2-Substituted estradiol bis-sulfamates, multitargeted antitumor agents: Synthesis, in vitro SAR, protein crystallography, and in vivo activity

Article abstract of DOI:10.1021/jm060705x

The anticancer activities and SARs of estradiol-17-O-sulfamates and estradiol 3,17-O,O-bis-sulfamates (E2bisMATEs) as steroid sulfatase (STS) inhibitors and antiproliferative agents are discussed. Estradiol 3,17-O,O-bis-sulfamates 20 and 21, in contrast t

Full text of DOI:10.1021/jm060705x

J. Med. Chem. 2006, 49, 7683-7696
7683
2-Substituted Estradiol Bis-sulfamates, Multitargeted Antitumor Agents: Synthesis, In Vitro
SAR, Protein Crystallography, and In Vivo Activity†
Mathew P. Leese,‡ Bertrand Leblond,‡ Andrew Smith,‡ Simon P. Newman,§ Anna Di Fiore,| Giuseppina De Simone,|
Claudiu T. Supuran, Atul Purohit,§ Michael J. Reed,§ and Barry V. L. Potter*,‡
Medicinal Chemistry, Department of Pharmacy and Pharmacology & Sterix Ltd., UniVersity of Bath, Bath BA2 7AY, United Kingdom,
Endocrinology and Metabolic Medicine and Sterix Ltd., Faculty of Medicine, Imperial College, St. Mary’s Hospital, London W2 1NY,
United Kingdom, Istituto di Biostrutture e Bioimmagini-CNR, Naples, Italy, and Laboratorio di Chimica Bioinorganica, UniVersita` degli
Studi di Firenze, Florence, Italy
ReceiVed June 12, 2006
The anticancer activities and SARs of estradiol-17-O-sulfamates and estradiol 3,17-O,O-bis-sulfamates
(E2bisMATEs) as steroid sulfatase (STS) inhibitors and antiproliferative agents are discussed. Estradiol
3,17-O,O-bis-sulfamates 20 and 21, in contrast to the 17-O-monosulfamate 11, proved to be excellent STS
inhibitors. 2-Substituted E2bisMATEs 21 and 23 additionally exhibited potent antiproliferative activity with
mean graph midpoint values of 18-87 nM in the NCI 60-cell-line panel. 21 Exhibited antiangiogenic in
vitro and in vivo activity in an early-stage Lewis lung model, and 23 dosed p.o. caused marked growth
inhibition in a nude mouse xenograft tumor model. Modeling studies suggest that the E2bisMATEs and
2-MeOE2 share a common mode of binding to tubulin, though COMPARE analysis of activity profiles was
negative. 21 was cocrystallized with carbonic anhydrase II, and X-ray crystallography revealed unexpected
coordination of the 17-O-sulfamate of 21 to the active site zinc and a probable additional lower affinity
binding site. 2-Substituted E2bisMATEs are attractive candidates for further development as multitargeted
anticancer agents.
Introduction
In previous studies we have established the ability of 3-O-
sulfamoylated estrogens such as estrone 3-O-sulfamate (EMATE)a
to act as potent irreversible inhibitors of steroid sulfatase
(STS),1,2 a clinical target for the treatment of hormone-
dependent breast cancer.3,4 This activity is believed to arise from
either nucleophilic attack of the sulfamate group onto the
conserved formylglycine residue in the STS active site or, most
likely, transfer of the sulfamate group either to a nucleophilic
residue in the active site of STS or by generation of a highly
electrophilic sulfonylamine species, to irreversibly arrest the
catalytic function of the enzyme.5 Further investigations on
the biological activities of these compounds revealed that
estrogen 3-O-sulfamates are also highly active reversible inhibi-
tors of carbonic anhydrase II, an enzyme which is highly
expressed in red blood cells, through a coordination of the
monoanionic form of the sulfamate moiety to the zinc atom in
the enzyme active site.6 This interaction is believed to underlie
the high oral bioavailability observed for estradiol 3-O-sulfamate
(1, E2MATE: Figure 1) (which is atypical of estrogen deriva-
tives), wherein reversible uptake by red blood cells and
interaction with carbonic anhydrase II results in a bypassing of
first-pass liver metabolism.7
Figure 1. Chemical structures of estradiol 3-O-sulfamate (E2MATE,
1), 2-methoxyestradiol (2), and 2-substituted E2MATE compounds 3-5.
The discovery of the antiproliferative and antiangiogenic
effects of 2-methoxyestradiol (2, 2-MeOE2; Figure 1) and a
number of closely related compounds has stimulated consider-
able interest in the potential of such molecules as clinical agents
for the treatment of cancer.8 The antiproliferative effects of
2-MeOE2 are independent of the estrogen receptor (ER) status
of the treated cancer cell line and, though a precise mechanistic
picture remains to be established, appear to stem from its ability
to disrupt the dynamics of tubulin polymerization.9 At high
concentrations 2-MeOE2 has been shown to down-regulate
HIF-1R and HIF-2R protein expression in tumor cells.10 This
causes subsequent down-regulation of the mRNA expression
for the HIF-1R-regulated genes Glut-1, VEGF165 and endothelin-
1. The effects on HIF, however, occur downstream of the
interaction with microtubules, and it has recently been suggested
that such effects occur on treatment with all microtubule-
disrupting agents.11 Various analogues of 2-MeOE2 modified
in the A-ring (C-2),12-15 B-ring (C-6),13 and D-ring (C-14, C-15,
C-16, C-17)14,16,17 display significantly enhanced in vitro
antiproliferative activity, though translation of their activity into
the in vivo context is complicated by conjugative inactiva-
tion (e.g. sulfation of the 3- and/or 17-hydroxyl groups) and
metabolic oxidation of their 17â-hydroxyl group to the inactive
C-17 ketones mediated by 17â-hydroxysteroid dehydrogenase
† Protein crystal data: Protein Data Bank accession code 2GD8.
* To whom correspondence should be addressed. Phone: +44 1225
386639. Fax: +44 1225 386114. E-mail: B.V.L.Potter@bath.ac.uk.
‡ University of Bath.
§ Imperial College.
| Istituto di Biostrutture e Bioimmagini-CNR.
Universita` degli Studi di Firenze.
a Abbreviations: EMATE, estrone 3-O-sulfamate; E2MATE, estradiol
3-O-sulfamate; E2bisMATE, estradiol 3,17-O,O-bis-sulfamate; STS, steroid
sulfatase; CA, carbonic anhydrase; hCAII, human carbonic anhydrase II;
ER, estrogen receptor; E2S, estradiol 3-O-sulfate; HDBC, hormone-
dependent breast cancer; HIF, hypoxia-inducible factor; DMA, N,N-
dimethylacetamide.
10.1021/jm060705x CCC: $33.50 © 2006 American Chemical Society
Published on Web 12/06/2006

7684 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Leese et al.
type II.18 The importance of conjugation and metabolism was
illustrated in a recent clinical trial where a dose escalation of
up to 6 g/day of 2-MeOE2 was used in an attempt to achieve
satisfactory plasma concentrations.19
Scheme 1. Synthesis of 3,17-O,O-Bis-sulfamate Derivatives of
Estradiola
Investigations on the effects of 2-substitution on sulfatase
inhibitory activity in the estrogen 3-O-sulfamate series and the
effects of 3-O-sulfamoylation on the antiproliferative activity
of 2-MeOE2 led us to discover a series of 2-substituted
estrogen 3-O-sulfamates as a class of antitumor agents notable
for their multiple mechanisms of action.20,21 Thus, in addition
to irreversibly inhibiting STS-mediated conversion of E1S to
E1 at concentrations in the nanomolar range, compounds such
as 2-methoxyestradiol 3-O-sulfamate (3, 2-MeOE2MATE;
Figure 1) displayed potent antiproliferative effects against a
range of estrogen-independent human cancer cell lines in vitro.
As with 2-MeOE2 the antiproliferative activities of the 2-sub-
stituted estrogen 3-O-sulfamates appear to arise from their ability
to disrupt the paclitaxel-induced polymerization of tubulin,
resulting in a G2/M phase cell cycle arrest and apoptosis.22 As
would be expected of microtubule-disrupting drugs, 2-substituted
estradiol 3-O-sulfamates, among other effects, induce tumor
suppressor protein p53 and phosphorylation of BCL-2 and BCL-
XL.22 Significantly, the antiproliferative effects of 2-MeO-
E2MATE were expressed at much lower concentrations than
that required for 2-MeOE2, and furthermore, the cell cycle arrest
induced by the sulfamate compound proved irreversible, whereas
the effects of 2-MeOE2 treatment were reversible.
a Reagents and conditions: (i) t-BuOK, H2NSO2Cl, 0 °C to rt; (ii) NaH,
DMF, then RX; (iii) H2, Pd/C, EtOH/THF; (iv) DBMP, H2NSO2Cl, CH2Cl2,
0 °C to rt.
of NMU-induced mammary tumors in rats,24 while a separate
study
showed
that
2-MeOE2MATE
and
2-
EtE2MATE dosed orally at 20 mg/kg inhibited the growth of
MDA-MB-435 xenografts (ER- human breast cancer cells) in
female mice by 33% and 40%, respectively, and that this
inhibition persisted after the cessation of dosing.21
Preliminary studies had established that D-ring modification
of 2-substituted estradiol 3-O-sulfamates could significantly
modulate both the antiproliferative and sulfatase inhibitory
activities of these compounds.25 In particular, it was found that
hydrogen-bonding interactions around C-17 were key to high
antiproliferative activity. As part of a wider structure-activity
program, we thus set out to establish the effect of introduction
of a second sulfamate group at C-17 on both STS inhibition
and antiproliferative activity, since an aliphatic sulfamate ester
moiety is unlikely to be rapidly removed in vivo. Such a
substitution was anticipated to both satisfy H-bonding require-
ments proximal to the D-ring and also block undesirable
conjugation at C-17. We report here synthetic routes to
2-substituted estradiol bis-sulfamates and those with various
simple substitutions at the 17-sulfamate group. In vitro and in
vivo results demonstrate the superiority of the simple 2-substi-
tuted bis-sulfamates with respect to both the corresponding
monosulfamates and their parent estradiol derivatives. In addi-
tion, a novel interaction of 2-methoxyestradiol 3,17-O,O-bis-
sulfamate with carbonic anhydrase II is demonstrated through
X-ray crystallography on a human carbonic anhydrase II
(hCAII)-ligand complex.
Structure-activity studies of 2-substituted estrogen 3-O-
sulfamates showed that optimal antiproliferative effects were
obtained with methoxy, ethyl, or methylsulfanyl substitution at
the 2-position (i.e. 2-CH3X, where X ) O, CH2, or S, e.g.,
compounds 3, 4, and 5; Figure 1).21 In contrast, in the absence
of a 3-O-sulfamate group 2-ethoxy and 2-(1-propenyl) substitu-
tion is reportedly optimal.12 The sulfamoylated estradiol deriva-
tives proved far more potent than the nonsulfamoylated deriva-
tives against the proliferation of a wide range of cancer cell
lines, with, for example, 2-MeOE2MATE proving to be 8-fold
more active against the proliferation of MCF-7 cells than
2-MeOE2. Comparison of the two molecules across the NCI
screening panel further highlighted the enhancement in anti-
proliferative activity afforded by incorporation of the 3-O-
sulfamate group, with 2-MeOE2MATE exhibiting a mean GI50
(mean graph midpoint, MGM) across the NCI panel of 110 nM
(cf. 2-MeOE2, 1.3 µM).21 Substitution of the sulfamate nitrogen
was not well tolerated and caused a dramatic reduction in
antiproliferative activity. In striking contrast to the nonsulfa-
moylated 2-substituted estrogen series the sulfamoylated estrone
derivatives also proved to be active against the proliferation of
cancer cells, with 2-ethylestrone 3-O-sulfamate (2-EtEMATE)
proving to be over 160-fold more active than 2-ethylestrone.
This result clearly indicated that, although 2-substituted estrogen
3-O-sulfamates exhibit mechanistic similarities with 2-substi-
tuted estradiols, they are not prodrugs of the parent estrogens
and are instead a novel class of anticancer agents with a
contrasting structure-activity relationship. Additionally, the
antiangiogenic effects of 2-substituted EMATEs have also been
shown to be superior to those of 2MeOE2 in in vitro model
systems, with significantly reduced concentrations of the sulfa-
mate derivatives (with respect to the corresponding estradiol
derivatives) being required to inhibit, for example, HUVEC
growth and in vitro angiogenesis.23 Evaluation of the anti-
tumor effects of 2-substituted estradiol 3-O-sulfamates in in
vivo models reinforced the findings of in vitro studies. 2-Meth-
oxyestrone 3-O-sulfamate (2-MeOEMATE) inhibited the growth
Chemistry
The synthesis of 3,17-O,O-bis-sulfamates was first investi-
gated in the estradiol series as we were interested to develop
the chemistry with readily available starting materials and also
to assess the STS inhibitory activities of these compounds.
Elaboration of these compounds was achieved in a straightfor-
ward and generally high-yielding manner (Scheme 1). Thus, to
generate differentially substituted sulfamate groups at the 3- and
17- positions, 3-O-benzylestradiol was sulfamoylated to afford
the 17-O-sulfamated estradiol 6 using potassium tert-butoxide
and sulfamoyl chloride in DMF solution. Alkylation of 6 by
reaction with the appropriate alkyl or benzyl halide proceeded
smoothly to give the N,N-dialkylsulfamate compounds 7-9 in
excellent yield (89-99%). Acylation was also achieved under
similar conditions, with acetyl chloride used to obtain the
monoacetylated sulfamate derivative 10, albeit in a modest 46%
yield. Compounds 6-10 were then converted into the corre-
sponding phenols 11-15 under standard hydrogenolytic condi-
tions, which in turn were sulfamoylated with the hindered
pyridine base 2,6-di-tert-butyl-4-methylpyridine (DBMP) and

