Acid dissociation constant, a potential physicochemical factor in the inhibition of the enzyme estrone sulfatase (ES)

Article abstract of DOI:10.1016/S0960-894X(01)00087-7

We report the initial results of the synthesis and biochemical evaluation of a series of aminosulfonate based compounds of phenol and the determination of the pK_a of the parent phenol in an attempt to investigate the role of this physicochemical factor in the irreversible inhibition of the enzyme estrone sulfatase (ES). The results of the study show that there is a strong correlation between the observed pK_a and inhibitory activity. We postulate that the stability of the phenoxide ion, as indicated by the acid dissociation constant, is an important factor in the irreversible inhibition of this enzyme.

Full text of DOI:10.1016/S0960-894X(01)00087-7

Bioorganic & Medicinal Chemistry Letters 11 (2001) 899–902
Acid Dissociation Constant, a Potential Physicochemical
Factor in the Inhibition of the Enzyme Estrone Sulfatase (ES)
Sabbir Ahmed,* Caroline P. Owen, Karen James
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 September 2000; revised 10 November 2000; accepted 2 February 2001
Abstract—We report the initial results of the synthesis and biochemical evaluation of a series of aminosulfonate based compounds
of phenol and the determination of the pKa of the parent phenol in an attempt to investigate the role of this physicochemical
factor in the irreversible inhibition of the enzyme estrone sulfatase (ES). The results of the study show that there is a strong
correlation between the observed pKa and inhibitory activity. We postulate that the stability of the phenoxide ion, as indicated by
the acid dissociation constant, is an important factor in the irreversible inhibition of this enzyme. # 2001 Published by Elsevier
Science Ltd.
Introduction
the proposed pharmacophore, we initiated a series of
structure–activity relationship(SAR) determination
studies. Initially, we undertook the determination of:
the transition state of the desulfatation reaction (Fig. 1)
and the charge density of EMATE and its derivatives
(2-nitro-, 4-nitro- and 2,4-dinitro-). Whilst the potential
transition state allowed us to design potentially new
compounds (involving the superimpositioning of poten-
tial inhibitors onto the derived transition state), the
calculation of the electron density and partial charges
(the electron density isosurface was generated by an
extended Huckel wavefunction, using a geometry deter-
mined by mechanics optimization using augmented
MM2 parameters) allowed us to consider the previously
proposed mechanisms for ES and thus the role of the
phenoxide ion (and therefore the involvement of pKa) as
a potential factor in the inhibition process. That is, the
results of the charge density calculation and partial
charge calculations showed that the sulfur atom of the
sulfamate group is susceptible to nucleophilic attack.
From this we concluded that the phenoxide ion stability
may be an important factor in aminosulfonate contain-
ing inhibitors (Fig. 2).
The enzyme estrone sulfatase (ES) converts the stored
(sulfated) form of the estrogens to the active forms and
has recently become a major therapeutic target against
hormone dependent breast cancer. A number of ster-
oidal inhibitors1ꢀ3 have been investigated as potent
inhibitors of this enzyme, including estrone-3-O-sulfa-
mate (EMATE). However, this compound has been
shown to possess potent estrogenic properties, and as a
result, the investigation of non-steroidal inhibitors has
intensified. The potent inhibitors, in general, contain an
aminosulfonate moiety which is involved in the irrever-
sible inhibition of ES. From the consideration of the
results obtained with the known sulfamate containing
steroidal and non-steroidal inhibitors, a ‘definitive
model’ was proposed4 where it was suggested that the
most fundamental and basic requirements for inhibition
was the phenolic ring attached via the phenolic oxygen
atom to the sulfamate group—it was presumed that the
oxygen atom was required for strong hydrogen bonding
to the active site with the result that the OꢀS bond of
the amino sulfonate groupwas weakened.
In an effort to overcome the lack of detailed informa-
tion regarding the active site and to probe the nature of
In an effort to verify our conclusion with respect to the
phenoxide ion, we designed and synthesized a number
of o-, m- and p-substituted phenol derivatives contain-
ing the aminosulfonate group. Here, we report the
initial results of the synthesis, biochemical evaluation,
*Corresponding author. Tel.: +44-20-8547-2000 ext. 2433; fax: +44-
20-8547-7562; e-mail: s.ahmed@kingston.ac.uk
0960-894X/01/$ - see front matter # 2001 Published by Elsevier Science Ltd.
PII: S0960-894X(01)00087-7

