The mechanism of the irreversible inhibition of estrone sulfatase (ES) through the consideration of a range of methane- and amino-sulfonate-based compounds

Article abstract of DOI:10.1016/S0960-894X(02)00137-3

We report the results of our study into a series of simple phenyl and alkyl sulfamates and alkyl methanesulfonates as potential inhibitors of the enzyme estrone sulfatase (ES). The results of the study show that the substituted phenyl sulfamates are good irreversible inhibitors; the alkyl sulfamate compounds were found to lack inhibitory activity; whilst the large alkyl chain containing methanesulfonate-based compounds were found to possess weak reversible inhibitory activity. Using the results of the inhibition study, we postulate the probable mechanism for ES and suggest that an attack by the gem-diol is a major requirement prior to the hydrolysis of the sulfamate group, following which, attack on the active site C=O occurs and which therefore leads to the production of an imine type functionality, resulting in irreversible inhibition.

Full text of DOI:10.1016/S0960-894X(02)00137-3

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1279–1282
The Mechanism of the Irreversible Inhibition of Estrone Sulfatase
(ES) Through the Consideration of a Range of
Methane- and Amino-Sulfonate-Based Compounds
Sabbir Ahmed,a,* Karen James,b Caroline P. Owen,a Chirag K. Patela
and Luther Sampsonc
aSchool of Chemical and Pharmaceutical Sciences, Kingston University, Penrhyn Road, Kingston upon Thames,
Surrey, KT1 2EE, UK
bInstitute of Cancer Research, Sutton, UK
cNovartis Pharma AG, CH-4002 Basel, Switzerland
Received 20 July 2001; revised 26 November 2001; accepted 21 February 2002
Abstract—We report the results of our study into a series of simple phenyl and alkyl sulfamates and alkyl methanesulfonates as
potential inhibitors of the enzyme estrone sulfatase (ES). The results of the study show that the substituted phenyl sulfamates are
good irreversible inhibitors; the alkyl sulfamate compounds were found to lack inhibitory activity; whilst the large alkyl chain
containing methanesulfonate-based compounds were found to possess weak reversible inhibitory activity. Using the results of the
inhibition study, we postulate the probable mechanism for ES and suggest that an attack by the gem-diol is a major requirement
prior to the hydrolysis of the sulfamate group, following which, attack on the active site C¼O occurs and which therefore leads to
the production of an imine type functionality, resulting in irreversible inhibition. # 2002 Elsevier Science Ltd. All rights reserved.
The enzyme estrone sulfatase (ES) converts the stored
(sulfated) form of estrogens to the active (non-sulfated)
form, thereby allowing the stimulation of estrogen-
dependent tumours via a non-aromatase (AR) pathway
(which is therefore not blocked by AR inhibitors). A
number of steroidal and non-steroidal inhibitors1,2 has
been investigated as potent inhibitors of this enzyme,
including estrone-3-O-sulfamate (EMATE) (a time- and
concentration-dependent irreversible steroidal inhibitor
with estrogenic properties) and coumarin-7-O-sulfamate
(COUMATE) (also a time- and concentration-depen-
dent irreversible non-steroidal inhibitor but lacking
estrogenic properties)—both compounds, along with
other potent inhibitors, contain a sulfamate moiety
which is believed to be involved in the irreversible
inhibition of ES (Fig. 1).
We argued that if the recently proposed mechanism5
was correct, a wide range of phenyl- and alkyl-sulfa-
mated compounds would be expected to possess inhibi-
tory activity involving the attack of the carbonyl group
at the active site of ES by the NH2 moiety of the sulfa-
mate-based compounds. From our study on pKa,6
however, we concluded that the hydrolysis of the S–OR
bond was an important step in the inhibition process
(Fig. 2). This contradicted the recently proposed
mechanism by Woo et al.5 (2000) (where the authors
suggested that the cleavage of the carbon backbone
occurred after the imine production).
The consequence of the conclusion from our earlier
study is, therefore, that if the hydrolysis of the sulfamic
From the results of our molecular modelling studies3,4
and a review of potential mechanisms for ES, we designed
a number of substituted phenyl- and alkyl-sulfamates.
*Corresponding author. Tel.: +44-20-8547-2000; fax: +44-20-8547-
7562; e-mail: ch_s534@kingston.ac.uk
Figure 1. Reaction catalysed by ES.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00137-3