2-Substituted Estradiol Bis-sulfamates
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7685
Table 1. STS Inhibitory Activity of 3-O-, 17-O-, and
3,17-O,O-Sulfamoylated Estradiol Derivatives in Placental Microsomes
Scheme 2. Synthesis of 2-Substituted Estradiol
3,17-O,O-Bis-sulfamatesa
compd
no.
inhibition (%)
at 10 µM
R1
R2
R3
IC50(STS)a
1
11
12
13
14
15
20
16
17
18
19
21
23
24
H
OSO2NH2 OH
16 nM
5 µM
H
H
H
H
H
H
H
H
H
H
OH
OH
OH
OH
OH
OSO2NH2
65
8.7 ( 3.2
0
1.4 ( 3.1
31 ( 3
OSO2NMe2
OSO2NPent2
OSO2NBn2
OSO2NHAc
a Reagents and conditions: (i) DMA, H2NSO2Cl, 0 °C to rt; (ii) NaH,
DMF, MeI, 38%.
OSO2NH2 OSO2NH2
OSO2NH2 OSO2NMe2
OSO2NH2 OSO2NPent2 190 nM
OSO2NH2 OSO2NBn2
18 nM
96 ( 0.3
93 ( 1
38.2 ( 2.9
90.9 ( 0.3
Scheme 3. Synthesis of 2-Substituted Estradiol
17-O-Sulfamates, 2-Substituted Estradiol
OSO2NH2 OSO2NHAc
17-O-(N,N-Dimethyl)sulfamates, and 2-Substituted
39 nM31
1 µM
320 nM
3-O-Sulfamoylestradiol 17-O-(N,N-Dimethyl)sulfamatesa
MeO OSO2NH2 OSO2NH2
Et OSO2NH2 OSO2NH2
MeS OSO2NH2 OSO2NH2
a IC50 figures are the mean values obtained from experiments performed
in triplicate, SEM < (7%.
derivatives 30 and 31. The 2-methoxy and 2-ethyl 17-O-
sulfamates 28-31 were then deprotected to give the free phenols
32-35 by catalytic hydrogenation. Sulfamoylation of the
3-hydroxy 17-O-(N,N-dimethyl)sulfamate derivatives 34 and 35
afforded the unsymmetrically substituted bis-sulfamate deriva-
tives 36 and 37. A set of 2-methoxy- and 2-ethyl-estradiol
analogues bearing 17-O-sulfamoyl or 17-O-(N,N-dimethyl)-
sulfamoyl groups in combination with a 3-hydroxyl or 3-O-
sulfamoyl group were thus available for evaluation.
a Reagents and conditions: (i) DMA, H2NSO2Cl, 0 °C to rt; (ii) NaH,
DMF, MeI (for 34 and 35 only); (iii) H2, Pd/C, THF, EtOH.
Results and Discussion
To ascertain the effect of C-17 sulfamoylation on the STS
inhibitory activity of the estradiol 3-O-sulfamate derivatives,
selected compounds were assayed using the radiometric assay
developed by Purohit et al.28 wherein the STS-mediated conver-
sion of tritiated E1S to E1 by placental microsomes is
determined. Unsurprisingly, the 17-O-sulfamate estradiol deriva-
tives 11-15 lacking a 3-O-sulfamate group and with the
sulfamate linked to a saturated ring displayed only minimal
inhibitory activity towards the enzyme, with the nonsubstituted
sulfamate 11 giving an IC50 of 5 µM, while the substituted
derivatives caused, at best, <10% inhibition at a concentration
of 10 µM (Table 1). Previous studies have shown that a
nonsubstituted aryl 3-O-sulfamate group that mimics the sulfate
group of the enzyme’s natural substrate is a requisite for potent
irreversible inhibition.1,5,28 The 3,17-O,O-bis-sulfamate deriva-
tives 16-20 proved more active than the 17-O-monosulfamates,
with E2bisMATE (20) proving to be a potent inhibitor of STS
(IC50 ) 18 nM). As can be seen from the activity of the 3,17-
O,O-(N,N-dimethyl)bis-sulfamate (16), 3,17-O,O-(N,N-dipen-
tyl)bis-sulfamate (18), and 3,17-O,O-(N-acetyl)bis-sulfamate
(19) derivatives substitution on the 17-O-sulfamate group is
relatively well tolerated, although the larger benzyl-substituted
compound 17 proved to be significantly less active. It was thus
apparent that in this series large substituents, though not
substitution per se, on the C-17 sulfamate are poorly tolerated
in the active site of this enzyme and, furthermore, that the 3-O-
sulfamate group is required for potent irreversible inhibition of
STS.5 In earlier studies we have shown that introduction of a
substituent at the 2-position can modulate the STS inhibitory
sulfamoyl chloride in dichloromethane solution26 to give the
bis-sulfamates 16-19.
Bis-sulfamoylation of estradiol proved to be straightforward,
with reaction of estradiol and excess sulfamoyl chloride in
dimethylacetamide27 delivering high yields of estradiol 3,17-
O,O-bis-sulfamate (20, E2bisMATE) directly in excellent yield
(Scheme 2). A series of 2-substituted estradiol 3,17-O,O-bis-
sulfamates was then synthesized in an analogous manner from
the appropriate 2-substituted estradiols. Thus, 2-methoxy-
estradiol 3,17-O,O-bis-sulfamate (21, 2-MeOE2bisMATE),
2-ethoxyestradiol 3,17-O,O-bis-sulfamate (22, 2-EtOE2bis-
MATE), 2-ethylestradiol 3,17-O,O-bis-sulfamate (23, 2-EtE2bis-
MATE), and 2-methylsulfanylestradiol 3,17-O,O-bis-sulfamate
(24, 2-MeSE2bisMATE) were prepared in a rapid manner and
in good yield as shown in Scheme 2. 2-MeOE2bisMATE (21)
was alkylated under basic conditions to give the bis(N,N-
dimethyl)sulfamate derivative 25 in a straightforward manner.
A small series of 17-O-sulfamate, 17-O-(N,N-dimethyl)-
sulfamate, and 3-O-sulfamoyl-17-O-(N,N-dimethyl)sulfamate
derivatives of 2-MeOE2 and 2-ethylestradiol (2-EtE2) was next
targeted. To sulfamoylate the 17-hydroxyl group in a selective
manner, the more acidic 3-hydroxyl group was benzylated by
reacting the appropriate 2-substituted estradiol with benzyl
bromide at room temperature in DMF solution. The resultant
2-substituted 3-O-benzylestradiol derivatives 26 and 27 were
then reacted with sulfamoyl chloride in DMA to give the 17-
O-sulfamates 28 and 29 (Scheme 3); samples of these com-
pounds were then methylated to give the N,N-dimethylsulfamate

