. M.;Reed, M. J.;Potter, B. V. L. J. Med. Chem. 1998,
2
2
4
(
4
5
1, 1068.
. Woo, L. W. L.;Lighttowler, M.;Purhoit, A.;Reed, M. J.;
Table 1. Showing the synthesised compound, the calculated logP (of
the non-sulfamated 4-hydroxybenzoate derivatives) and the mean IC50
values (n=3)
Potter, B. V. L. J. Steroid Biochem. Mol. Biol. 1996, 57, 79.
6. Ahmed, S.;James, K.;Patel, C. K. Biochem. Biophys. Res.
Commun. 2000, 272, 583.
7
. Ahmed, S.;James, K.;Owen, C. P.;Patel, C. K.;Patel, M.
Bioorg. Med. Chem. Lett. 2001, 11, 841.
2 4
. Methyl-4-hydroxybenzoate (1): Concd H SO (3 mL) was
Compd
R
Calculated Percentage inhibition % IC50/mM
logP
8
[I]=50 mM [I]=10 mM
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 (ꢁ15 mL, pH
13) 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
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
3.80
1.60
74.7
63.4
76.9
ND
86.2
ND
83.7
80.4
76.2
ND
ND
83.1
25.4
25.6
42.6
48.3
64.4
72.6
69.5
63.0
31.6ꢄ1.23
31.6ꢄ1.95
13.2ꢄ0.4
10.5ꢄ0.28
5.9ꢄ0.44
3.8ꢄ0.16
3.4ꢄ0.25
5.0ꢄ0.26
4.8ꢄ0.17
22.4ꢄ0.48
0.5ꢄ0.001
12ꢄ0.16
C
C
C
2
3
4
H
H
H
5
7
9
1
1
1
1
1
2
0
2
4
6
8
0
C
5
H
H
H
H
H
11
13
15
17
19
C
C
C
C
6
7
8
9
ꢂ
precipitate was filtered, and dried (80 C), to give (1) (3.3 g,
ꢂ
9
9.9%) as a white crystalline solid [mp 112–115 C; R
ꢂ
f
0.47
diethyl ether/petroleum ether 40–60 C (50/50)]. n(max.) (film)
ND
ꢀ
1
cm : 3263.0 (OH) 1688.2 (C¼O). dH (CDCl ): 7.95 (2H, d,
J=8 Hz, ArH), 6.89 (2H, d, J=8 Hz, ArH), 6.06 (1H, s, OH),
C
10 21
H
—
—
36.5
68.6%
47.6%
3
EMATE
COUMATE
3.90 (3H, s, CH
1
3
). d
c
(CDCl
22.3, 115.1 (CAr), 52.0 (CH
3
): 167.2 (C¼0), 160.0, 131.8,
3
). GCMS t 9.176 m/z 152
R
ND=not determined.
+
(
9
M ).
. 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 atmosphere of
a
Table 2. Calculated pK of parent phenols of compounds
ꢂ
Compd
R
lmax Ab (nm)
pK
a
nitrogen gas at 0 C. After evolution of hydrogen had ceased
(
ꢁ
after 30 min), aminosulfonyl chloride in toluene (10 mL,
10 mmol) was added in one portion and the reaction allowed
to stir for 10 h. The reaction was then quenched with saturated
sodium bicarbonate (NaHCO ) solution (50 mL), extracted
2
4
6
8
CH
3
296
296
296
295
296
296
295
8.28ꢄ0.07
8.22ꢄ0.09
8.03ꢄ0.11
8.07ꢄ0.09
8.47ꢄ0.13
8.52ꢄ0.15
8.27ꢄ0.1
C
C
C
2
3
4
H
H
H
5
7
9
3
into dichloromethane (DCM) (2 ꢃ 50 mL), washed with water
1
1
1
0
2
4
C
C
C
5
H
H
H
11
13
15
(3 ꢃ 30 mL) and dried over anhydrous magnesium sulfate
6
7
4
(MgSO ). The mixture was filtered and the solvent removed
under vacuum to give a yellow oil which was purified using
flash chromatography to give (2) (0.24 g, 31.6%) as a pure
white solid [mp 118–121 C; R 0.24 diethyl ether/petroleum
f
ꢂ
ꢂ
ꢀ1
ether 40–60 C (50/50)]. n(max.) (film) cm : 3376.1, 3274.0
(
(
NH
2
), 1704.3 (C¼O), 1376.7, 1156.9 (S¼O). d
H 3
(CDCl ): 8.08
2H, d, J=9 Hz, ArH), 7.41(2H, d, J=9 Hz, ArH), 5.10 (2H,
s, NH ), 3.93 (3H, s, H C–). d (CDCl ): 165.2 (C¼O), 154.0,
2
3
c
3
+
1
2
1
49.8, 131.6, and 121.9 (CAr), 52.4 (CH
3
). MS m/z found: M
S) requires 231.0201.
0. ES assay: The total assay volume was 1 mL. H-estrone
+
8 9 5
31.0198, (C H NO
3
sulfate (25 mL, 20 mM/tube;300,000 dpm) and the inhibitors
50 mM/tube) dissolved in ethanol were added to a 10 mL assay
tube, and the ethanol removed with a stream of nitrogen. Tris–
(
Figure 4. Plot of IC50 versus logP of the synthesised compounds.
HCl buffer (0.05 M, pH 7.2, 0.2 mL) was added to each tube.

2394
S. Ahmed et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2391–2394
Placental microsomes were then diluted with Tris–HCl buffer
determined using EMATE (10 mM), COUMATE (100 mM), 12
(700 mM) and 14 (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 buf-
(
bated for 5 min at 37 C in a shaking water bath prior to the
115 mg/mL). The microsomes and assay tubes were pre-incu-
ꢂ
addition of the microsomes (0.8 mL) to the tubes. After 20 min
ꢂ
ꢂ
incubation (at 37 C), toluene (4 mL) was added to quench the
fer (50 mM, pH 7.2, 945 mL) at 37 C for 10 min. A control
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 scintillation cocktail
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
(
TRITONX). The aliquots were counted for 3 min. All sam-
ples were run in triplicate. Control samples with no inhibitor
were incubated simultaneously. Blank samples were obtained
by incubating with boiled microsomes. It should be noted that
EMATE and COUMATE were synthesised within our
laboratories using published procedures and used within our
assays as the standard compounds.
ꢂ
subjected 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 descri-
bed above.
12. Harwood, L. M.;Moody, C. J. In Experimental Organic
Chemistry, 1989;p. 716.
1
1. Irreversible ES assay: The irreversible inhibition was