A-ring-substituted estrogen-3-O-sulfamates: Potent multitargeted anticancer agents

Article abstract of DOI:10.1021/jm050066a

Efficient and flexible syntheses of 2-substituted estrone, estradiol and their 3-O-sulfamate (EMATE) derivatives have been developed using directed ortho-lithiation methodology. 2-Substituted EMATEs display a similar antiproliferative activity profile to the corresponding estradiols against a range of human cancer cell lines. 2-Methoxy (3, 4), 2-methylsulfanyl (20, 21) and 2-ethyl EMATEs (32, 33) proved the most active compounds with 2-ethylestradiol-3-O-sulfamate (33), displaying a mean activity over the NCI 55 cell line panel 80-fold greater than the established anticancer agent 2-methoxyestradiol (2). 2-Ethylestradiol-3-O-sulfamate (33) was also an effective inhibitor of angiogenesis using three in vitro markers, and various 2-substituted EMATEs also proved to be inhibitors of steroid sulfatase (STS), a therapeutic target for the treatment of hormone-dependent breast cancer. The potential of this novel class of multimechanism anticancer agents was confirmed in vivo with good activity observed in the NCI hollow fiber assay and in a MDA-MB-435 xenograft mouse model.

Full text of DOI:10.1021/jm050066a

J. Med. Chem. 2005, 48, 5243-5256
5243
A-Ring-Substituted Estrogen-3-O-sulfamates: Potent Multitargeted Anticancer
Agents
Mathew P. Leese,† Hatem A. M. Hejaz,† Mary F. Mahon,§ Simon P. Newman,‡ Atul Purohit,‡
Michael J. Reed,‡ and Barry V. L. Potter†,*
Medicinal Chemistry, Department of Pharmacy and Pharmacology & Sterix Ltd., and Department of Chemistry,
University of Bath, Bath BA2 7AY, U.K., and Endocrinology and Metabolic Medicine & Sterix Ltd., Faculty of Medicine,
Imperial College, St. Mary’s Hospital, London W2 1NY, U.K.
Received January 24, 2005
Efficient and flexible syntheses of 2-substituted estrone, estradiol and their 3-O-sulfamate
(EMATE) derivatives have been developed using directed ortho-lithiation methodology.
2-Substituted EMATEs display a similar antiproliferative activity profile to the corresponding
estradiols against a range of human cancer cell lines. 2-Methoxy (3, 4), 2-methylsulfanyl (20,
21) and 2-ethyl EMATEs (32, 33) proved the most active compounds with 2-ethylestradiol-3-
O-sulfamate (33), displaying a mean activity over the NCI 55 cell line panel 80-fold greater
than the established anticancer agent 2-methoxyestradiol (2). 2-Ethylestradiol-3-O-sulfamate
(33) was also an effective inhibitor of angiogenesis using three in vitro markers, and various
2-substituted EMATEs also proved to be inhibitors of steroid sulfatase (STS), a therapeutic
target for the treatment of hormone-dependent breast cancer. The potential of this novel class
of multimechanism anticancer agents was confirmed in vivo with good activity observed in
the NCI hollow fiber assay and in a MDA-MB-435 xenograft mouse model.
Introduction
2-MeOE2 in various cancer cell lines have been pro-
posed. 2-MeOE2 has been shown to interact with the
colchicine binding site primarily on â-tubulin (although
evidence has also been presented for interaction with
R-tubulin) and appears to induce metaphase arrest by
functioning as an antimicrotubule agent.10 Several
studies have shown that 2-MeOE2 inhibits tubulin
assembly in vitro. However, Attalla et al. have shown
that in intact cells, 2-MeOE2, like paclitaxel, promotes
tubulin polymerization.11 Given that the concentrations
required to achieve biological effects are far lower that
those required to affect microtubule structure, it may
well be that 2-MeOE2 affects the kinetics of mitotic
spindle dynamics in vivo rather than altering micro-
tubule polymerization per se.12 Cytotoxicity may also
arise from the effects of 2-MeOE2 on apoptosis regula-
tors such as p538 or BCL-2,11 or alternatively from
interference with DNA synthesis, an effect atypical of
microtubule disruptors.13,14 A recent study has demon-
strated that at a concentration of 50 µM, 2-MeOE2
inhibits HIF-1R at the posttranscriptional level, pre-
vents HIF-1 target gene expression in tumor cells and
inhibits HIF-2R in human endothelial cells.15 These
effects occur downstream from the interaction of
2-MeOE2 and microtubules and, inter alia, inhibit the
transcriptional activation of the angiogenic promoter
vascular endothelial growth factor (VEGF).15
The discovery that the endogenous estrogen metabo-
lite 2-methoxyestradiol 2 (2-MeOE2) could inhibit can-
cer cell proliferation and angiogenesis1 by a mechanism
independent of the estrogen receptor has stimulated
considerable interest in the mechanism underlying
these activities and the potential application of this
molecule as a therapeutic agent. The promise displayed
by 2-MeOE2 in preclinical studies has led to its evalu-
ation in ongoing Phase I/II clinical trials as a treatment
for various cancers although a precise understanding
of its mechanism of action has not yet been established.
Metabolism of estradiol (E2) by cytochrome P450
generates 2-hydroxyestradiol which, by catechol O-
methyl transferase mediated methylation, is then con-
verted to 2-MeOE2.2 The affinity of 2-MeOE2 for both
estradiol receptors ERR and ERâ is approximately one
hundredth that of E2.3 2-MeOE2 inhibits the prolifera-
tion of a range of estrogen-dependent (ER+) and estro-
gen independent (ER-) cell lines both in vitro4 and in
vivo1,5 indicating that its underlying mechanism of
action is independent of the estradiol receptors. Simi-
larly, when assayed against the proliferation of brain-
derived capillary endothelial cells as a measure of
antiangiogenic activity, 2-MeOE2 proved 90-times more
potent than 2-methoxyestrone (2-MeOE1) and 250-times
more potent than estradiol.1
Alongside its evaluation as an anticancer agent,
2-MeOE2 has also been proposed as a potential therapy
in a wide range of other areas including arthritis,16
asthma,14 atherosclerosis,17 inflammation and cardio-
vascular disease.18 The biological activities of 2-MeOE2
have stimulated several groups to investigate novel
2-substituted estradiols as potential therapeutic agents;
A-,13,19-26 B-27,28 and D-ring24,29,30 modified analogues of
2-MeOE2 have shown enhanced in vitro activity as
A number of potential mechanisms underlying the
reversible mitotic arrest6 and apoptosis7-9 induced by
* To whom correspondence should be addressed. Tel. +44 1225
386639. Fax +44 1225 383763. E-mail: B.V.L.Potter@bath.ac.uk.
† Department of Pharmacy and Pharmacology
University of Bath.
& Sterix Ltd.,
§ Department of Chemistry, University of Bath.
‡ Endocrinology and Metabolic Medicine & Sterix Ltd., Imperial
College.
10.1021/jm050066a CCC: $30.25 © 2005 American Chemical Society
Published on Web 07/01/2005