7686 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Leese et al.
Table 2. Antiproliferative Activities of Estradiol Derivatives Against
MCF-7 Human Breast Cancer Cells
corresponding 3-O-sulfamates. 2-MeOE2bisMATE (21) (GI50
) 0.25 µM) was found to be nearly 10-fold more active in this
assay than 2-MeOE2 (2) and also slightly more active than
2-MeOE2MATE (3) (GI50 ) 0.36 µM). Similarly, 2-MeS-
E2bisMATE (24) (GI50 ) 0.19 µM) proved to be 20-fold more
active than 2-MeSE2 and 2-fold more active than the corre-
sponding monosulfamate 5. As in the 3-O-sulfamate series, the
most potent compound in vitro proved to be the 2-ethyl
derivative, with 2-EtE2bisMATE (23) causing 50% inhibition
of cell proliferation at 70 nM; in the in vitro context the mono-
and bis-sulfamate derivatives of 2-EtE2 proved to be equipotent.
In contrast, 2-EtOE2bisMATE (22) (GI50 ) 10 µM) proved to
compd
no.
GI50(MCF-7)
2-substituent 3-substituent 17-substituent
(µM)
20
2a
H
OSO2NH2
OH
OSO2NH2
OH
OSO2NH2
OH
OSO2NH2
OSO2NMe2
OSO2NH2
OH
OSO2NH2
OH
OSO2NH2
OH
OSO2NH2
OH
OSO2NH2
OSO2NH2
OSO2NH2
OH
OH
OSO2NH2
OSO2NH2
OSO2NMe2
OSO2NMe2
OSO2NMe2
OSO2NH2
OH
>10
2.35
0.36
>10
0.25
>10
>10
>100
10
10.5
0.07
>10
0.07
>10
4.41
3.96
0.43
0.19
MeO
MeO
MeO
MeO
MeO
MeO
MeO
EtO
Et
Et
Et
Et
Et
3a
32
21
34
36
25
22
4aa
4a
33
23
35
37
5aa
5a
be less active than the corresponding monosulfamate (GI50
)
OH
0.61 µM21), a result which illustrates the steric limitations on
the size of the 2-substituent which we had previously observed
in the monosulfamate series.21
OSO2NH2
OSO2NH2
OSO2NMe2
OSO2NMe2
OH
Et
MeS
MeS
MeS
The effect of C-17 sulfamate substitution on the activity of
the bis-sulfamates was evident from the activities of 2-methoxy-
and 2-ethylestradiol-3-O-sulfamoyl-17-O-(N,N-dimethyl)sulfa-
mates (36 and 37) whose respective activities were >40- and
63-fold less than those of the corresponding unsubstituted
bisMATEs 21 and 23. Unsurprisingly, the tetramethylsulfamate
25 also proved devoid of antiproliferative activity, as we had
previously observed that alkylation of the 3-O-sulfamate group
was detrimental to in vitro antiproliferative activity in the
monosulfamate series,21 although we would anticipate activity
of such a compound in vivo. It thus appears that incorporation
of a nonsubstituted sulfamate at C-17 is well tolerated at the
site of action of the 2-substituted estradiol 3-O-sulfamates,
though alkylation of this group is not tolerated. Whether the
detrimental effect of alkylation is attributable to the removal of
H-bond donor capabilities of the group or simply due to the
increased steric size is not clear, although precedent from 17-
oximo derivatives of 2-ethylestrone 3-O-sulfamates suggests that
steric considerations are likely the most important factor.25
OH
OSO2NH2
24
a Data for these compounds are taken from ref 20. GI50 figures are the
mean values obtained from experiments performed in triplicate, SEM )
(7%.
activity of estrone 3-O-sulfamate derivatives, with high activity
being conferred by electron-withdrawing groups.29,30 The high
STS inhibitory activity displayed by 2-MeOE2bisMATE (21)
was thus not surprising, nor was the decrease in activity observed
across the 2-substituted series 21 > 24 > 23 on the basis of the
electronic properties of these substituents. It would thus appear
that, in addition to their antiproliferative and antiangiogenic
effects discussed below, estradiol 3,17-O,O-bis-sulfamates could
have therapeutic potential for the treatment of hormone-depend-
ent breast cancer (HDBC) by inhibition of STS and the resultant
lowering of circulatory and intratumoral estrogen levels.4
As a first-stage predictor of anticancer activity the compounds
were evaluated for their ability to inhibit the proliferation of
MCF-7 cells, an ER+ human breast cancer cell line. Previous
experience had shown that results from this assay were
predictive of the antiproliferative effects against ER+ and ER-
cancer cells already obtained with 2-MeOE2MATE. The results
of this assay are presented in Table 2; data for 2-substituted
estradiols and selected 3-O-sulfamate derivatives are included
for comparison.21 As can be seen estradiol 3,17-O,O-bis-
sulfamate (20) showed only modest activity as an antiprolif-
erative agent, with a concentration of 10 µM causing a 32%
inhibition of MCF-7 proliferation. The 3-O-sulfamate derivatives
3, 4, and 5 are, respectively, 8-, 150- and 9-fold more active
than the corresponding estradiol derivatives 2, 4a, and 5a.21 In
contrast, introduction of a single sulfamate group at the 17-
position proved deleterious to activity, with 2-methoxyestradiol
17-O-sulfamate (32, 2-MeOE2-17MATE) proving to be over 4
times less active than 2-MeOE2. 2-Ethylestradiol 17-O-sulfa-
mate (33) also proved to be inactive. The N-alkylated 17-
monosulfamates 2-methoxyestradiol 17-N,N-dimethylsulfamate
(34) and 2-ethylestradiol 17-N,N-dimethylsulfamate (35) also
proved to be devoid of significant antiproliferative activity. It
could thus be concluded that although 17-O-sulfamoylation of
2-methoxyestradiol may serve to block inactivating conjugation
and metabolism at this position, its deleterious effects on
antiproliferative activity are profound. It is also apparent, albeit
not surprisingly, that estradiol 17-O-sulfamates do not act as
prodrugs of the parent estradiols under the conditions of the
assay.
A selection of these compounds was made for screening in
the NCI human cancer cell line panel, thus allowing an
evaluation of activity across various cancer phenotypes.32 Data
obtained in eight individual cell lines from this assessment are
presented in Table 3. The cell line data presented are on the
grounds of the availability of comparative data for 2-MeOE2
which were obtained from the literature.12 The MGM data
represent the mean concentration required to cause 50% growth
inhibition in all the cell lines successfully evaluated and thus
indicate the relative activity of compounds across the panel.
MGM (µM) values are restricted by definition by the range of
concentrations at which the assays are performed, in this case
10-4 to 10-8 M; thus, a numerical value of 0.01 is maximal
and would be obtained for a compound which causes 50%
growth inhibition at concentrations of 10 nM or less in all cell
lines.
As can be seen, the relative activities of these compounds
across the panel correlate well with those obtained in the
preliminary MCF-7 assay discussed above as illustrated in the
2-methoxy-substituted series of compounds, wherein mean
concentrations of 1.3 µM, 110 nM, and 87 nM of the estradiol
2, 3-O-sulfamate 3, and bis-sulfamate 21 compounds, respec-
tively, were required to effect 50% growth inhibition across the
panel. The activity of the 2-alkoxybisMATEs was seen to
decrease with an increase in the steric size of the alkoxy group,
with the 2-ethoxy compound 22 proving to be 34-fold less potent
than the 2-methoxy compound 21. As in the MCF-7 assay the
2-ethyl compounds proved to be the most potent, with an MGM
value of 18 nM obtained for 2-EtE2bisMATE (23). Signifi-
The bis-sulfamoylated derivatives 21, 23, and 24, in contrast,
displayed equal or greater activity in this assay than the

2-Substituted Estradiol Bis-sulfamates
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7687
Table 3. GI50 (µM) and MGM (µM) Values Obtained from the NCI Screening Panela
lung
HOP-62
colon
HCT-116
CNS
SF-539
melanoma
UACC-62
ovarian
OVCAR-3
renal
SN12-C
prostate
DU-145
breast
MDA-MB-435
MGM
2
3
0.7
0.21
15
0.051
1.3
<0.01
<0.01
23.5
2.41
0.47
0.066
3.82
0.045
2.3
<0.01
N/A
0.32
0.044
4.81
0.036
6.5
<0.01
<0.01
12.4
0.36
0.039
4.2
<0.01
1.8
<0.01
<0.01
17.4
0.21
0.026
2.5
<0.01
15.6
<0.01
<0.01
18.1
0.95
0.32
11.8
0.126
7.2
0.04
<0.028
22.8
7.53
1.8
0.19
18.3
0.083
2.7
<0.01
<0.01
38.7
0.08
0.02
0.646
<0.01
34.5
<0.01
<0.01
16.6
1.3
0.11
6.4
0.087
2.95
0.016
0.018
14.5
32
21
22
4
23
20
18
18.7
10.5
2.44
2.39
10.8
3.42
4.57
a Data for 2,12 3, and 421 were taken from the literature.
Figure 3. Activity of 2-MeOE2bisMATE (21) in the NCI Lewis lung
model.
Having obtained highly promising activity in the NCI panel,
2-MeOE2bisMATE was selected for further evaluation by the
NCI. The NCI antiangiogenesis resource uses in vitro HUVEC
growth inhibition, cord formation, and chemotaxis assays to
select compounds with antiangiogenic activity.34 2-MeO-
E2bisMATE (21) gave IC50 values of 0.70, 0.13, and 0.40 µM
in these respective assays, thus indicating the potential antian-
giogenic activity of this molecule is in agreement with results
reported elsewhere.23
On the basis of its in vitro activity, 2-MeOE2bisMATE was
also selected for evaluation in the NCI hollow fiber assay, which
allows an assessment of the ability of subcutaneous and
intraperitoneally dosed candidate compounds to inhibit prolif-
eration of a selection of human cancer cells in vivo. This assay
entails intraperitoneal (ip) and subcutaneous (sc) implanting of
hollow poly(vinylidene fluoride) containing a culture of one of
twelve human tumor cell line fibers in mice.35 Each tumor line
is thus examined in the ip and sc environments. The mice are
then treated ip at two dose levels (25 and 37.5 mg/kg, 25% and
37.5%, respectively, of the MTD) on a daily basis for 4 days,
after a further day the fibers are collected for analysis, and the
analyzed compound is awarded a score of 2 if a 50% reduction
in viable cell mass compared to that of the vehicle control
samples is observed. In this assay 2-MeOE2bisMATE obtained
an ip score of 22 and an sc score of 6 (i.e., 50% growth
inhibition was observed in eleven of the ip implanted fibers
and three of the sc implanted fibers; the maximum possible score
is 96), thus surpassing the threshold score of 20 assigned as a
positive indication of in vivo efficacy.
Figure 2. In vitro SAR of E2bisMATE derivatives as (a) STS inhibitors
and (b) antiproliferative agents.
cantly, the compounds identified in the preliminary MCF-7 assay
proved to have high activity against the whole range of cancer
cell types present in the panel. In contrast to the 2-substituted
bis-sulfamate derivatives the C-2 unsubstituted estradiol bis-
sulfamates 18 and 20 displayed only modest activity. A
COMPARE analysis33 of data obtained for 23 against the
publicly available NCI screening data afforded only 12 positive
correlations, with the highest Pearson correlation coefficient
being 0.67 (>0.6 is considered a positive correlation), which
was obtained for the known microtubule disruptor Auristatin
A though, significantly, no correlation was seen with the
2-substituted estradiol derivatives. Similar results were obtained
for the COMPARE analysis of 21.
A summary of the in vitro SAR of the E2bisMATE
derivatives as STS inhibitors and antiproliferative agents is
presented in Figure 2. STS activity is conferred by the 3-O-
sulfamate group in conjunction with either no substituent at C-2,
thus allowing unhindered interaction/reaction of the sulfamate
group at the enzyme active site, or electronegative C-2 substit-
uents, which can either participate in electrostatic interactions
with the proximal residues of the active site or render the
sulfamate group more reactive to nucleophilic attack. Substitu-
tion of the C-17 sulfamate group with alkyl groups is relatively
well tolerated. In contrast, substitution at C-2 is seen to be
essential for high antiproliferative activity in the bisMATE
series, with the optimal substituent being the CH3X group (X
) O, S, CH2). Here, lipophilic interactions appear to dominate
as the observed in vitro activity is inversely related to the H-bond
acceptor capability of the 2-substituent and, furthermore, a
radical reduction in antiproliferative activity is seen with
increasing steric size. Removal of the 3-O-sulfamate group
results in a drastic reduction in antiproliferative activity as does
methylation of the 17-O-sulfamate nitrogen.
2-MeOE2bisMATE (21) was then selected for evaluation in
the early-stage Lewis lung carcinoma model, a mouse model
of non-small cell lung cancer.36 A plot of the data obtained
is presented in Figure 3 and clearly shows the ability of
2-MeOE2bisMATE to inhibit tumor growth. In this study dosing
was commenced 3 days after implantation and maintained for
15 days. The ip cohort (2.5, 5, and 10 mg/kg) was dosed daily