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S.Ahmed et al./ Bioorg.Med.Chem.Lett.11 (2001) 899–902
Figure 1. The transition-state for the desulfatation reaction catalysed by ES.
pKa determination and the SAR determination study
for these compounds.
yield without any major problems—the synthetic pro-
cedures for the phenyl sulfamate (1) is outlined below as
an example.9 In the synthesis of the 2-substituted sulfa-
mate compounds, a number of compounds proved dif-
ficult to synthesize (presumably due to steric hindrance
or intramolecular hydrogen bonding) and as a result the
2-substituted compounds were not produced.
In the synthesis of the 3- and 4-substituted phenol deri-
vatives, a modified literature procedure5ꢀ7 (Scheme 1)
was followed and was found to proceed well and in good
Although the pKa values exist for a small number of the
phenols, we concluded that a better, more consistent
approach would be the determination of the pKa values
of all of the parent phenols considered within our pre-
sent study. The determination of the pKa of the starting
phenols involved a spectroscopic technique8 which con-
sidered the change in UV absorption by the phenolic
groupunder acidic, pH 9, and basic conditions.
Scheme 1. Synthesis of aminosulfonate derivatives of the phenol based
compounds [a=NaH (or anhydrous K2CO3)/toluene/H2NSO2Cl].
Figure 2. To show partial charges for 2,4-dinitroEMATE.
Table 1. Showing the synthesized compound and the determined pKa, IC50 values and relative potency as compared to COUMATE
Compound number
Group
Substitution
pKa
IC50/mM
Relative potency
1
2
3
4
5
6
7
8
H
CH3
F
Cl
Br
CN
NO2
CH3
F
Cl
Br
—
3
3
3
3
3
3
4
4
4
4
4
4
—
—
9.98
10
>10,000
>10,000
2089
537
—
—
174
45
21
16
10
—
—
132
76
25
27.5
1
9.16
9
8.95
8.54
8.28
10.2
9.8
9.5
9.29
8.02
7.15
—
257
190.5
120
>10,000
>10,000
1584.8
912
9
10
11
12
13
CN
300
330
12
NO2
COUMATE
EMATE
—
0.1
0.008

