Mechanism of the hydrolysis of the sulfamate EMATE-an irreversible steroid sulfatase inhibitor

Article abstract of DOI:10.1016/j.tetlet.2010.02.065

The kinetics of hydrolysis of the medicinally important sulfamate ester EMATE have been probed over a wide pH range and into moderately strong base (H_ region). Analysis of the pH/H_-rate profile, measurements of pK_as, solvent-reactivity, kinetic isotope effects and determination of activation data reveal that in the pH range from ～1 to ～8 an S_N2 (S) solvolytic mechanism is followed and after the pK_a of EMATE (pK_a ～9) is passed, a second pathway showing a first-order dependence on base operates and an E1cB mechanism is supported.

Full text of DOI:10.1016/j.tetlet.2010.02.065

Tetrahedron Letters 51 (2010) 2059–2062
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Tetrahedron Letters
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Mechanism of the hydrolysis of the sulfamate EMATE—an irreversible steroid
sulfatase inhibitor
*
William J. Spillane , Jean-Baptiste Malaubier
School of Chemistry, National University of Ireland, Galway, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
The kinetics of hydrolysis of the medicinally important sulfamate ester EMATE have been probed over a
wide pH range and into moderately strong base (H_ region). Analysis of the pH/H_-rate profile, measure-
Received 3 December 2009
Revised 4 February 2010
Accepted 12 February 2010
Available online 16 February 2010
ments of pK
in the pH range from ꢀ1 to ꢀ8 an S
pK
ꢀ9) is passed, a second pathway showing a first-order dependence on base operates and an E1cB
mechanism is supported.
a
s, solvent-reactivity, kinetic isotope effects and determination of activation data reveal that
N
2 (S) solvolytic mechanism is followed and after the pK of EMATE
a
(
a
Ó 2010 Elsevier Ltd. All rights reserved.
The importance of certain types of sulfamate esters in medicinal
chemistry has been outlined1 and numerous authoritative re-
views have appeared in this area.3 However the mechanism(s)
of action of these medicinally-important esters while speculated
cause it represents a good generic example from the field of medic-
inally relevant sulfamate esters. A sample of EMATE was readily
available (see Acknowledgments) and a sample was subsequently
synthesized in this laboratory by standard procedures.14
An array of techniques has been used viz., (pH/H_)-rate profile,
solvent-reactivity studies (Grunwald–Winstein, extended Grun-
wald–Winstein and nucleophilicity tests), kinetic isotope effects
and measurement of thermodynamic data. Some data have also
been collected under non-biological conditions, for example, at
alkaline pHs and the lower H_ region in order to probe the mech-
anism over the whole range of the pH/H_-rate profile.
The (pH/H_)-rate profile data (see Supplementary data) for 1a is
plotted in Figure 1. Rates were measured in water at 50 °C over the
pH range 1.74–13.69 and in strong alkali up to 3 M KOH (H_ re-
gion). The rates of the formation of estrone 2a (at k = 275 nm), or
under more alkaline conditions, of anionic estrone 2b (at
k = 296 nm) were followed. Rates did not vary more than 5% based
,2
–6
7
–9
on in the literature
remains uncertain, though very recent
1
0,11
work
has thrown new light on the mechanism(s) through
which they inactivate arylsulfatases. In earlier work the hydrolysis
and aminolysis of simpler sulfamate esters12,13 have been exam-
ined thoroughly in aqueous media and in acetonitrile and the prin-
cipal reaction pathways have been eliminative in character with
E1cB and E2 mechanisms being supported. However, recently a
non-eliminative mechanism1 has been found under conditions
(
pH ꢀ2.7) that do not allow the removal of a hydrogen from the es-
ter, NH SO O–.
2
2
O
Z
on the average of three runs. Constant ionic strength (l = 1.0) was
1
1
1
a: Z = NH SO O
2 2
-
b: Z = NHSO O
1
0
2
c: Z = 2-NSO O
2
2a: Z = HO
-
-1
2b: Z = O
-
2
The purpose of this Letter is to explore, especially under condi-
tions that are as close as possible to those employed in clinical
application, the physical organic chemistry of an important biolog-
ically active sulfamate ester, namely, estrone 3-O-sulfamate
-3
-4
(
EMATE) (1a). EMATE was the first active site directed irreversible
-5
sulfatase inhibitor to be discovered. It was chosen for this study be-
-6
0
2
4
6
8
10
12
14
16
Measured pH/H-
*
Corresponding author. Tel.: +353 91 492475; fax: +353 91 525700.
E-mail address: william.spillane@nuigalway.ie (W.J. Spillane).
Figure 1. (pH/H_)-rate profile data at 50 °C for 1a.
