Hydrophobicity, a physicochemical factor in the inhibition of the enzyme estrone sulfatase (ES)

Article abstract of DOI:10.1016/S0960-894X(01)00482-6

We report the initial structure-activity relationship study (SAR) (in particular log P) of a series of compounds based upon 4-sulfamated phenyl ketones as potent inhibitors of the enzyme estrone sulfatase (ES). The results of the study show that these compounds are irreversible inhibitors of ES and that they are more potent than COUMATE, but weaker than EMATE. Analysis of the SAR data shows a strong correlation between IC₅₀ and log P but also supports our previous study, which suggests a very strong relationship between pK_a and IC₅₀.

Full text of DOI:10.1016/S0960-894X(01)00482-6

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2525–2528
Hydrophobicity, a Physicochemical Factor in the Inhibition of the
Enzyme Estrone Sulfatase (ES)
Sabbir Ahmed,* Karen James, Caroline P. Owen, Chirag K. Patel and Mijal Patel
School of Chemical and Pharmaceutical Sciences, Kingston University, Penrhyn Road, Kingston upon Thames, Surrey, KT1 2EE, UK
Received 16March 2001; revised 9 July 2001; accepted 10 July 2001
Abstract—We report the initial structure–activity relationship study (SAR) (in particular log P) of a series of compounds based
upon 4-sulfamated phenyl ketones as potent inhibitors of the enzyme estrone sulfatase (ES). The results of the study show that these
compounds are irreversible inhibitors of ES and that they are more potent than COUMATE, but weaker than EMATE. Analysis of
the SAR data shows a strong correlation between IC50 and log P but also supports our previous study, which suggests a very strong
relationship between pKa and IC50. # 2001 Elsevier Science Ltd. All rights reserved.
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
(AR).1,2 However, the use of AR inhibitors does not
result in the inhibition of all of the biosynthetic pro-
cesses which lead to estrogen formation. That is, the
enzyme estrone sulfatase (ES) converts the stored (sul-
fated) form of the estrogens to the active (non-sulfated)
form (Fig. 1), thereby allowing the stimulation of
tumours via a non-AR pathway (which is not blocked
by AR inhibitors).
model’ was proposed where it was suggested that the
most fundamental and basic requirements for inhibition
were the phenolic ring, and a bridging oxygen atom
joining the phenyl ring to the sulfamate group.5 We
have recently shown that the stabilisation of the phen-
oxide ion6 and the incorporation of electron-with-
drawing groups within the phenyl ring can result in a
significant increase in the inhibitory activity of com-
pounds, thus alkyl sulfamates, being poorer acids (in
comparison to phenols), are weaker inhibitors of ES.6
In an effort to overcome the lack of detailed informa-
tion regarding the active site of ES, probe the nature of
A number of steroidal inhibitors3,4 has been investi-
gated as potent inhibitors of this enzyme, including
estrone-3-sulfamate (EMATE) (a time- and concentra-
tion-dependent irreversible steroidal inhibitor) and the
4-methylcoumarin-7-O-sulfamate derivative (COU-
MATE) (an irreversible non-steroidal inhibitor) (Fig. 2).
However, since EMATE has been shown to possess
potent estrogenic properties, the investigation into non-
steroidal inhibitors has intensified.
Figure 1. Action of the enzyme ES on estrone sulfate.
In general, the potent inhibitors contain an amino-
sulfonate moiety which is believed to be involved in the
irreversible inhibition of ES. From the consideration of
the results obtained with the known sulfamate contain-
ing steroidal and non-steroidal inhibitors, a ‘definitive
*Corresponding author. Tel.: +44-208-547-2000; fax: +44-208-547-
7562; e-mail: ch_s534@kingston.ac.uk
Figure 2. Structures of EMATE and COUMATE.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00482-6