7688 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Leese et al.
Figure 4. In vivo effect of 2-EtE2bisMATE (23) on the growth of
MDA-MB-435 tumor xenografts in female nude mice. Vehicle (THF/
propylene glycol) or compound was administered orally (20 mg/kg)
once daily for 28 days. Tumor volumes were monitored at weekly
intervals for the duration of drug administration and continued for a
further 4 weeks postdosing (means ( SEM, n ) 12) (p < 0.01 vs
controls at the end of the study, Student’s t test).
(vehicle 10% DMSO in saline), and the iv (30 mg/kg) cohort
was dosed every 3 days (vehicle 12.5% EtOH, 12.5% cremo-
phore, 75% saline). Inhibition of tumor growth was seen for
all doses apart from the 2.5 mg/kg ip dose. Significantly, no
weight loss was observed on treatment with 21.
An assessment of the ability of 2-EtE2bisMATE (23) to
inhibit tumor growth in vivo was performed using xenografts
derived from MDA-MB-435 (ER-) human breast cancer cells
transplanted into female mice. As can be seen in Figure 4, oral
administration of 2-EtE2bisMATE (20 mg/kg, qd, 28d) caused
an 84% inhibition of tumor growth over the dosing period. No
significant weight loss or other signs of toxicity were observed
in the treated animals. Interestingly, significant tumor growth
inhibition was still observed after cessation of drug treatment
at 4 weeks. The in vivo activity of 23 in this model was
essentially equivalent to that observed for 2-MeOE2bisMATE
(21), which inhibited tumor growth by 86%.37 The bis-sulfamates
were thus significantly more active than the analogous 3-O-
monosulfamates 3 and 4, which, in the same model, at the same
dose, caused 33% (2-MeOE2MATE, 3) and 40% (2-EtE2MATE,
4) inhibition of tumor growth over the treatment period.21
2-MeOE2 caused no inhibition of tumor growth in this model
at this dose.37 The enhanced activity of 2-EtE2bis-
MATE (23) with respect to the corresponding monosulfamate
4 may well reflect the susceptibility of the monosulfamate
compound to accelerated clearance and deactivation by conjuga-
tion of the C-17 hydroxyl group. These preliminary in vivo
studies with nonoptimized dosing underline the potential of the
2-substituted estradiol bis-sulfamates for further development
as therapeutic agents for the treatment of cancer.
Figure 5. (a) Overlays of the highest scoring docks of 2-MeOE2 (2)
(violet), 2-MeOE2bisMATE (21) (silver), and 2-EtE2bisMATE (23)
(amber) into the colchicine binding site of tubulin. (b) Overlay of the
highest scoring dock of 21 (silver) and the crystallographic ligand
DAMA (violet). (c) Residues with the potential to H-bond to the 3-O-
sulfamate group of 21. (d) Residues with the potential to H-bond to
the 17-O-sulfamate group of 21.
drawn to study the computational docking of 2-MeOE2bisMATE
(21) and 2-EtE2bisMATE (23), as well as 2-MeOE2 (2), to
tubulin around the colchicine binding site, an approach facili-
tated by the publication of the crystal structure (Protein Data
Bank (PDB) accession code 1SA0) of a colchicine derivative
bound to a microtubule stabilized on a stathmin-like domain.39
Comparison of the best scoring docks (Figure 5a) of 2-meth-
oxyestradiol (violet) (2) and the 2-methoxyestradiol (silver) (21)
and 2-ethylestradiol (amber) (23) bis-sulfamate derivatives
shows that all three share the same binding mode. Unsurpris-
ingly, the sulfamate groups as well as the steroid skeleton of
21 and 23 occupy the same regions. The steroidal D-ring of 21
overlies the seven-membered aromatic ring of the colchicine-
derivedligandN-deacetyl-N-(2-mercaptoacetyl)colchicine(DAMA),
with the sulfamate 21 and hydroxyl group of DAMA apparently
occupying the same region of space (Figure 5b). The possibility
for H-bonding around the steroidal A-ring is shown for 21 in
An important mechanistic element of the antiproliferative
effects of both the 2-substituted estradiol 3-O-sulfamates and
their estradiol counterparts is their ability to disrupt normal micro-
tubule dynamics.22 Furthermore, the 2-substituted estradiol 3-O-
sulfamates are believed, like 2-methoxyestradiol,9 to bind to
tubulin in a competitive manner with colchicine. It has previ-
ously been established that treatment of MDA-MB-231 cells with
21 caused a complete loss of microtubule structure, thus support-
ing the postulate that this compound, like the monosulfamate
derivatives, functions as a microtubule disruptor.38 We were thus

2-Substituted Estradiol Bis-sulfamates
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7689
Figure 5c; here the proximal Asn249 and Lys254 residues are
available for H-bonding to the sulfamate NH2 (and for the OH
of 2-MeOE2 (2) (not shown)), while Gln11 could potentially
form a H-bond donor/acceptor pair interaction with the sulfamate
group. The residues proximal to the C-17 sulfamate of 21 are
shown in Figure 5d; once again potential H-bond acceptor
functions are available for interaction with the sulfamate NH2
group (Asn349 and Val315), while Lys352 and Thr314 appear
well placed to donate H-bonds to the oxygen atoms of the
sulfamate group (Lys 352 appears the most likely to interact
with the 17-hydroxyl group of 2-MeOE2 (2) (not shown)).
with the hydroxyl group of Thr199 (N---ThrOG ) 2.75 Å),
which in turn interacts with the Glu106OE1 atom (2.58 Å). On
the other hand, one of the oxygen atoms of the coordinated
sulfamate moiety is hydrogen bonded to the backbone amide
of Thr199 (ThrN---O ) 3.01 Å), whereas the other one is at a
distance of 3.15 Å from the Zn2+ ion. The organic scaffold of
the inhibitor is oriented toward the hydrophobic part of the active
site cleft, establishing a large number of strong Van der Waals
interactions (<4.5 Å) with residues Gln92, Val121, Phe131,
Val135, Leu198, Thr200, and Pro202. Finally, the 3-O-sulfamate
group interacts poorly with the enzyme and is very poorly
defined in the electron density maps (Figure 6b).
While the results of this docking study are not unequivocal
and are speculative in the absence of experimental support for
interaction of 21 and 23 with the colchicine binding site, the
potential of the sulfamate group to offer further H-bonding
interactions with respect to 2-MeOE2 around both the A- and
D-rings is clear and is reflected by the higher docking scores
obtained in this computational study. Perhaps more importantly,
insights obtained from this docking could direct point mutation
studies with the aim of generating resistant cell lines, especially
when the in vitro results on analogous molecules indicate that
the H-bond acceptor properties of the C-17 substituent are key
to antiproliferative activity.25
The observation that this inhibitor molecule was bound to
the zinc ion in the active site with the 17-O-sulfamate group,
rather than the 3-O-sulfamate group (Figure 6c), as elsewhere
as the monoanion, is rather surprising. In fact, the ionization of
the alkyl sulfamate of C-17 is disfavored relative to ionization
of the aryl sulfamate of the 3-position. This may arise because
the 2-substituent can potentially render less favorable the
interaction of the 3-O-sulfamate group with the zinc ion, causing
a steric hindrance with proximal residues in the active site;
indeed, we noticed in the earlier structures of the o-bromo
derivative of a dual sulfatase aromatase inhibitor that the affinity
of the brominated ligand was 5-fold lower than that of the non-
brominated compound.42
The ability of estrogen 3-O-sulfamates to reversibly inhibit
carbonic anhydrase (CA) is now well established and arises from
the coordination of the monoanionic form of the sulfamate
moiety to the zinc ion of the active site.6 This interaction
therefore offers the possibility of sequestration of the estrogen
sulfamate into red blood cells and the consequent bypassing of
first-pass liver metabolism.7 Having ascertained that 2-MeO-
E2bisMATE (21) reversibly inhibits hCAII (IC50 ) 379 nM)40
and also that this compound displays high oral bioavailability
(85%) in rats, we were drawn to investigate its interaction with
carbonic anhydrase II37 in more detail.
Comparison of the CAII inhibitory activity of 2-MeO-
E2bisMATE (21) (IC50 ) 379 nM) with that of EMATE (IC50
) 42 nM,40 which binds through its aryl sulfamate6), 2-MeO-
EMATE (IC50 ) 376 nM40), and 2-MeOE2-17MATE (32) (IC50
) 526 nM40) shows that the inhibitory activity of 2-MeO-
E2bisMATE (21) is greater than that of 2-MeOE2-17MATE
(32), where coordination can occur solely through the cyclic
alkyl sulfamate at C-17 as in that observed in the corresponding
structure of a loosely related sugar sulfamate.44 Furthermore,
since 2-MeOEMATE is significantly less active than EMATE
as a CAII inhibitor, methoxy substitution at C-2 hinders the
interaction of aryl sulfamates with CAII. Finally, since 2-MeO-
EMATE acts as an inhibitor of CAII through its 3-O-sulfamate
group, it seems likely that 2-MeOE2bisMATE binds to CAII
through both its 3-O- and 17-O-sulfamate groups and that these
interactions are likely to underlie the high oral bioavailability
of 21 that is atypical of estradiol derivatives and contrasts
strongly with that of 2-MeOE2.
We recently reported the X-ray crystal structures of two
classes of sulfamate ester drugs bound to carbonic anhydrase
II, STX64, a compound from clinical trial,4,41 and two examples
of a dual sulfatase aromatase inhibitor,42 and were thus drawn
to examine the interaction of 2-MeOE2bisMATE (21) and
hCAII (Figure 6) using the same method. Crystals of the
hCAII-21 adduct obtained by cocrystallization were isomor-
phous with those of the native protein, allowing for the
determination of the crystallographic structure by difference
Fourier techniques. The model was refined using the CNS
program to crystallographic R-factor and R-free values of 0.195
and 0.212, respectively. The statistics for data collection and
refinement are presented in the Supporting Information. The
overall quality of the model was high, with 100% of the non-
glycine residues located in the allowed regions of the Ram-
achandran plot. Inspection of the electron density maps in the
enzyme active site, at various stages of the crystallographic
refinement, allowed location of two inhibitor molecules in the
active cavity of the enzyme (Figure 6a,b), with one interacting
with the zinc ion of the active site and a second lying close to
the protein surface. Unexpectedly, the first ligand was not
coordinated to Zn through its 3-O-arylsulfamate, as had been
previously observed,6,41,42 but through the 17-O-sulfamate.
The second binding site located for the ligand close to the
usual binding pocket does not include sulfamate coordination
and was not seen in the earlier study on the related ligand
EMATE.6 Presumably, while unique, this represents a much
lower affinity interaction with the enzyme, as confirmed by the
rather low occupation factor (0.61), and is most likely a function
of the high ligand concentration used in the crystallization.
Thus, in the present study we have synthesized a series of
novel estradiol bis-sulfamate derivatives substituted at C-2 and
on the nitrogen of both the 3-O- and 17-O-sulfamate groups.
Introduction of a second sulfamate group on the 17-hydroxyl
group of the potent irreversible STS inhibitor E2MATE afforded
E2bisMATE, a molecule with slightly reduced STS inhibitory
activity relative to that of the parent compound. Substitution of
the C-17 sulfamate group nitrogen of E2bisMATE with large
alkyl or benzyl groups proved deleterious to the STS inhibitory
activity. The STS inhibition effected by the novel C-2-
substituted E2bisMATEs varied with the electronic properties
of the substituent, though the IC50 values remained in the
nanomolar range. It could thus be concluded that the STS
inhibitory properties of bis-sulfamoylated estradiol derivatives
Several polar and hydrophobic interactions stabilized the first
inhibitor molecule within the hCAII active site (Figure 6c).
Indeed, the ionized N atom of the 17-O-sulfamate moiety of
21 was coordinated to the catalytic Zn2+ ion with a tetrahedral
geometry (N---Zn2+ ) 1.93 Å), displacing the hydroxide ion
usually present in the active site of the uninhibited enzyme.43
In addition, this nitrogen atom also makes a hydrogen bond

7690 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Leese et al.
Figure 6. (a) Ribbon diagram of the hCAII-21 complex. The two inhibitor molecules, metal-coordinating residues His94, His96, and His119, and
the zinc ion are represented in ball and stick format. (b) Stereoview of the active site region in the hCAII-21 complex. The simulated annealing
omit |2Fo - Fc| electron density map, relative to the two inhibitor molecules, is shown. Residues coordinating the metal ion and participating in
recognition of the inhibitor molecules are also reported. (c) Detailed schematic representation of 21 within the hCAII active site.
highlight their potential for application against hormone-
dependent breast cancer.
the NCI panel for 2-MeOE2bisMATE (21) and 2-EtE2bisMATE
(23) (cf. 2-MeOE2, 1.3 µM). In addition to antiproliferative
activity 2-MeOE2bisMATE also exhibited significant inhibi-
tory activity in in vitro assays of angiogenesis. The antiprolif-
erative activity of these molecules, believed to principally derive
from their ability to disrupt normal microtubule dynamics, was
also evidenced in the in vivo context, with significant growth
inhibition observed with ip dosing of 2-MeOE2bisMATE in
In addition to these findings a number of 2-substituted
estrogen 3,17-O,O-bis-sulfamates exhibited high antiproliferative
activity in vitro against a wide range of hormone-independent
tumor cell lines. Notably, these compounds were typically over
10-fold more potent than the parent estradiols as evidenced by
the respective MGM values of 87 and 18 nM obtained from