0
040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.02.065

2
060
W. J. Spillane, J.-B. Malaubier / Tetrahedron Letters 51 (2010) 2059–2062
maintained with KCl over the pH range studied and solutions were
buffered with a stock solution of 0.01 M TRIS-cacodylate. In some
cases at pHs ꢀ11–12 the increase in both 2a and 2b could be fol-
lowed simultaneously and the variation in rate was at maximum
Log kobs ¼ mYOTs þ lNOTs
where the second term measures the sensitivity (l) of the hydrolysis
to nucleophilicity through the use of the NOTs parameter which is a
measure of the nucleophilicity of each solvent mixture. A priori
since m is low, one would expect for this hydrolysis that the nucle-
ophilicity would be important and this is the case; an l value of 0.93
and an m value of 0.24 being found from the plot for the extended
equation (r = 0.98). These values strongly support a mechanism
involving nucleophilic attack by water in the hydrolysis of 1a and
a lesser role for the ionizing power of the solvent. In the extended
equation the m value is expected to be somewhat different to that
found in the basic Grunwald–Winstein equation. Kevill has sug-
gested that l/m ratios of greater than 1.6 indicate a bimolecular
7
%. Product studies carried out at several points on the pH/H_ pro-
file showed that 2a/2b formed almost quantitatively. For example,
the absorbance of a ‘spent’ kinetic solution of 1a in 1.5 M KOH was
within 4% of the absorbance measured for a freshly made-up
ꢂ4
1
ꢁ 10 M solution of 2b. The agreement between spent and
spiked solutions was always within 6%.
The first pK of 1a (loss of the first proton from NH
be derived from the pH/H_-rate profile below as 9.45. This kinetic
pK is in good agreement with the UV value of 9.5 in 70% aqueous
MeOH. In water, the second pK
based on these pK values and spectrophotometric determinations
in this laboratory in MeCN, which gave pK values of 15.1 and 21.4
for the first and second ionizations, respectively, it should be ꢀ15–
a
2 2
SO O–) can
a
1
5
a
has not been determined but
mechanism and values between 0 and 1 may be found for unimo-
a
22,23
lecular reactions.
In the present study the l/m value is 3.9.
a
22
Hydrolysis studies by the Kevill group on series of carbonyl-
23
and sulfonyl- chlorides showed that l/m values falling into both
1
6
1
6. The hydrogen
a
a to the carbonyl group in 1a will have a pK in
categories were found for the former and they were assigned to
water of ꢀ16.5 since this is the reported value for cyclopenta-
‘
ionization’ or addition–elimination mechanisms, respectively,
whereas for the latter all the values were ꢀ2 and they were inter-
preted as indicating an S 2 solvolytic displacement mechanism.
For an N-acylsulfamate ester of the type, R-CONHSO O-Ar, which
was found to undergo an S 2 type reaction, an l/m value of 3.3
1
7
a
none. This pK should be virtually the same for both 1b and 1c
since the distance from the nitrogen centre to the carbonyl centre
is considerable and any effect on the ionization would be minimal.
The pH/H_-rate profile displays two distinct regions reminis-
N
2
N
cent of those obtained for the simpler sulfamate NH
2
SO
–p. However, the change in the slope from zero to unity for
this ester occurs at about pH 5 whereas for 1a this change occurs
at about pH 9.5. This lateral shift is due to the fact that the pK
of the former is 7.29 while the corresponding pK for 1a is about
pK units higher. In the pH-independent region the reacting spe-
cies will be 1a, and above the pK
2 6 4-
OC H
may be calculated from data for its hydrolysis in similar aqueous
1
2
NO
2
24
EtOH mixtures.
Another type of solvent-reactivity mechanistic probe that can
be applied here involves the use of two solvent mixtures with iden-
tical ionizing power (same YOTs) but very different nucleophilicities
a
a
2
a
(
differing NOTs). If the hydrolysis is significantly slowed down in
a
of 1a ꢀ9.5, it will be 1b and even-
the solvent of lower nucleophilicity, this suggests that the solvent
is playing an important role as a nucleophile. The results of the
application of this method to the hydrolysis of 1a are given in Table
tually 1c. Two important conclusions can be made from
examination of Figure 1. First the upward curvature indicates that
a change in mechanism occurs,18,19 and second, in the pH-indepen-
2. It can be readily seen that employing 97% trifluoroethanol (TFE),
dent region there is no catalysis and the upward line has a slope of
8
5% TFE and 97% hexafluoro-i-propanol (HFIP) results in a moder-
ꢂ
1
indicating that the process occurring is first order in [OH ].