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S. Ahmed et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1279–1282
potent inhibitors but to evaluate the inhibitory activity
of the alkyl- and phenyl-sulfamates in an attempt to
gain further insight into the mechanism of inhibition by
sulfamate based compounds. Detailed consideration of
the inhibitory activity suggests that a marked difference
is observed between the phenyl- and alkyl-sulfamate
based compounds. That is, the former is found to pos-
sess greater inhibitory activity compared to the alkyl
sulfamates, the latter being, in general, non-inhibitors
(Table 1). A trend is also observed within the alkyl sul-
famates, however, where a-substituted compounds such
as 1 are found to be more potent than non-a-substituted
compounds. Consideration of the pKa of the parent
alcohols of the a-substituted alkyl alcohols and phenols
shows that these compounds possess a lower pKa value
than the non-substituted alkyl alcohols—the latter
would therefore be expected to produce a less stable
ROÀ ion when compared to the a-substituted alcohols
or indeed the phenols. The investigation into the mode
of action10,11 of all of the inhibitors which were initially
observed to possess some inhibition, shows that the
sulfamate-based inhibitors (both phenolic and alkyl
alcohol-based compounds) are irreversible, that is, the
enzyme did not recover activity after incubation with the
synthesised sulfamate (alkyl or phenyl)-based com-
pounds, whereas the methanesulfonate-based com-
pounds, although very weak inhibitors, are found to be
reversible in nature. This observation demonstrates that
the sulfamate-based compounds that show a degree of
inhibition do so through an irreversible interaction with
the active site.
Figure 2. Hydrolysis of phenyl aminosulfonate containing compounds.
moiety (step 1, Fig. 2) occurs prior to the irreversible
inhibiting step (step 2, Fig. 2), then the phenyl sulfamate
based compounds should possess good inhibitory activ-
ity whilst the alkyl sulfamates would be expected to lack
inhibitory activity (since the phenoxide ion would be
expected to be more stable than the alkoxide anion
thereby favouring the hydrolysis reaction). However, if
the proposed mechanism of Woo et al.5 was correct,
then all sulfamate-based compounds would be expected
to possess inhibitory activity since they all contain the
NH2 moiety using which they would be able to under-
take attack of the C¼O group in the formylglycine
(FGly 69) residue. We therefore argued that the syn-
thesis of a range of phenyl- and alkyl-sulfamated com-
pounds would allow us to resolve the problem and to
determine whether the hydrolysis of the S–OR bond
plays any part in the inhibitory activity of compounds
as irreversible inhibitors of ES.
Here, we report the synthesis and biochemical evalu-
ation of a range of alkyl- and phenyl-sulfamates and a
small range of methanesulfonate-based compounds of
straight chain alkyl alcohols. From the consideration of
the structure–activity relationship (SAR) determination
of these compounds, we suggest an alternative and
novel mechanism for inhibition of ES, a mechanism that
can be used in the rationalisation of a wide range of
irreversible and reversible inhibitors.
Table 1. Showing the initial screening inhibition data and IC50 values
for a range of the synthesised compounds
Compd
R
R0 Percentage
inhibition (mmoles/litre)
IC50
1
2
3
4
5
6
7
8
Cl3C–CH2
Cl2HC–CH2
ClH2C–CH2
NH2
NH2
NH2
60.0b
30.0b
15.0b
0a
750
ND
In the synthesis of the 4-aminosulfonated derivatives of
4-hydroxy phenol and alkyl alcohols, modified literature
procedure3,5 (Scheme 1) was followed and was found to
proceed well and in good yield without any major prob-
lems. The synthesis of 2,2,2-trichloroethyl sulfamate7
and 4-bromophenyl sulfamate8 are given as examples.
ND
ND
ND
ND
ND
ND
CH3-(CH2)5–CH2– NH2
CH3-(CH2)6–CH2– NH2
CH3-(CH2)7–CH2– NH2
CH3-(CH2)7–CH2– CH3
CH3-(CH2)10–CH2– CH3
Phenyl
4-Methylphenyl NH2
3-Methylphenyl NH2
4-Fluorophenyl
3-Fluorophenyl
4-Chlorophenyl
3-Chlorophenyl
4-Bromophenyl
3-Bromophenyl
4-Iodophenyl
0a
0a
28.0b
17.0b
29.7b
27.4b
39.5b
37.0b
79.6b
37.6b
62.0b
50.8b
75.1b
66.0b
89.4b
74.4b
84.3b
82.5b
90.4b
99.5b
99.8b
9
NH2
>10,000
>10,000
10
11
12
13
14
15
16
17
18
19
20
21
22
23
The results of the biochemical evaluation9À11 (Table 1)
shows that, in general, all of the compounds considered
within the present study are less potent than COU-
MATE and EMATE—it should be noted that the aim
of the present study was not the synthesis of highly
2089ꢀ50
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
537ꢀ21.2
2089ꢀ50.0
1585ꢀ66.1
537ꢀ21.2
912ꢀ12.4
257ꢀ6.3
560ꢀ16.2
120ꢀ1.2
3-Iodophenyl
4-Cyanophenyl
3-Cyanophenyl
4-Nitrophenyl
3-Nitrophenyl
300ꢀ3.3
191ꢀ4.3
330ꢀ10.3
120ꢀ3.9
COUMATE 4-Methyl coumarin NH2
EMATE Estrone NH2
12ꢀ0.16
0.5ꢀ0.001
aAt inhibitor concentration of 10 mM.
bAt inhibitor concentration of 1 mM.
ND, not determined.
Scheme 1. Synthesis of the phenyl and alkyl-sulfamates (R=alkyl
alcohol or phenol; R0=Me, NH2, CH3–Ph; a=NaH or K2CO3/
toluene).