2-Substituted Estradiol Bis-sulfamates
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7691
minimization as implemented within Sybyl7.1. The ligands were
then docked into this cleaned form of the 1SA0 crystal structure
using the GOLD version 2.2 docking package from the Cambridge
Crystallographic Data Centre (CCDC).48-52 The site for docking
was defined as an 11 Å radius centered on atom number 5208 (the
Cd2 atom of Leu 255) of the crystal structure which fully
encapsulates the colchicine binding site. Each ligand was docked
into the protein a total of 25 times and the docking fit scored with
the GoldScore scoring function; early termination based on the rmsd
of the docked poses was disabled. Validation was carried out by
docking the crystallographic ligand DAMA into the protein, with
the highest scoring dock proving to be consistent with that of the
crystallographic ligand (rmsd between the crystallographic and
docked colchicine 0.75 Å).
Protein Crystallography. The hCAII-21 complex was obtained
by adding a 5 M excess of the inhibitor to a 10 mg/mL protein
solution in 100 mM Tris-HCl, pH 8.5. Crystals of the complex
were obtained at 22 °C using the hanging drop vapor diffusion
method. Drops of 2 µL, prepared by mixing 1 µL of complex
solution with an equal volume of precipitant solution (2.6 M (NH4)2-
SO4, 0.3 M NaCl in 100 mM Tris-HCl (pH 8.4), and 5 mM
4-(hydroxymercury)benzoate), were suspended over wells contain-
ing 1.0 mL of precipitant. In about 2 days crystals of about 0.2
mm × 0.2 mm × 0.3 mm were grown. A complete dataset was
collected at 1.46 Å resolution from a single crystal, at 100 K, at
the synchrotron source Elettra in Trieste, Italy, using a Mar CCD
detector. Prior to cryogenic freezing, the crystals were transferred
to the precipitant solution with the addition of 15% (v/v) glycerol.
Diffracted intensities were processed using the HKL crystal-
lographic data reduction package (Denzo/Scalepack).53 A total of
142133 reflections were measured and reduced to 40728 unique
reflections. Crystal parameters and data processing statistics are
summarized in tabular form in the Supporting Information.
The structure of the native hCAII (PDB code 1CA2)43 was used
as a starting model for rigid body refinement in CNS.54 Water
molecules were removed from the starting model prior to structure
factor and phase calculations. Fourier maps calculated with 3Fo -
2Fc and Fo - Fc coefficients showed prominent electron density
features in the active site region. After an initial refinement, limited
to the enzyme structure, a model for two inhibitor molecules was
easily built up and introduced into the atomic coordinate set for
further refinement. Many cycles of manual rebuilding using the
program O55 and positional and temperature refinement using the
program CNS54 were necessary to reduce the crystallographic
R-factor and R-free values (in the 20.00-1.46 Å resolution range)
to 0.195 and 0.212, respectively. The statistics for refinement are
summarized in the Supporting Information.
Biology. The effects of compounds on cell growth were
determined using a microtiter plate assay (Promega, Madison, WI).
STS inhibition was assayed by the method of Purohit et al.28 The
ability of 2-EtE2bisMATE (23) to inhibit tumor growth in vivo
was examined using xenografts derived from MDA-MB-435 (ER-)
human breast cancer cells transplanted into female mice with 12
mice per group. These studies were carried out by AntiCancer Inc.
(San Diego, CA). Treatments were initiated when tumor volumes
reached 100-200 mm.3 Drugs were dissolved in a minimum
volume of tetrahydrofuran (THF), diluted with propylene glycol,
and administered at 20 mg/kg orally, daily for 28 days. The length
(l) and width (w) of the tumors were measured at weekly intervals,
from which tumor volumes were calculated using the formula (lw2/
2). Monitoring of tumor volumes continued for a further 28 day
period after the end of drug administration. Lewis lung studies
were carried out by the developmental therapeutics program of the
NCI.56
3-O-Benzylestradiol 17-O-sulfamate (6). An ice cold solution
of 3-O-benzylestradiol57 (1.20 g, 3.31 mmol) in DMF (35 mL) was
treated with potassium tert-butoxide solution (4 mL, 4 mmol, 1 M
in THF) and then, after 0.25 h, sulfamoyl chloride (24 mL, 16.3
mmol, 0.7 M in toluene) in a dropwise manner. The reaction was
allowed to warm to rt over 2 h and then recooled to 0 °C before
addition of saturated ammonium chloride (15 mL) and water (60
the NCI hollow fiber assay and the early-stage Lewis lung
model. Furthermore, oral administration of 2-MeOE2bisMATE
and 2-EtE2bisMATE caused tumor regression and prolonged
growth inhibition upon cessation of dosing in an MDA-MB-
435 xenograft model. Computational docking studies suggest
that the 2-substituted estradiol bis-sulfamates share a mode of
binding to tubulin which is common with 2-MeOE2 but can
exploit further H-bonding interactions around the steroidal A-
and D-rings. To further investigate the origins of the high oral
bioavailability displayed by this class of molecule,37 a crystal
structure of 2-MeOE2bisMATE with CAII was obtained, and
it was confirmed for the properly bound ligand of the two
located in the structure that one of the sulfamate groups interacts
with the zinc atom in the active site of CAII, although the
orientation was unexpected and against established precedent
for aryl sulfamate derivatives.
In conclusion, the studies detailed herein highlight compounds
such as 2-MeOE2bisMATE (21) and 2-EtE2bisMATE (23) as
potent inhibitors of in vivo tumor growth that may target the
tumor cells directly by microtubule disruption. Translation of
the antiangiogenic activity observed in in vitro screens to the
in vivo context could extend the therapeutic potential of these
molecules, as could their ability to inhibit steroid sulfatase. The
compounds are straightforward to synthesize, display high oral
bioavailability, and are resistant to deactivating metabolism and
conjugation. Studies are presently under way to progress these
agents toward clinic trial.
Experimental Section
Chemistry. All chemicals were purchased from Aldrich Chemi-
cal Co. (Gillingham, U.K.), Fluka (Gillingham, U.K.), or Lancaster
Synthesis (Morecambe, U.K.). Organic solvents of AR grade were
supplied by Fisher Scientific (Loughborough, U.K.) and used as
supplied. Anhydrous N,N-dimethylformamide (DMF) and N,N-
dimethylacetamide (DMA) were purchased from Aldrich and stored
under a positive pressure of N2 after use. THF was distilled from
sodium with benzophenone indicator. Sulfamoyl chloride was
prepared by an adaptation of the method of Appel and Berger45
and was stored in the refrigerator under a positive pressure of N2
as a solution in toluene as described by Woo et al.46 An appropriate
volume of this solution was freshly concentrated in vacuo im-
mediately before use. E1 was purchased from Sequoia Research
Products (Oxford, U.K.). Reactions were carried out at room
temperature (rt) unless otherwise stated. Flash column chromatog-
raphy was performed on silica gel (MatrexC60).
1H NMR and 13C NMR spectra were recorded with a Varian
Mercury VX400 NMR spectrometer at 400 and 100 MHz,
respectively, and chemical shifts are reported in parts per million
(ppm, δ) relative to the peak for tetramethylsilane (TMS) as an
internal standard. Mass spectra were recorded at the Mass
Spectrometry Service Center, University of Bath, U.K., or at the
EPSRC National Mass Spectrometry service, Swansea, U.K.
FAB-MS were carried out using m-nitrobenzyl alcohol (NBA) as
the matrix. Elemental analyses were performed by the Micro-
analysis Service, University of Bath. Melting points were deter-
mined using a Reichert-Jung Thermo Galen Kofler block and are
uncorrected.
Molecular Modeling. The ligands for docking were built using
the Sybyl7.147 molecular modeling environment from Tripos Inc.,
assigned with Gasteiger-Huckel charges, and then minimized using
the MMFF94s force field as implemented within Sybyl7.1. The
crystal structure 1SA039 was cleaned also using Sybyl7.1 by first
removing the crystallographic ligand and cofactors, then adding
the requisite hydrogen atoms to the protein, and finally carrying
out a backbone-constrained minimization of all hydrogen atoms
and side chain atoms to allow for internal hydrogen bond formation
and removal of any internal clashes. The AMBER FF99 force field
with Gasteiger-Huckel charges was used for the constrained