S.Ahmed et al./ Bioorg.Med.Chem.Lett.11 (2001) 899–902
901
The results of the pKa determination and biochemical
evaluation are shown in Table 1. In an effort to consider
the inhibitory activity of the compounds under con-
sideration, we undertook the evaluation of two of the
known potent inhibitors against ES, namely, EMATE
and coumarin-7-O-sulfamate (COUMATE). From the
IC50 values, we can clearly see that a large range of
inhibitory activity exists against ES, from compounds
which are clearly non-inhibitors (e.g., the 3- and 4-
methyl derivatives), to compounds (3-nitrophenyl sulfa-
mate) which are only 10 times weaker than the most
potent non-steroidal compound COUMATE (which
possesses an IC50 of 12 mM). All the synthesized inhibi-
tors were observed to possess irreversible inhibition (the
method used in irreversible inhibition determination is
outlined within the References and Notes).
suggest that compounds containing groups (i.e., elec-
tron withdrawing groups such as nitro and cyano
groups) that are able to stabilize the phenoxide ion may
result in more potent inhibitors than those containing
groups which destabilize the phenoxide ion (i.e., elec-
tron donating groups such as methyl). This trend can be
observed clearly when the two series of compounds are
considered separately, that is, a higher correlation coef-
ficient is obtained for the plot of IC50 versus pKa for the
4-substituted compounds alone (R2=0.99) (Fig. 4),
compared to R2=0.73 for both series of data (Fig. 3).
In conclusion, the results of the biochemical evaluation
show that a relationshipmay exist between the inhibitory
activity and the pKa of the parent phenol. That is, when
the hydrolysis reaction occurs (Fig. 5), the resulting
phenoxide ion (and in particular its stability) is possibly
a factor in determining the inhibitory activity of the
sulfamate containing inhibitors.
From the consideration of the overall results for both 3-
and 4-substituted phenols, it can be observed that a
relationshipexists between the inhibitory activity of the
sulfamate compounds and the pKa of the parent phenol
(Fig. 3). Furthermore, the data (Table 1) may also
References and Notes
1. Purohit, A.; Potter, B. V. L.; Parker, M. G.; Reed, M. J.
Chemico-Biological Interactions 1998, 109, 183.
2. Howarth, N. M.; Purohit, A.; Reed, M. J.; Potter, B. L. V.
Steroids 1997, 62, 346.
3. Selcer, K. W.; Jagannathan, S.; Rhodes, M. E.; Li, P.-K. J.
Steroid Biochem.Mol.Biol. 1996, 59, 83.
4. 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.
5. Li, P. K.; Pillai, R.; Dibbelt, L. Steroids 1995, 60, 299.
6. Anderson, C.; Freeman, J.; Lucas, L. H.; Farley, M.; Dal-
houmi, H.; Widlanski, T. S. Biochemistry 1997, 36, 2586.
7. Woo, L. W. L.; Lighttowler, M.; Purhoit, A.; Reed, M. J.;
Potter, B. V. L. J.Steroid Biochem.Mol.Biol. 1996, 57, 79.
8. Harwood L. M.; Moody C. J. Experimental Organic
Chemistry; Blackwell: Oxford, 1989, pp 716–719.
Figure 3. Plot of IC50 versus pKa for all the 3- and 4-substituted
phenols.
9. Chemistry: Synthesis of phenyl sulfamate (1): NaH (80%
dispersion in mineral oil, 0.12 g, 4.0 mmol) was added to a
stirred solution of phenol (0.30 g, 3.19 mmol) in DMF (20 mL)
under nitrogen 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
overnight. The reaction was then quenched in NaHCO3
(50 mL), extracted into DCM (2ꢃ50 mL), washed (3ꢃ30 mL
water) and dried (MgSO4). Removal of the solvent under
vacuum yielded a yellow oil, which was run through a column
to give 1 (0.14 g, 25.4%) as a pure white solid mp 77.6–81.2 ꢁC.
Rf=0.32 [diethyl ether/petroleum ether 40–60 ꢁC (6:4)]. n(max)
(Film) cmꢀ1 : 3421.1 and 3307.8 (NH), 1367.5 and 1177.2
(S¼O). 300 MHz dH (CDCl3) 7.43–7.25 (5H, m, ArH), 5.24 (2H,
s, NH2). dC (CDCl3) 150.024, 129.923, 127.306, 122.142. MS
(M+) calculated mass 173.014665, actual mass 173.015633.
ES assay: The biochemical evaluation of the series of com-
pounds was undertaken in triplicate using the previously
reported method of Selcer.3 The total assay volume was 1 mL.
3H estrone sulfate (25 mL, 20 mM/tube; 300,000dpm) and the
inhibitors (of varying 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 micro-
somes and assay tubes were pre-incubated for 5 min at 37 ꢁC in
a shaking water bath prior to the addition of the microsomes
Figure 4. Plot of log IC50 versus pKa for the 4-substituted phenols.
Figure 5. Hydrolysis of aminosulfonates.

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S.Ahmed et al./ Bioorg.Med.Chem.Lett.11 (2001) 899–902
(0.8 mL) to the tubes. After 20 min incubation (at 37 ꢁC), tol-
uene (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 (TRI-
TONX). 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.
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
estrone sulfatase 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 duplicate, was subjected to dialysis at 4ꢁC for
16 h, with regular changes of Tris–HCl buffer. The microsomes
were then removed from the dialysis tubing and tested for estrone
sulfatase activity as described above.
Irreversible ES assay: The irreversible inhibition was deter-
mined using the procedure described in ref 1 using EMATE
(10 mM), COUMATE (100 mM), 12 (400 mM) and 13 (400 mM).