In order to probe the mechanism on the non-catalyzed section
ate reduction in rate thus pointing to an important role for water as
a nucleophile in the hydrolysis of 1a. Curiously, though the ratios
in Table 2 are considerably lower than those observed for the
of the pH/H_ profile a Grunwald–Winstein plot (see Supplemen-
tary data) was constructed using twelve rate constants at 50 °C
for reaction of 1a in aqueous EtOH mixtures containing from 90%
to 20 water content and using YOTs values20 for these solutions
hydrolysis of other compounds including the simpler ester
1
NH
2
SO
2
OC
6
H NO
4
2
-p. The hydrolysis of the ester 667-COUMATE
(
3), which is the subject of considerable interest, gives ratios in line
(
(
Table 1). The pH ranged from 5.68 (90% water content) to 6.45
20% water content).
25
with those in Table 2.
Kinetic solvent isotope effects (KSIEs) kH2O/kD2O for 1a of 3.93
The plot yields an m value of 0.13 (correlation coefficient,
(
pD = 6.61, at 60 °C) and 5.48 (pD = 12.90, at 15 °C) have been
determined. The former value supports a bimolecular S 2 path-
way in the flat region of the pH/H_ rate profile. The reacting spe-
cies will be d -1a because of rapid exchange of the protons for
deuterons in D O however, this will not contribute to the isotope
effect since the ND centre is not involved in the slow step of the
2 reaction at sulfur and the value of 3.93 is a KSIE. At pD 12.90
r = 0.97). Values of m at this level are interpreted as indicating sup-
port for a bimolecular mechanism; in this case, most likely an S
N
N
2
1
mechanism involving attack by water at the sulfur of 1a. The data
can be scrutinized in another way using the extended Grunwald–
Winstein equation (see Supplementary data),21
2
2
2
S
N
Table 1
Rate data for hydrolysis of 1a in aqueous EtOH and parameters for Grunwald–
Winstein plots
on the ascending part of the profile d-1b will be deuterated on
nitrogen and the isotope effect observed (5.48) is a KIE effect
reflecting extensive cleavage of the deuterium-nitrogen bond in
b
Log (kobs)c (sꢂ1
)
Water–Ethanol
Y
OTs
N
OTs
2
6
the slow step of an E1cB reaction.
9
8
7
7
6
5
5
5
4
3
2
2
0–10
0–20
5–25
0–30
3.78
3.32
3.11
2.84
1.97
1.92
1.83
1.29
0.92
0.47
0.23
0.00
ꢂ0.41
ꢂ0.34
ꢂ
ꢂ4.73
ꢂ4.78
ꢂ4.86
ꢂ4.95
ꢂ5.04
ꢂ5.06
ꢂ5.07
ꢂ5.09
ꢂ5.12
ꢂ5.20
ꢂ5.22
ꢂ5.24
O
a
O
ꢂ0.35
ꢂ0.23
—
0–40
6.8–43.2
5.2–44.8
0–50
0–60
0–70
NH SO O
2
2
—
ꢂ0.09
ꢂ0.08
ꢂ0.05
—
3
a
5–75
0–80
Activation data were determined for the hydrolysis of 1a at var-
ious pHs and in 1.5 M and 3 M KOH. The energy of activation E
was obtained from the Arrhenius equation and the heat of activa-
0.00
a
a
b
c
20
These two YOTs values were interpolated from the literature.
N
à
à
OTs values were taken from the literature.20
tion
DH and the change in entropy DS were calculated from the
The rates are the average of three runs with less than 4% deviation.
Eyring equation. Table 3 tabulates the values obtained and the

W. J. Spillane, J.-B. Malaubier / Tetrahedron Letters 51 (2010) 2059–2062
2061
Table 2
-
-
OH
OH
2-
-
Nucleophilicity data for the hydrolysis of 1a at 50 °C
NH SO OR
NHSO OR
NSO2 OR
2
2
2
6
ꢂ1
Solv. mixture
w/w)
N
OTs
Y
OTs
k
obs ꢁ 10 (s
)
Rate ratio
ksolv mixture/ksolv. low nucl.
1a
1b
1c
(
H O
4
5
3
4
5
3
1
1
1
4.8% EtOH
2.4% MeOH
6.0% MeCN
3.2% EtOH
0.5% MeOH
4.5% MeCN
0% EtOH
ꢂ0.19
ꢂ0.17
ꢂ1.16
ꢂ0.20
ꢂ0.18
ꢂ1.16
ꢂ0.41
ꢂ0.41
ꢂ1.29
1.83
1.83
1.83
1.92
1.92
1.92
3.78
3.78
3.60
8.51
6.51
5.89
8.71
8.56
4.65
18.8
16.4
16.1
6.03a
2
a
4.61
4.17
a
O
O
b
-
2-
N
4.58
4.50
NH2
-
NH
S
OR
S
OR
b
H
H
δ
+
S
.