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S.Ahmed et al./ Bioorg.Med.Chem.Lett.11 (2001) 2525–2528
the proposed pharmacophore and rationalise the inhi-
bitory activity of the aminosulfonate based compounds,
we initiated a series of structure–activity relationship
(SAR) determination studies. From the results of our
initial molecular modelling study and a consideration of
potential mechanisms for ES, we concluded that log P
may also be an important factor in the inhibition pro-
cess. That is, we concluded that the role of the carbon
backbone is to favour the formation of the ROꢀ ion
(hence the requirement for the aromatic moiety) and the
expulsion of the ROꢀ due to the high log P of the car-
bon backbone within ROꢀ. We also hypothesised that
as a result of the high log P requirement, the reaction
catalysed by ES ‘appears’ to be an irreversible reaction.
weaker than EMATE. In comparison to COUMATE,
we observe that a number of the synthesised compounds
are equipotent or are stronger inhibitors than COU-
MATE; indeed compounds 5, 6 and 7 are 2.4, 2.1 and
3.5 times more potent, respectively. These three com-
pounds would therefore appear to be some of the most
potent non-steroidal compounds known to date. The
synthesised compounds were further evaluated to deter-
mine their mode of action.12 It was discovered that the
enzyme did not recover after the incubation with the
synthesised compounds, that is the compounds are irre-
versible inhibitors of ES (Fig. 3).
From the consideration of the initial SAR, the data
from the biochemical evaluation of these inhibitors
suggest that log P is indeed a factor in the inhibitory
activity of compounds against ES—this is therefore the
first report to show a high level of correlation between
the inhibitory activity and the log P of the parent phenol
(Fig. 4). Furthermore, the data suggest that there is an
apparent optimum log P and thus there is alkyl chain
length limit (between 6and 8) beyond which the
potency of the inhibitors decreases. The decrease in
inhibitory activity with further increase in alkyl chain
length may be as a result of: steric interaction between
the alkyl chain and a part of the enzyme active site,
thereby resulting in the destabilisation of the enzyme–
inhibitor complex; or the log P of the overall sulfamated
compound being too large, that is the high log P of the
sulfamated compound disfavours the entry of the sulfa-
mated compound into the ES active site, thereby low-
ering the inhibitory activity. That the hypothesis
regarding the size of compound 8 may be a potential
factor in the decreasing inhibitory activity can be
observed when the compounds under consideration are
superimposed onto estrone (Fig. 5). It is found that the
overall length of compounds, such as 8, exceeds that of
estrone, resulting in a potential increase in interactions
between parts of the active site and the alkyl chain.
Whilst the steric factor may be important, we strongly
believe that within the range of compounds synthesised,
it is the greater hydrophobicity of 8, in comparison to
the lower alkyl chain containing inhibitors, which
results in reduced inhibitory activity, that is the high
hydrophobicity of this inhibitor disfavours its entry into
the active site.
In order to verify our hypothesis with respect to the
importance of log P, we undertook a design process so
as to incorporate the increasing log P, whilst restricting
the pKa of the parent phenol, and we predicted that
sulfamated phenyl ketones would possess the appro-
priate characteristics. Here, we report the initial results
of our study where we have undertaken: the synthesis of
derivatives of 4-sulfamated phenyl ketones; the in vitro
biochemical evaluation of the synthesised compounds;
and the evaluation of the mode of action, that is rever-
sible or irreversible inhibition.
Chemistry
In the synthesis of the 4-aminosulfonated derivatives of
4-hydroxyphenyl ketones,
a
modified literature
procedure7ꢀ9 (Scheme 1) was followed and was found to
proceed well and in good yield without any major pro-
blems. The synthesis of 4-nonanoylphenyl sulfamate is
given as an example10—it should be noted that since 4-
hydroxynonanophenone is commercially available, it
was not synthesised via the initial Friedel–Crafts acyla-
tion reaction.
The results of the biochemical evaluation11 of the syn-
thesised sulfamated phenyl ketones (as well as EMATE
and COUMATE within our assays for comparison) are
shown in Table 1 together with the relative potencies
against the latter two compounds. Consideration of the
results shows that these compounds are potent inhibi-
tors of ES, with compound 7 being only 6.8 times
Compounds 9 and 10 appear to possess unusually weak
inhibitory activity compared to, for example, com-
pounds 4 and 5, although all four inhibitors possess
similar log P values. We believe that whilst the hydro-
phobicity factor is an important one, the pKa factor is of
even greater importance and highlights our previous
hypotheses6 regarding the stability of the phenoxide ion
resulting from the hydrolysis of the sulfamate group.
That is, within compounds 9 and 10, the electron with-
drawing C¼O group attached directly to the sulfamated
phenyl ring is connected to an electron-rich phenyl ring.
In the case of 10, this involves the conjugation of the
overall p system. We suggest that the ability of the C¼O
group to attract electrons from a ‘second’ and alter-
native source of electrons lowers the stability of the
phenoxide ion in comparison to compounds such as 8.
Scheme 1. Synthesis of the 4-sulfamate derivative of the substituted
benzoic acid [a=acid chloride/AlCl3/DCM; b=NaH/H2NSO2Cl/tol-
uene].