S. Ahmed et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1279–1282
1281
As previously mentioned, the mechanism of Woo et al.5
(2000) suggests that all sulfamated compounds (both
alkyl and phenyl) would be expected to possess inhibi-
tory activity resulting from the attack of the C¼O group
within the active site by the NH2 moiety of the sulfa-
mate group. However, consideration of the results
shows that the non-a-substituted alkyl sulfamate-based
compounds possessed no inhibitory activity whatsoever,
even up to concentrations of inhibitor exceeding 10
mmol/L. We can therefore only conclude that the pro-
posed attack of the aldehydic group by the NH2 group,
as the first step of the mechanism, does not occur. It may
be argued that the larger alkyl chains may be involved
in some steric interaction which may result in weak (or a
lowering of) inhibitory activity, however, we have
recently shown good inhibitory activity in similar long
alkyl chain containing phenolic compounds,12 as such,
we propose that the steric factor is not of great sig-
nificance.
3. Ahmed, S.; James, K.; Sampson, L. Pharm. Pharmacol.
Commun. 1998, 4, 481.
4. Ahmed, S.; James, K.; Sampson, L.; Mastri, C. Biochem.
Biophys. Res. Commun. 1999, 254, 811.
5. Woo, L. W. L.; Purohit, A.; Malini, B.; Reed, M. J.; Potter,
B. V. L. Chem. Biol. 2000, 7, 773.
6. Ahmed, S.; James, K.; Patel, C. K. Biochem. Biophys. Res.
Commun. 2000, 272, 583.
7. Synthesis of 2,2,2-trichloroethyl sulfamate (1). Potassium
carbonate (0.58 g, 4.20 mmol, 2 equiv) was added to a stirred
solution of trichloroethanol (0.40 g, 4.2mmol) in toluene (20
mL) under nitrogen, and warmed to 40 ꢁC. Aminosulfonyl
chloride in toluene (20 mL, ꢂ20 mmol) was then added, and
the reaction allowed to stir for 4 days at room temperature.
The reaction was quenched in NaHCO3 (50 mL), extracted
into DCM (2ꢃ50 mL), washed (3ꢃ30 mL water) and dried
(MgSO4). Removal of the solvent under vacuum produced 8
as an orange solid. Mp 46.5–49.2 ꢁC (Yield 10%). n(max) (Film)
cmÀ1: 3397.5 and 3294.4 (NH), 1371.8 and 1189.1 (S¼O).
300 MHz dH (CDCl3) 5.45 (2H, s, NH2) 4.46 (2H, s, CH2–). dC
(CDCl3) 93.1567 (–CCl3), 78.6485 (–CH2CCl3).
8. Synthesis of 4-bromophenyl sulfamate (16). Compound 16
was synthesised following the same procedures as for com-
pound 1 except that NaH (60% dispersion in mineral oil, 0.2
g, 5.00 mmol) was added to a stirred solution of 4-bromo-
phenol (0.5 g, 2.89 mmol) in DMF (10 mL). Aminosulphonyl
chloride in toluene (10 mL, ꢂ10 mmol) was added after 30
min. Removal of the solvent under vacuum yielded a clear
yellow oil, which was purified using flash chromatography to
give (16) (0.29 g, 39.8%) as a white solid [mp 113–116 ꢁC; Rf
0.37 ether/petroleum ether 40–60 ꢁC (60/40)]. n(max) (Film)
cmÀ1: 3377.0, 3274.6 (NH2), 1349.6, 1172.7 (S¼O). dH
(CDCl3): 7.56 (2H, d, J=9 Hz, ArH), 7.19 (2H, d, J=9 Hz,
ArH), 5.02(2H, s, NH 2). dc (CDCl3): 150.6, 133.4, 125.1, 120.1
(C–Ar). MS m/z found: MH+ 250.9254, (BrC6H6NO3S)H+
requires 250.9252.
9. ES assay. 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 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 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 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.
The consideration of the inhibitory activity and SAR
data for a wide range of alkyl- and phenyl-sulfamate-
based compounds, and more importantly the non-inhi-
bitors, suggests that the initial step in the inhibition of
ES by sulfamate based compounds is indeed the clea-
vage of the S–OR bond and not, as suggested by Woo et
al.,5 the attack of the C¼O group by the NH2 moiety of
the sulfamate group (Fig. 2). We therefore propose that
the order of events involves the cleavage of the S–OR
bond, resulting in the formation of the sulfamate moiety
as well as the ROÀ ion (which is therefore related to the
pKa of the parent ROH, as such, phenol and a-sub-
stituted alkyl alcohol-based sulfamated compounds
possess inhibitory activity). The production of the sul-
famic acid moiety is then followed by the attack of the
aldehydic group by the NH2 of sulfamic acid resulting
in the formation of an imine type structure (with the
loss of a molecule of water) resulting in the irreversible
inhibition of ES.
In conclusion, through the consideration of the inhibi-
tion data of a wide range of compounds, we have
rationalised the potent (as well as the lack of) inhibitory
activity within a range of sulfamate and methanesulfo-
nate-based inhibitors of ES, thereby allowing a new
mechanism for the irreversible inhibition of ES by
sulfamate containing compounds to be proposed.
Acknowledgement
10. Irreversible ES assay. The irreversible inhibition was
determined using the procedure described by Purohit et al.
(1998)14 using EMATE (10 mM), COUMATE (100 mM) and
sulfamated 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, pH 7.2, 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 proce-
dure above, except that 900 mL of Tris–HCl buffer was added
to the assay tubes. A second aliquot (100 mL) in triplicate, was
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 described above.
The high resolution mass spectra were undertaken by
the EPSRC National Mass Spectrometry centre at the
University of Wales College Swansea.
References and Notes
1. Howarth, N. M.; Purohit, A.; Reed, M. J.; Potter, B. L. V.
Steroids 1997, 62, 346.
2. Woo, L. W. L.; Howarth, N. M.; Purohit, A.; Hejaz,
H. A. M.; Reed, M. J.; Potter, B. V. L. J. Med. Chem. 1998,
41, 1068.