5244 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Leese et al.
MATE remains to be elucidated its spectrum of biologi-
cal activity encouraged us to explore closely related
structures in the search for new antitumor compounds.
We thus set out to determine the structural require-
ments for potency in the 2-substituted estrogen sulfa-
mates, which required an efficient and flexible synthetic
approach to 2-substituted estrones and their sulfamate
derivatives. Although efficient approaches to 2-substi-
tuted estradiols are well-established, we reasoned that
direct access to the 2-substituted estrone would afford
greater synthetic efficiency by avoidance of the protec-
tion, oxidation and deprotection sequence required for
transformation of estradiol to estrone. We report here
our synthetic approach to such A-ring-substituted es-
trogen sulfamates with their biological activity in vitro
and in vivo and the structure-activity relationship for
these compounds. A preliminary account of the work on
2-alkyl sulfanyl derivatives been published.19
Figure 1. Chemical structures of 2-MeOE1 1, 2-MeOE2 2,
2-MeOEMATE 3 and 2-MeOE2MATE 4.
inhibitors of cancer cell proliferation and tubulin po-
lymerization. To truly improve on the natural product,
however, a consideration of the in vivo fate of the
molecule must be undertaken, since metabolic oxidation
of the 17-hydroxyl group of 2-MeOE2 by 17â-hydroxys-
teroid dehydrogenase Type 2 generates inactive 2-MeOE1
1 (Figure 1). In addition, rapid formation of sulfate
conjugates of the 3- and 17-hydroxyl groups of 2-MeOE2
leads to a efficient clearance of 2-MeOE2. Simple
2-MeOE2 analogues are therefore equally likely to suffer
from metabolic inactivation and rapid clearance. An
appreciation of the importance of these factors can be
drawn from a recent clinical trial where a dose escala-
tion up to 6 g/day of 2-MeOE2 was tested in an attempt
to achieve a satisfactory plasma concentration.31
Our initial focus on 2-MeOE2-like molecules arose
from a long standing interest in the development of
inhibitors of steroid sulfatase32-34 which led to the
discovery that 2-substituted estrone sulfamates
(EMATEs), in addition to being potent nonestrogenic
inhibitors of steroid sulfatase,35 also exhibit antiprolif-
erative effects on human breast cancer cells.36 Initial
work showed that at a concentration of 1 µM 2-meth-
oxyestrone-3-O-sulfamate (2-MeOEMATE) 3, in contrast
to 2-MeOE1 1, could cause cell cycle arrest in the G2/M
phase; furthermore, this arrest was irreversible while
that triggered by 2-MeOE2 was reversible.36 The effect
of 2-MeOEMATE on cancer cells was shown to be
independent of the estrogen receptor. A key finding of
this study was that 2-MeOEMATE also induced apop-
tosis in these cell lines; a number of potential pro-
apoptotic actions were identified including phosphory-
lation of BCL-2 and BCL-XL.37 2-MeOEMATE also
proved to be significantly more active than 2-MeOE1
and 2-MeOE2 in the induction of the tumor suppressor
protein p53 and inhibition of the paclitaxel-induced
polymerization of tubulin in vitro.37 Furthermore, initial
in vivo work showed that 2-MeOEMATE caused regres-
sion of nitrosomethylurea-induced mammary tumors in
rats.36 A noteworthy property of estrogen sulfamates is
their high observed oral bioavailability, believed to be
due to their uptake by red blood cells and consequent
bypassing of first pass liver metabolism.38 This effect
appears to stem from the reversible interaction of these
steroidal sulfamates with the catalytic site of carbonic
anhydrase II, an enzyme which is highly expressed in
red blood cells, through a coordination of the monoan-