7692 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Leese et al.
29.0, 27.5, 26.7, 25.7, 22.6, 11.6; MS [ES-] m/z 350.5 ((M+
mL). The solution was extracted into ethyl acetate (3 × 100 mL),
and the combined organic layers were washed with brine (3 × 100
mL), dried, and evaporated to give a yellow solid (1.48 g).
Recrystallization (ethyl acetate/hexane) gave 6 as white crystals
(1.29 g, 88% yield): mp 108-109 °C; 1H NMR δ 7.26-7.45 (5H,
m), 7.19 (1H, d, J ) 8.6 Hz), 6.78 (1H, dd, J ) 8.6 and 2.3 Hz),
6.71 (1H, d, J ) 2.3 Hz), 5.03 (2H, s), 4.55-4.80 (2H, br), 4.51
(1H, dd, J ) 8.2 and 8.2 Hz), 2.80-2.90 (2H, m), 1.15-2.40 (13H,
m), 0.87 (3H, s); MS [FAB+] m/z 441.2 (M+, 41), 91.1 (100);
HRMS [FAB+] m/z found 441.1977, calcd 441.1974. Anal. (C25H31-
NO4S) C, H, N.
3-O-Benzylestradiol 17-O-(N,N-dimethyl)sulfamate (7). A
solution of 6 (300 mg, 0.68 mmol) in DMF (10 mL) was treated
with sodium hydride (60 mg, 1.36 mmol) and then methyl iodide
(169 µL, 2.72 mmol). The reaction was stirred for 16 h and then
diluted with ethyl acetate (50 mL) and water (30 mL). The organic
layer was then separated, washed with brine (3 × 50 mL), dried,
and evaporated to give sulfamate 7 as a pale yellow solid (310
mg, 99%): mp 117-121 °C; 1H NMR δ 7.25-7.45 (5H, m), 7.19
(1H, d, J ) 8.4 Hz), 6.78 (1H, dd, J ) 8.4 and 2.7 Hz), 6.72 (1H,
d, J ) 2.7 Hz), 5.03 (2H, s), 4.46 (1H, dd, J ) 8.2 and 7.9 Hz),
2.87 (6H, s), 2.75-2.90 (2H, m), 1.15-2.40 (13H, m), 0.86 (3H,
s); MS [FAB+] m/z 469.2 (M+, 50), 345.2 (46), 91.1 (100); HRMS
[FAB+] m/z found 469.2284, calcd 469.2287.
-
H)-, 100). Anal. (C18H25NO4S‚0.5H2O) C, H, N.
17â-O-(N,N-Dimethylsulfamoyl)estradiol (12). A solution of
7 (310 mg, 0.66 mmol) in methanol (20 mL) and THF (5 mL) was
treated with 10% Pd/C (150 mg) and then stirred under a H2
atmosphere for 16 h. The catalyst was removed by filtration, and
the solvent was evaporated to afford 12 as a white solid (205 mg,
1
82%): mp 170-173 °C; H NMR δ 7.11 (1H, d, J ) 8.6 Hz),
6.63 (1H, dd, J ) 8.6 and 2.7 Hz), 6.56 (1H, d, J ) 2.7 Hz), 4.63
(1H, dd, J ) 9.4 and 7.8 Hz), 3.48 (2H, br), 2.87 (6H, m), 2.74-
2.90 (2H, m), 1.18-2.36 (14H, m), 0.86 (3H, s); MS [FAB+] m/z
379.2 (M+, 16); HRMS [FAB+] m/z found 379.1818, calcd
379.1817.
17â-O-(N,N-Dipentylsulfamoyl)estradiol (13). A solution of 8
(460 mg, 0.79 mmol) in methanol (20 mL) and THF (7 mL) was
treated with 10% Pd/C (150 mg) and then stirred under a H2
atmosphere for 2.25 h at rt. The catalyst was removed by filtration,
and the solvent was evaporated to afford 13 as a white solid (310
1
mg, 80%): mp 93-96 °C; H NMR δ 7.14 (1H, d, J ) 8.2 Hz),
6.62 (1H, dd, J ) 8.2 and 2.7 Hz), 6.56 (1H, d, J ) 2.7 Hz), 4.58
(1H, br), 4.42 (1H, dd, J ) 8.5 and 8.3 Hz), 3.12-3.24 (4H, m),
2.76-2.86 (2H, m), 1.18-2.34 (15H, m), 0.91 (6H, t, J ) 7.0 Hz),
0.83 (3H, s); MS [FAB+] m/z 491.3 (M+, 16), 255.2 (100); HRMS
[FAB+] m/z found 491.3074, calcd 491.3069.
3-O-Benzylestradiol 17-O-(N,N-dipentyl)sulfamate (8). Sulfa-
mate 6 (300 mg, 0.68 mmol) in DMF (10 mL) was reacted with
pentyl bromide (0.45 mL, 3.62 mmol) and sodium hydride (73 mg,
1.81 mmol) as described for the synthesis of 7. Column chroma-
tography (hexane to 9:1 hexane/ethyl acetate) gave 8 as a white
solid (490 mg, 92%): mp 55-56 °C; 1H NMR δ 7.26-7.48 (5H,
m), 7.19 (1H, d, J ) 8.2 Hz), 6.78 (1H, dd, J ) 8.2 and 2.7 Hz),
6.71 (1H, d, J ) 2.7 Hz), 5.03 (2H, s), 4.42 (1H, dd, J ) 8.5 and
8.4 Hz), 3.10-3.25 (4H, m) 2.75-2.90 (2H, m), 1.10-2.40 (25H,
m), 0.91 (6H, t, J ) 7.8 Hz), 0.83 (3H, s); MS [FAB+] m/z 581.2
(M+, 28); HRMS [FAB+] m/z found 581.3522, calcd 581.3539.
3-O-Benzylestradiol 17-O-(N,N-dibenzyl)sulfamate (9). Sulfa-
mate 6 (300 mg, 0.68 mmol) in DMF (10 mL) was reacted with
benzyl bromide (0.33 mL, 2.72 mmol) and sodium hydride (60 mg,
1.36 mmol) as described for the synthesis of 7. Column chroma-
tography (5:1 hexane/ethyl acetate) gave 9 as a white solid (380
17â-O-(N,N-Dibenzylsulfamoyl)estradiol (14). A solution of
9 (200 mg, 0.38 mmol) in methanol (20 mL) was treated with 10%
Pd/C (100 mg) and then stirred under a H2 atmosphere for 7 h at
rt. The catalyst was removed by filtration, and the solvent was
evaporated to afford 14 as a white solid (230 mg, 84%): mp 60-
63 °C; 1H NMR δ 7.28-7.39 (10H, m), 7.14 (1H, d, J ) 8.2 Hz),
6.63 (1H, dd, J ) 8.2 and 2.7 Hz), 6.56 (1H, d, J ) 2.7 Hz), 4.49
(1H, dd, J ) 8.5 and 8.3 Hz), 4.32 (4H, s), 2.76-2.84 (2H, m),
1.27-2.34 (14H, m), 0.78 (3H, s); MS [FAB-] m/z 530.2 ((M+
-
H)-, 56%), 276.1 (100); HRMS [FAB+] m/z found 531.2423, calcd
531.2443.
Estradiol-17â-O-(N-acetyl)sulfamate (15). A solution of 10 (110
mg, 0.23 mmol) in methanol (10 mL) was treated with 10% Pd/C
(50 mg) and then stirred under a H2 atmosphere for 5 h at rt. The
catalyst was removed by filtration, and the solvent was evaporated
to afford 15 as a white solid (90 mg, 87%): mp 130-134 °C dec;
1H NMR δ 7.11 (1H, d, J ) 8.6 Hz), 6.63 (1H, dd, J ) 8.6 and 2.7
Hz), 6.56 (1H, d, J ) 2.7 Hz), 4.63 (1H, dd, J ) 9.4 and 7.8 Hz),
3.48 (2H, br), 2.70-2.90 (2H, m), 2.13 (3H, s), 1.18-2.34 (13H,
m), 0.88 (3H, s); MS [FAB+] m/z 393.2 (M+, 86); HRMS [FAB+]
m/z found 393.1613, calcd 393.1610.
1
mg, 89%): mp 92-93 °C; H NMR δ 7.28-7.45 (15H, m), 7.19
(1H, d, J ) 8.6 Hz), 6.78 (1H, dd, J ) 8.6 and 2.3 Hz), 6.71 (1H,
d, J ) 2.3 Hz), 5.03 (2H, s), 4.48 (1H, dd, J ) 9.0 and 8.8 Hz),
4.40 (2H, d, J ) 15.6 Hz), 4.26 (2H, d, J ) 15.6 Hz), 2.78-2.93
(2H, m), 1.16-2.34 (13H, m), 0.78 (3H, s); MS [FAB+] m/z 621.3
(M+, 34), 245.2 (56), 91.1 (100); HRMS [FAB+] m/z found
621.2906, calcd 621.2913.
3-O-Sulfamoylestradiol 17â-O-(N,N-dimethyl)sulfamate (16).
A solution of 12 (180 mg, 0.47 mmol) in dichloromethane (5 mL)
was treated with DBMP (292 mg, 1.42 mmol) and then sulfamoyl
chloride (3.49 mL, 2.37 mmol). The reaction mixture was stirred
for 19 h and then diluted with ethyl acetate (50 mL) and water (40
mL). The separated organic layer was then washed with brine (3
× 40 mL), dried, and evaporated. Column chromatography (3:1
hexane/ethyl acetate) gave 16 as a white solid (195 mg, 90%): mp
3-O-Benzylestradiol 17-O-(N-acetyl)sulfamate (10). A solution
of 6 (300 mg, 0.68 mmol) in dichloromethane (20 mL) was treated
with sodium hydride (27 mg, 0.68 mmol) and acetyl chloride (53
µL, 0.75 mmol). The reaction mixture was stirred for 16 h before
evaporation of solvent. Column chromatography (2:1 to 1:1 hexane/
ethyl acetate) afforded recovered starting material (100 mg, 33%)
and the desired sulfamate 10 (150 mg, 46%) as a white solid: mp
1
140-142 °C; H NMR δ 7.31 (1H, d, J ) 8.6 Hz), 7.08 (1H, dd,
1
82-85 °C; H NMR δ 8.70 (1H, br), 7.28-7.46 (5H, m), 7.18
J ) 8.6 and 2.7 Hz), 7.04 (1H, d, J ) 2.7 Hz), 4.89 (2H, br), 4.69
(1H, dd, J ) 8.6 and 8.6 Hz), 2.88 (6H, s), 2.80-2.92 (2H, m),
1.20-2.38 (13H, m), 0.86 (3H, s); MS [FAB+] m/z 458.2 (M+,
18), 334.2 (100); HRMS [FAB+] m/z found 458.1537, calcd
458.1545. Anal. (C20H30N2O6S2) C, H, N.
(1H, d, J ) 8.6 Hz), 6.78 (1H, dd, J ) 8.6 and 2.9 Hz), 6.72 (1H,
d, J ) 2.9 Hz), 5.03 (2H, s), 4.68 (1H, dd, J ) 8.4 and 8.4 Hz),
2.77-2.93 (2H, m), 2.23 (3H, s), 1.20-2.36 (13H, m), 0.89 (3H,
s); MS [FAB-] m/z 482.2 ((M+ - H)-, 100); HRMS [FAB+] m/z
found 483.2079, calcd 483.2079.
3-O-Sulfamoylestradiol 17â-O-(N,N-dipentyl)sulfamate (17).
Phenol 13 (260 mg, 0.54 mmol) in dichloromethane (15 mL) was
reacted with DBMP (330 mg, 1.61 mmol) and sulfamoyl chloride
(3.95 mL, 2.69 mmol) as described for the synthesis of 16. Column
chromatography (5:1 hexane/ethyl acetate) gave 17 as a white solid
(270 mg, 89%): mp 105-107 °C; 1H NMR δ 7.31 (1H, d, J ) 8.6
Hz), 7.08 (1H, dd, J ) 8.6 and 2.7 Hz), 7.04 (1H, d, J ) 2.7 Hz),
4.89 (2H, br), 4.43 (1H, dd, J ) 8.3 and 7.8 Hz), 3.12-3.26 (4H,
m), 2.82-2.93 (2H, m), 1.18-2.38 (25H, m), 0.91 (6H, t, J ) 7.0
Hz), 0.83 (3H, s); MS [FAB-] m/z 569.2 ((M+ - H)-, 100); HRMS
[FAB+] m/z found 569.2739, calcd 569.2719. Anal. (C28H45N2O6S2)
C, H, N.
Estradiol 17-O-sulfamate (11). A solution of 6 (205 mg, 0.46
mmol) in THF (1 mL) and ethanol (5 mL) was treated with 10%
Pd/C (30 mg) and then stirred under a H2 atmosphere for 16 h.
The catalyst was removed by filtration, and the solvent was
evaporated to afford 11 as a white solid (160 mg, 98%). A sample
of this material was crystallized from ethyl acetate/hexane: mp
1
168-169 °C; H NMR (DMSO-d6) δ 9.00 (1H, s), 7.39 (2H, s),
7.04 (1H, d, J ) 8.4 Hz), 6.50 (1H, dd, J ) 8.4 and 2.5 Hz), 6.44
(1H, d, J ) 2.5 Hz), 4.33 (1H, dd, J ) 8.4 and 8.2 Hz), 2.65-2.76
(2H, m), 1.15-2.32 (13H, m), 0.76 (3H, s); 13C NMR δ 155.0,
137.1, 130.0, 126.1, 114.9, 112.7, 87.4, 48.6, 43.3, 42.6, 39.3, 35.9,