δ
b
O
.
OR
2.44
2.01
O
O
c
O
O
0% MeOH
0% MeCN
1.74c
1.71c
slow
slow
-
N
-
-
HN SO2 + OR
SO2 + OR
a
Rate ratio against hydrolysis of 1a in 97% TFE (NOTs = ꢂ3.25, YOTs = 1.83),
slow
ꢂ6 ꢂ1
k
k
k
obs = 1.41 ꢁ 10
s .
fast
b
Rate ratio against hydrolysis of 1a in 85% TFE (NOTs = ꢂ2.01, YOTs = 1.92),
fast
ꢂ6 ꢂ1
obs = 1.90 ꢁ 10
s .
+
c
-
Rate ratio against hydrolysis of 1a in 97% HFIP (NOTs = ꢂ4.27, YOTs = 3.60),
NH SO OH
+
HOR
a
OR + H
2
2
ꢂ6 ꢂ1
obs = 9.4 ꢁ 10
s .
2
2b
Table 3
Activation data for the hydrolysis of 1a
O
R =
pH/H_ Temp. rangea (K)
(kJ mol ) DH (kJ mol ) DS (J K mol )
E
ꢂ1
à
ꢂ1
à
ꢂ1
ꢂ1
a
Scheme 1. Proposed mechanisms of hydrolysis of 1a, 1b and 1c.
1
9
.74 333–358
.59 313–353
95
88
78
76
75
92
86
76
74
73
ꢂ52
ꢂ69
ꢂ25
ꢂ19
ꢂ15
1
1
1
3.69 278–318
by about two units. The incursion of a path involving the dianionic
b
4.33 283–308
4.85c 278–298
sulfamate, 1c, arises in the strongest base as the second pK
a
of 1a,
2
that is, 1b ¡ 1c is approached.
a
Rates were measured at 5 K intervals over the range. The correlation coeffi-
cients (r) of the Arrhenius and Eyring plots were P0.98.
A range of physical organic mechanistic techniques have indi-
cated that the hydrolysis of EMATE (1a) occurs by two different
b
This H_ corresponds to 1.5 M KOH.
This H_ corresponds to 3 M KOH.
c
mechanisms: an S
mechanisms involving N-sulfonylamines at higher pHs.
2 (S) mechanism below pH ꢀ9.5 and E1cB
N
Acknowledgements
footnotes, the number of points used and the range of tempera-
tures for each determination. The entropies at pHs 1.74 and 9.59
are reasonably negative and would support a bimolecular mecha-
nism involving exclusively 1a (at pH 1.74) and 1a/1b (at pH
Professor B. V. L. Potter, Department of Pharmacy and Pharma-
cology, University of Bath and Sterix Ltd. is thanked for kindly pro-
viding a gift of EMATE. Professor Potter is also thanked for many
helpful suggestions during the preparation of this Letter. Professor
S. Thea, University of Genova, Italy is thanked for advice regarding
9
.59). At pH 13.69 and in stronger base 1b and 1c will be the spe-
cies present and since the first step of an elimination path (removal
of a proton) has been realized the mechanism here is likely to be
E1cB. The negative entropies measured at pHs 1.74 and 9.59 sup-
a
pK values. The NUI, Galway Millennium Fund is thanked for finan-
cial support.
port an S
unimolecular slow decomposition of 1b and 1c.
In Scheme 1 the slow S 2 solvolytic pathway for the decompo-
N
2 mechanism and the less negative entropies suggest a
N
Supplementary data
sition of 1a in the acidic pH region is shown on the left, in the cen-
tre an E1cB mechanism involving the hydrolysis of monoanionic 1b
and on the right a similar mechanism involving dianionic 1c is
shown. Various N-sulfonylamines are envisaged in the eliminative
routes (Scheme 1) as previously proposed for the hydrolysis of
Supplementary data (a table containing kinetic data for the pH/
H_-rate profile for 1a at 50 °C, a figure showing the Grunwald–
Winstein plot for 1a in aqueous EtOH at 50 °C and a figure showing
the extended Grunwald–Winstein plot for 1a) associated with this
article can be found, in the online version, at doi:10.1016/
j.tetlet.2010.02.065.
1
2
2 2 6 4
NH SO OC H X. All routes give the same final products, namely,
sulfamic acid and estrone, as either 2a or 2b.
Recently the kinetics of inactivation of an arylsulfatase (from
Pseudomonas aeruginosa) by simple sulfamates NH
have shown that there is a high degree of cleavage of the
2 2 6 4
SO OC H X
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