S.Ahmed et al./ Bioorg.Med.Chem.Lett.11 (2001) 2525–2528
2527
Table 1. Inhibition data for compounds 1–11 and the relative potencies of some of the synthesised compounds in comparison to EMATE and
COUMATE
Compound
R
IC50 (mM)
Potency wrt
EMATE
Potency wrt
COUMATE
Calculated log P
of parent phenol
1
2
3
4
5
6
7
8
H
CH3
254ꢄ10.1
302ꢄ6.7
0.0019
0.0016
0.01260.3015
0.0239
0.0472
0.0397
1.4
1.1
C3H7
C4H9
C6H13
C7H15
C8H17
C9H19
Ph
CH¼CHPh
CH2Ph
—
39.8ꢄ1.4
20.9ꢄ0.38
5.0ꢄ0.360.1000
5.6ꢄ0.19
3.4ꢄ0.13
13ꢄ0.05
2.1
3.3
0.5742
2.5
2.4000
0.0893
0.1471
0.0385
0.0079
0.0019
0.0152
1
2.1429
3.5294
0.9231
0.1905
0.04563.4
0.3636
24
3.7
4.1
4.4
2.9
9
10
63ꢄ1.83
263ꢄ5.5
11
EMATE
COUMATE
33ꢄ1.1
2.9
3.9
0.5ꢄ0.001
12ꢄ0.160.0417
—
1
1.7
date; only the recently reported 667-COUMATE is
known to be of greater potency. The compounds are
therefore good lead compounds in the search for more
potent non-steroidal inhibitors and have allowed us
further insight into the structural features required for
increased inhibitory activity.
Figure 3. Plot of counts per min (CPM) versus time to show the time-
and concentration-dependent inhibition of ES by compound 5:
0 mmol/L (^), 2 mmol/L (&) and 20 mmol/L (~) concentrations.
References and Notes
1. Feutrie, M. L.; Bonneterre, J. Bulletin Cancer 1999, 86, 821.
2. Brodie, A. M. H.; Njar, V. C. O. Steroids 2000, 65, 171.
3. Howarth, N. M.; Purohit, A.; Reed, M. J.; Potter, B. L. V.
Steroids 1997, 62, 346.
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. Woo, L. W. L.; Lighttowler, M.; Purhoit, A.; Reed, M. J.;
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.
Figure 4. Plot of IC50 versus calculated log P of parent phenol.
7. Anderson, C.; Freeman, J.; Lucas, L. H.; Farley, M.; Dal-
houmi, H.; Widlanski, T. S. Biochemistry 1997, 36, 2586.
8. Selcer, K. W.; Jagannathan, S.; Rhodes, M. E.; Li, P.-K. J.
Steroid Biochem.Mol.Biol. 1996, 59, 83.
9. Purohit, A.; Potter, B. V. L.; Parker, M. G.; Reed, M. J.
Chemico-Biological Interactions 1998, 109, 183.
10. Chemistry: 4-Nonanoylphenyl sulfamate (7): Sodium
hydride (NaH) (60% dispersion in mineral oil, 0.18 g,
4.5 mmol) was added to a stirred solution of 4-hydroxy-
nonophenone (1 g, 4.27 mmol) in dimethyl formamide (DMF)
(10 mL) under an atmosphere of nitrogen gas 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 for 10 h. The reaction was then quen-
ched with saturated sodium bicarbonate (NaHCO3) solution
(50 mL), extracted into dichloromethane (DCM) (2ꢃ50 mL),
washed with water (3ꢃ30 mL) and dried over anhydrous
magnesium sulfate (MgSO4). The mixture was filtered and the
solvent removed under vacuum to give a yellow oil which
solidified on addition of water. The crude product was purified
using flash chromatography to give 7 (0.32 g, 23.9%) as a
white solid [mp 102–104 ꢁC; Rf 0.57 ether/petroleum ether 40–
60 ꢁC (70/30)]. n(max) (Film) cmꢀ1: 3389.0, 3289.0 (NH2),
1682.3 (C¼O), 1377.9, 1181.8 (S¼O). dH (CDCl3): 7.99 (2H, d,
J=9 Hz, ArH), 7.42 (2H, d, J=9 Hz, ArH), 5.17 (2H, s, NH2),
2.94 (2H, t, J=7 Hz, COCH2CH2), 1.72 (2H, m,
COCH2CH2CH2), 1.35 (10H, m, COCH2CH2[CH2]5CH3),
Figure 5. Superimpositioning of compound 8 onto estrone to show the
length of the alkyl chain.
That our hypothesis may be valid is supported by the
greatly increased inhibitory activity observed in com-
pound 11 (compared to 9 and 10), where the two p sys-
tems are now separated by a CH2 spacer group which
discontinues the conjugation, thereby allowing the C¼O
to withdraw electrons solely from the sulfamated phenyl
ring system. However, the slightly weaker inhibitory
activity of compound 11 compared to compound 4
suggests that a steric factor may also be involved.
In conclusion, we have synthesised a range of sulfa-
mated phenyl ketones, which have proved to be some of
the most potent non-steroidal compounds known to

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S.Ahmed et al./ Bioorg.Med.Chem.Lett.11 (2001) 2525–2528
0.88 (3H, t, J=7 Hz, –CH3). dC (acetone-d6): 154.6(C ¼O),
138.3, 136.1, 130.4, 122.8 (C–Ar) 38.8, 32.3, 29.7, 23.0 (CH2),
14.1 (CH3). MS m/z obtained MH+ 314.1422,
(C15H23NO4S)H+ requires 314.1426.
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.
11. ES assay: In the biochemical evaluation, the standard lit-
erature method was used.8 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 dilu-
ted 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
12. Irreversible ES assay: The irreversible inhibition was
determined using the procedure described by Purohit et al.
(1998)9 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 16h, with regular changes of
Tris–HCl buffer. The microsomes were then removed from the
dialysis tubing and tested for ES activity as described above.