2-Substituted Estradiol Bis-sulfamates
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7693
3-O-Sulfamoylestradiol 17â-O-(N,N-dibenzyl)sulfamate (18).
Phenol 14 (200 mg, 0.38 mmol) in dichloromethane (10 mL) was
reacted with DBMP (230 mg, 1.13 mmol) and sulfamoyl chloride
(2.77 mL, 1.88 mmol) as described for the synthesis of 16. Column
chromatography (eluant 4:1 to 3:2 hexane/ethyl acetate) gave 18
as a white foam (194 mg, 84.5%): mp 58-62 °C; 1H NMR δ 7.28-
7.38 (11H, m), 7.08 (1H, dd, J ) 8.4 and 2.3 Hz), 7.04 (1H, d, J
) 2.3 Hz), 4.88 (2H, br), 4.49 (1H, dd, J ) 8.4 and 8.3 Hz), 4.32
(4H, s), 2.85-2.92 (2H, m), 1.28-2.32 (13H, m), 0.78 (3H, s);
MS [FAB+] m/z 611.1 (M+ + H, 7), 334.1 (100); HRMS [FAB-]
m/z found 609.2091, calcd 609.2093 ((M+ - H)-). Anal.
(C32H38N2O6S2) C, H, N.
3-O-Sulfamoylestradiol 17â-O-(N-acetyl)sulfamate (19). A
solution of 15 (67 mg, 0.17 mmol) in DMF (5 mL) was treated
with DBMP (105 mg, 0.51 mmol) and then sulfamoyl chloride (1.25
mL, 0.85 mmol) and was stirred for 7 h. The reaction mixture was
then partitioned between ethyl acetate (50 mL) and water (40 mL)
andthe organic layer separated, then washed with brine (3 × 40
mL), dried, and evaporated. Column chromatography (2:1 hexane/
ethyl acetate) gave 19 as a pale orange solid (27 mg, 34%): mp
76-80 °C dec; 1H NMR δ 8.12 (1H, br), 7.28 (1H, d, J ) 8.6 Hz),
7.08 (1H, dd, J ) 8.6 and 2.7 Hz), 7.04 (1H, d, J ) 2.7 Hz), 4.95
(2H, br), 4.69 (1H, dd, J ) 8.7 and 8.4 Hz), 2.80-2.86 (2H, m),
2.24 (3H, s), 1.20-2.38 (13H, m), 0.89 (3H, s); MS [FAB+] m/z
472.2 (M+, 20); HRMS [FAB-] m/z found 471.1257 calcd 471.1260
((M+ - H)-).
workup the crude product was purified by column chromatography
(9:1 chloroform/acetone) and gave the desired bis-sulfamate 23 as
a white crystalline solid (334 mg, 91%): mp 190-192 °C; 1H NMR
(DMSO-d6) δ 7.92 (2H, s), 7.38 (2H, s), 7.19 (1H, s), 6.98 (1H, s),
4.32 (1H, dd, J ) 9.0 and 7.8 Hz), 2.75-2.81 (2H, m), 2.62 (2H,
q, J ) 7.4 Hz), 1.18-2.40 (H, m), 1.12 (3H, t, J ) 7.4 Hz), 0.77
(3H, s); 13C NMR δ 145.9, 137.7, 134.8, 133.3, 126.4, 121.4, 87.3,
48.6, 43.5, 42.6, 37.8, 35.9, 28.6, 27.5, 26.5, 25.5, 23.6, 22.5, 14.8,
11.5; MS [ES-] m/z 457.4 ((M+ - H)-, 100). Anal. (C20H30N2O6S2)
C, H, N.
2-Methylsulfanylestradiol 3,17-O,O-bis-sulfamate (24). 2-
Methylsulfanylestradiol (279 mg, 0.87 mmol) was reacted with
sulfamoyl chloride (2.2 mmol) in DMA (1.5 mL) as described for
the synthesis of 21. Column chromatography (9:1 chloroform/
acetone) gave the desired bis-sulfamate 24 (250 mg, 60%) as a
foam. Crystallization (ethyl acetate/hexane) gave white crystals:
1
mp 176-178 °C; H NMR (DMSO-d6) δ 8.01 (2H, s), 7.39 (2H,
s), 7.17 (1H, s), 7.06 (1H, s), 4.32 (1H, dd, J ) 8.2 and 8.2 Hz),
2.75-2.82 (2H, m), 1.20-2.45 (16H, m including 2.40 (3H, s)),
0.77 (3H,s); 13C NMR δ 145.0, 138.2, 134.1, 128.2, 124.0, 121.1,
87.3, 48.5, 43.5, 42.6, 37.7, 35.9, 28.5, 27.5, 26.4, 25.5, 22.6, 14.8,
11.6; MS [ES-] m/z 475.4 ((M+ - H)-, 100); HRMS [ES-] m/z
found 475.1041, calcd 475.1037 ((M - H)-). Anal. (C19H28N2O6S3‚
H2O) C, H, N.
2-Methoxy-3,17-O,O-bis(N,N-dimethylsulfamoyl)estradiol (25).
A solution of 21 (150 mg, 0.33 mmol) in DMF (10 mL) was treated
with sodium hydride and then, after 10 min, methyl iodide (178
µL, 2.87 mmol). The reaction was stirred for 16 h, then diluted in
ethyl acetate (25 mL), and quenched with water (25 mL). The
organic layer was separated, then washed with water (2 × 25 mL)
and brine (25 mL), dried, and evaporated. The crude product was
crystallized from acetone/hexane to give 25 as white crystals (64
mg, 38%): mp 140-143 °C; 1H NMR (CDCl3, TMS ) 0) δ 7.04
(1H, s), 6.86 (1H, s), 4.45 (1H, dd, J ) 7.9 and 7.8 Hz), 3.86 (3H,
s), 2.97 (6H, s), 2.87 (6H, s), 2.76-2.84 (2H, m), 1.23-2.35 (13H,
m), 0.87 (3H, s); 13C NMR δ 149.0, 139.0, 137.0, 129.2, 123.5,
110.1, 89.2, 56.2, 49.4, 44.3, 43.4, 38.7, 38.6, 38.1, 36.6, 28.6, 27.9,
27.0, 26.2, 23.1, 11.8; HRMS [FAB+] m/z found 516.1964, calcd
516.1964. Anal. (C23H36N2O7S2) C, H, N.
Estradiol 3,17-O,O-bis-sulfamate (20). Estradiol (270 mg, 1
mmol) was added to an ice cold solution of sulfamoyl chloride (3
mmol) in DMA (1.5 mL). The resulting mixture was stirred for 3
h at room temperature, then diluted with ethyl acetate (20 mL),
then washed with water (3 × 20 mL) and brine (20 mL), dried,
and evaporated to give a white foam. Column chromatography
(CHCl3/acetone 9:1) afforded the desired bis-sulfamate 20 as a
colorless oil which was crystallized from ethyl acetate/hexane to
1
give white crystals (300 mg, 70%): mp 200-201 °C; H NMR
(DMSO-d6) δ 7.95 (2H, s), 7.40 (2H, s), 7.35 (1H, d, J ) 8.7 Hz),
7.01 (1H, dd, J ) 8.7 and 2.4 Hz), 6.96 (1H, d, J ) 2.4 Hz), 4.34
(1H, dd, J ) 8.9 and 7.9 Hz), 2.78-2.88 (2H, m), 1.20-2.40 (13H,
m), 0.77 (3H, s); 13C NMR δ 148.0, 138.1, 126.7, 121.9, 119.3,
87.4, 48.7, 43.5, 42.6, 37.8, 35.9, 30.7, 29.0, 27.5, 26.4, 25.5, 22.6,
11.6; MS [ES-] m/z 429.5 ((M+ - H)-, 100). Anal. (C18H26N2O6S)
C, H, N.
2-Methoxy-3-O-benzylestradiol (26). A solution of 2-MeOE2
(5.48 g, 18.2 mmol) in ethanol (100 mL) was refluxed with
potassium carbonate (22.7 g, 7.5 mmol) and benzyl bromide (6.51
mL, 55 mmol) for 8 h. The reaction was then diluted with ethyl
acetate (150 mL), washed with water (3 × 100 mL) and brine (50
mL), dried, and evaporated. Column chromatography (9:1 to 3:1
hexane/ethyl acetate) afforded 26 as a white foam (6.2 g, 86%)
which was then precipitated from chloroform/hexane to give a white
2-Methoxyestradiol 3,17-O,O-bis-sulfamate (21). 2-MeOE2 (2)
(1.00 g, 3.31 mmol) was added to an ice cold solution of sulfamoyl
chloride (13.2 mmol) in DMA (6.5 mL). The resulting mixture was
stirred for 3 h at room temperature and then cooled to 0 °C, at
which stage a dense white suspension formed. The reaction mixture
was then transferred into a separating funnel, diluted with ethyl
acetate (100 mL), then washed with water (2 × 100 mL) and brine
(2 × 100 mL), dried, and evaporated to give a white foam. Column
chromatography (8:1, then 4:1, and then 1:1 CHCl3/acetone)
afforded the desired bis-sulfamate 21. Crystallization (ethyl
acetate/hexane) gave white crystals (1.22 g, 79%): mp 180-182
1
powder: mp 75-78 °C (lit.58 mp 89-90 °C); H NMR (DMSO-
d6) δ 8.32 (1H, s), 7.28-7.47 (5H, m), 6.84 (1H, s), 6.71 (1H, s),
5.01 (2H, s), 4.51 (1H, m), 3.73 (3H, s), 2.65-2.75 (2H, m), 1.10-
2.38 (13H, m), 0.68 (3H, s, 18-CH3); MS [APCI+] m/z 392.5 (M+,
100). Anal. (C26H32O3) C, H, N.
2-Ethyl-3-O-benzylestradiol (27). A solution of 2-EtE2 (1.2 g,
4 mmol) in ethanol (30 mL) was refluxed with potassium carbonate
(4.69 g, 34 mmol) and benzyl bromide (1.42 mL, 12 mmol) for 8
h as described for the synthesis of 26. The crude oil was purified
by column chromatography (3:1 hexane/ethyl acetate) to give 27
1
°C; H NMR (DMSO-d6) δ 7.83 (2H, s), 7.42 (2H, s), 6.99 (1H,
s), 6.99 (1H, s), 4.34 (1H, dd, J ) 8.4 and 8.2 Hz), 3.77 (3H, s),
2.70-2.80 (2H, m), 1.15-2.45 (13H, m), 0.78 (3H, s); MS [ES-]
m/z 459 ((M+ - H)-, 100), 379 (25). Anal. (C19H28N2O7S2) C, H,
N.
1
as a colorless glassy solid (1.2 g, 76%): mp 73-75 °C; H NMR
2-Ethoxyestradiol 3,17-O,O-bis-sulfamate (22). 2-Ethoxyestra-
diol (180 mg, 0.57 mmol) was reacted with sulfamoyl chloride (2.5
mmol) in DMA (1.5 mL) as described for the synthesis of 21.
Column chromatography (chloroform/acetone gradient) gave the
desired sulfamate 22 (230 mg, 85%) as a white powder: mp 180-
δ 7.29-7.44 (5H, m), 7.09 (1H, s), 6.61 (1H, s), 5.02 (2H, s), 3.68-
3.76 (1H, m), 2.75-2.90 (2H, m), 2.66 (2H, q, J ) 7.4 Hz), 1.14-
2.39 (17H, m including 1.21 (3H, t, J ) 7.4 Hz)), 0.77 (3H, s); 13
C
NMR δ 154.3, 137.6, 134.8, 132.1, 130.1, 128.3, 127.5, 126.9,
126.1, 111.8, 81.9, 69.8, 50.1, 44.1, 433, 39.0, 36.8, 30.7, 29.8,
1
181 °C; H NMR δ 7.02 (1H, s), 6.90 (1H, s), 5.11 (2H, s), 4.84
27.4, 26.5, 23.6, 23.2, 14.8, 11.2; MS [ES-] m/z 389.6 ((M+
-
(2H, s), 4.51 (1H, dd, J ) 8.4 and 7.5 Hz), 4.07-4.15 (2H, m),
2.78-2.83 (2H, m), 1.20-2.34 (16H, m including 1.42 (3H, t, J )
3.5 Hz)), 0.87 (3H, s); MS [APCI-] m/z 473.19 ((M+ - H)-, 100).
Anal. (C20H30N2O7S2) C, H, N.
H)-, 100). Anal. (C27H34O2) C, H, N.
2-Methoxy-3-O-benzylestradiol 17-O-sulfamate (28). 26 (900
mg, 2.3 mmol) was added to a solution of sulfamoyl chloride (4.6
mmol) in DMA (3 mL) as described for the synthesis of 21. Column
chromatography (3:2 hexane/ethyl acetate) gave the desired sulfa-
2-Ethylestradiol 3,17-O,O-bis-sulfamate (23). 2-Ethylestradiol
(240 mg, 0.80 mmol) was reacted with sulfamoyl chloride (2.4
mmol) in DMA (1.5 mL) as described for the synthesis of 21. After
1
mate 28 (860 mg, 79%) as white crystals: mp 169-171 °C; H
NMR δ 7.26-7.46 (5H, m), 6.82 (1H, s), 6.62 (1H, s), 5.10 (2H,