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S. Ahmed et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1279–1282
11. Time and concentration dependency assay. An experiment
was conducted to determine whether the active compounds
were inhibiting estrone sulfatase in a time and concentration
dependent manner. Dilutions of each compound (in ethanol)
were placed in 5 mL assay tubes and the ethanol removed with
a stream of nitrogen. Tris–HCl buffer (50 mM, pH 7.2, 0.2
mL) was added and the tubes warmed in a shaking water bath
(37 ꢁC, 5 min). At the appointed time the microsomes were
added (800 mL, 192 mg), and the assay tubes incubated for
between 0 and 60 min. After incubation, dextran coated char-
coal (1% w/v in Tris–HCl buffer, 500 mL) was added and the
tubes allowed to shake at 37ꢁC for a further 10 min. After this
time the tubes were immediately centrifuged (1000 rpm, 3
min), and a portion of the supernatant (750 mL) added to pre-
incubated 10 mL assay tubes containing estrone sulfate (25 mL
(ethanol removed) to give final concentration 50 mM/tube,
750,000 dpm/tube) and Tris–HCl buffer (50 mM, pH 7.2, 250
mL). After 30 min of incubation at 37 ꢁC, the assay tubes were
quenched by the addition of toluene (4 mL) and placed on ice.
Each tube was vortexed for 45 s, and centrifuged (3000 rpm)
for 10 min. Aliquots (1 mL) of each toluene layer were added
to Cocktail T (5 mL) and counted by liquid scintillation.
12. Ahmed, S.; James, K.; Owen, C. P.; Patel, C. K.; Patel, M.
Bioorg. Med. Chem. Lett. 2001, 11, 841.