7694 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Leese et al.
s), 4.51 (1H, dd, J ) 8.2 and 8.2 Hz), 3.86 (3H, s), 2.67-2.84
(2H, m), 1.19-2.40 (13H, m), 0.87 (3H, s); MS [FAB-] m/z 470.3
((M+ - H)-, 100); HRMS [FAB+] m/z found 471.2084, calcd
471.2079. Anal. (C26H33NO4S) C, H, N.
chromatography (4:1 hexane/ethyl acetate) afforded 34 as a white
solid (175 mg , 59%): mp 151-155 °C; 1H NMR δ 6.76 (1H, s),
6.63 (1H, s), 5.43 (1H, s), 4.46 (1H, dd, J ) 9.2 and 8.1 Hz), 3.85
(3H, s), 2.86 (6H, s), 2.74-2.82 (2H, m), 1.17-2.32 (13H, m),
0.85 (3H, s); 13C NMR δ 144.6, 143.5, 131.4, 129.3, 114.6, 108.0,
89.5, 56.0, 49.2, 44.0, 43.4, 38.6, 38.6, 36.5, 28.9, 27.8, 27.1, 26.3,
23.0, 11.7; MS [FAB+] m/z 409.3 (M+, 100); HRMS [FAB+] m/z
found 409.1938, calcd 409.1923. Anal. (C21H31NO5S) C, H, N.
2-Ethylestradiol 17-O-(N,N-dimethyl)sulfamate (35). A solu-
tion of 31 (340 mg, 0.68 mmol) in THF (10 mL) and methanol (20
mL) was reacted with 10% Pd/C (30 mg) and hydrogen (1 atm)
for 16 h as described for the synthesis of 11. 35 was obtained as a
2-Ethyl-3-O-benzylestradiol 17-O-sulfamate (29). 27 (900 mg,
2.3 mmol) was added to a solution of sulfamoyl chloride (4.6 mmol)
in DMA (6 mL) as described for the synthesis of 19. Column
chromatography (2:1 hexane/ethyl acetate) gave the desired sulfa-
1
mate 29 (950 mg, 88%) as a white powder: mp 144-147 °C; H
NMR δ 7.28-7.45 (5H, m), 7.09 (1H, s), 6.63 (1H, s), 5.04 (2H,
s), 4.67 (2H, s), 4.51 (1H, dd, J ) 8.8 and 7.7 Hz), 2.80-2.88
(2H, m), 2.66 (2H, q, J ) 7.4 Hz), 1.15-2.40 (16H, m including
1.21 (3H, t, J ) 7.4 Hz)), 0.87 (3H, s); MS [ES-] m/z 468.3 ((M+
- H)-, 100). Anal. (C27H35NO4S‚H2O) C, H, N.
1
white solid (270 mg, 98%): mp 163-165 °C; H NMR δ 7.04
(1H, s), 6.50 (1H, s), 4.65 (1H, s), 4.45 (1H, dd, J ) 8.0 and 7.8
Hz), 2.87 (6H, s), 2.76-2.84 (2H, m), 2.60 (2H, q, J ) 7.4 Hz),
1.25-2.38 (13H, m), 1.23 (3H, t, J ) 7.4 Hz) and 0.86 (3H, s);
MS [ES-] m/z 406.2 ((M - H)-, 20); HRMS [FAB+] m/z found
407.2130, calcd 407.2130. Anal. (C22H33NO4S) C, H, N.
2-Methoxy-3-O-benzylestradiol 17-O-(N,N-dimethyl)sulfa-
mate (30). A solution of 28 (300 mg, 0.63 mmol) in DMF (10
mL) at rt was treated with sodium hydride (55 mg, 1.38 mmol)
and then methyl iodide (133 µL, 2.14 mmol). After 16 h the reaction
was diluted with ethyl acetate (50 mL) and water (30 mL). The
organic layer was separated, washed with water (3 × 50 mL), then
dried, and evaporated. The crude oil was then crystallized (ethyl
acetate/hexane) to give the desired product 30 as a white solid (300
mg, 94%): mp 153-156 °C; 1H NMR δ 7.25-7.46 (5H, m), 6.83
(1H, s), 6.62 (1H, s), 5.10 (2H, s), 4.46 (1H, dd, J ) 8.2 and 8.2
Hz), 3.86 (3H, s), 2.87 (6H, s), 2.66-2.85 (2H, m), 1.19-2.38
(13H, m), 0.86 (3H, s); 13C NMR δ 147.5, 146.4, 137.3, 132.4,
128.6, 128.4, 127.7, 127.2, 114.5, 109.6, 89.4, 71.0, 56.3, 49.2,
44.0, 43.4, 38.6, 38.6, 36.5, 29.0, 27.8, 27.1, 26.2, 23.0, 11.6; MS
[FAB+] m/z 499.3 (M+, 100); HRMS [FAB+] m/z found 499.2393,
requires 499.2392. Anal. (C28H37NO5S) C, H, N.
2-Methoxy-3-O-sulfamoylestradiol 17â-O-(N,N-dimethyl)-
sulfamate (36). A solution of 34 (133 mg, 0.325 mmol) in
dichloromethane (10 mL) was added at rt to DBMP (200 mg, 0.975
mmol) and then sulfamoyl chloride (2.39 mL, 1.625 mmol) as
described for the synthesis of 16. Chromatography (eluent hexane/
ethyl acetate, 3:2) afforded 40 mg (30%) of recovered starting
material and the desired sulfamate 36 (105 mg, 66%) as a white
1
solid: mp 181-186 °C; H NMR δ 7.04 (1H, s), 6.92 (1H, s),
4.95 (2H, br), 4.46 (1H, dd, J ) 9.4 and 7.8 Hz), 3.86 (3H, s),
2.88 (6H, s), 2.76-2.84 (2H, m), 1.20-2.34 (13H, m), 0.87 (3H,
s); MS [FAB+] m/z 488.2 (M+, 100); HRMS [FAB+] m/z found
488.1661, calcd 488.1651.
2-Ethyl-3-O-sulfamoylestradiol 17-O-(N,N-dimethyl)sulfa-
mate (37). Sulfamoyl chloride (0.56 mmol) was dissolved in DMA
(1 mL) at 0 °C before addition of 35 (50 mg, 0.12 mmol) as
described for the synthesis of 19. Column chromatography (5%
acetone in chloroform) gave 37 as a clear colorless oil which was
precipitated from acetone/hexane to give a white powder (31 mg,
2-Ethyl-3-O-benzylestradiol 17-O-(N,N-dimethyl)sulfamate
(31). A solution of sulfamate 29 (520 mg, 1.11 mmol) in DMF (10
mL) was reacted with sodium hydride (98 mg, 2.43 mmol) and
methyl iodide (234 µL, 2.4 mmol) as described for the synthesis
of 30. Column chromatography (4:1 hexane/ethyl acetate) yielded
a colorless oil which solidified on contact with ethyl acetate to give
31 (440 mg, 80%) as a white solid: mp 152-154 °C; 1H NMR δ
7.26-7.44 (5H, m), 7.09 (1H, s), 6.62 (1H, s), 5.03 (2H, s), 4.45
(1H, dd, J ) 9.2 and 7.9 Hz), 2.86 (6H, s), 2.78-2.86 (2H, m),
2.65 (2H, q, J ) 7.4 Hz), 1.20-2.38 (13H, m), 1.20 (3H, t, J )
7.4 Hz), 0.85 (3H, s); 13C NMR δ 154.5, 137.7, 134.8, 131.8, 130.3,
128.5, 127.6, 127.0, 126.2, 111.8, 89.5, 69.8, 49.2, 43.8, 43.5, 38.6,
38.6, 36.6, 29.6, 27.8, 27.1, 26.1, 23.5, 23.0, 14.7, 11.7; MS [APCI+]-
m/z 498.4 (M+ + H, 7), 373.5 (100). Anal. (C29H39NO4S) C, H, N.
2-Methoxyestradiol 17-O-sulfamate (32). A solution of 28 (103
mg, 0.22 mmol) in THF (1 mL) and ethanol (6 mL) was treated
with Pd/C (18 mg, 10%) and hydrogen (1 atm) for 16 h as described
for the synthesis of 11. The resultant solid was crystallized (ethyl
acetate/hexane) to give 30 as fine needles (65 mg, 77%): mp 180-
1
52%): mp 178-180 °C; H NMR δ 7.16 (1H, s), 7.06 (1H, s),
4.98 (2H, s), 4.46 (1H, dd, J ) 8.6 and 8.2 Hz), 2.87 (6H, s), 2.80-
2.86 (2H, m), 2.69 (2H, q, J ) 7.4 Hz), 1.25-2.36 (13H, m), 1.21
(3H, t, J ) 7.4 Hz) and 0.85 (3H, s); 13C NMR δ 146.0, 138.9,
135.6, 133.6, 126.9, 121.3, 89.3, 49.4, 44.0, 43.4, 38.6, 38.2, 36.5,
29.2, 27.9, 26.0, 23.2, 23.1, 14.8, 11.8; HRMS [FAB+] m/z found
486.1858, calcd 486.1858. Anal. (C22H34N2O6S2‚0.5H2O) C, H, N.
Acknowledgment. This work was supported by Sterix Ltd.,
a member of the IPSEN group. We thank Sincrotrone Trieste
CNR/Elettra for giving us the opportunity to collect data at the
Crystallographic Beamline, the EPSRC National Mass Spec-
trometry Service, Swansea, U.K., for high-resolution mass
spectra, and the NCI Developmental Therapeutic Program for
in vitro and in vivo screening of the compounds described in
this paper.
1
182 °C; H NMR δ 6.76 (1H, s), 6.63 (1H, s), 5.43 (1H, s) 4.70
(2H, s), 4.50 (1H, dd, J ) 9.4 and 8.2 Hz), 3.85 (3H, s), 2.72-
2.82 (2H, m), 1.16-2.34 (14H, m), 0.86 (3H, s); 13C NMR δ 144.6,
143.5, 131.2, 129.3, 114.6, 108.0, 90.8, 56.0, 49.1, 44.0, 43.3, 38.4,
36.4, 28.8, 27.7, 27.1, 26.3, 23.0, 11.7; MS [ES-] m/z 380.5 ((M+
- H)-, 100). Anal. (C19H27NO5S) C, H, N.
Supporting Information Available: Elemental analysis results
and crystallographic data for the hCAII-21 complex. This material
is available free of charge via the Internet at http://pubs.acs.org.
2-Ethylestradiol 17-O-sulfamate (33). A solution of 29 (156
mg, 0.33 mmol) in THF (1 mL) and methanol (5 mL) was reacted
with 10% Pd/C (30 mg) and hydrogen (1 atm) for 16 h as described
for the synthesis of 11. The resultant white powder was crystallized
(ethyl acetate/hexane) to give 33 as white crystals (98 mg, 78%):
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