ionic form of the sulfamate moiety to the zinc atom.39
The potential of any such active delivery mechanism
acting for 2-substituted estrogen sulfamates would be
highly beneficial given the generally poor oral bioavail-
ability of 2-substituted estrogens (vide supra).
Results and Discussion
Chemistry. An initial series of A-ring modified 3-O-
sulfamoylated estrogens was targeted. Maximum flex-
ibility in this route was to be achieved by initial
elaboration of the parent estrone 5 (E1), which could
then be sulfamoylated prior to adjustment of the oxida-
tion state at C-17. A number of reports had appeared
on the synthesis of 2-methoxyestrogens, primarily in the
estradiol series, and these could be classified into two
distinct strategies: introduction of an oxidizable func-
tion by electrophilic means40 or activation of the 2-posi-
tion toward nucleophilic substitution.41,42 The latter
approach involves intrinsically fewer steps since no
intermediate protection of the 3-hydroxyl function is
required. The major drawback of this approach is,
however, the lack of selectivity of the reactions used to
activate the ring; iodination, for example, gives a
mixture of the 2-, 4-, and 2,4-iodinated estrones with
literature reports indicating isolated yields of the de-
sired 2-iodo compounds to be around 50%.41,43 This
approach was also hampered by the need to use heavy
metal salts as catalysts to obtain optimal yield and
selectivity. An elegant approach to 2-MeOE2 involves
formation of a η5-iridium oxo-complex on the A-ring
which can then undergo nucleophilic substitution with
methoxide at the 2-position.44 Unfortunately, this ap-
proach is stoichiometric in iridium thus making it
prohibitively expensive for scale-up purposes.
The first synthesis of 2-methoxyestrogens reported by
Fishman involves construction of an oxidizable xan-
thylium salt onto the estrane A-ring.40 To obtain
2-MeOE1 it proved necessary to first access protected
2-MeOE2 since the 17-keto function did not tolerate the
oxidative conditions of the key rearrangement. However,
this route only yields small amounts of the desired
compound after a convoluted synthesis. Acylation at the
2-position by Friedel-Crafts and Fries rearrangement
chemistry was explored by Nambara and co-workers
with oxidation of this group by the Baeyer-Villiger
reaction giving access to the key 2-hydroxy intermedi-
ate.45 Although successful, this synthesis is lengthened
by the protecting group chemistry required to access the
target estrone and gives a low overall yield. This
strategy has subsequently been refined in a recent
synthesis of 2-MeOE2 using a Fries rearrangement to
Although, as with 2-MeOE2, a full mechanistic pic-
ture of the multiple targeted activity of the 2MeOE-

A-Ring-Substituted Estrogen-3-O-sulfamates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5245
Scheme 1a
Scheme 2a
a Reagents and conditions: (i) H2NSO2Cl, DBMP, DCM, 0 °C
to room temperature, 84%; (ii) NaBH4, THF/IPA, 85%; (iii) NaH,
MeI, DMF 58%; (iv) Ac2O, pyridine, 78%.
performed on a -78 °C THF solution of the MOM/ketal
7 with sec-butyllithium was then followed by a DMF
quench to deliver the 2-formyl compound 8 in 86% yield
accompanied by a small amount (ca. 3%) of the 4-formyl
isomer which was removed by flash chromatography.
The ortho-lithiation step proved highly dependent on
reagent quality, and hence the use of freshly distilled
DMF and good quality sec-butyllithium solution was key
to successful reaction. Completion of the synthesis was
achieved in much the same manner as the synthesis of
2-MeOE2.22 The formyl group was transformed into the
formate intermediate by Baeyer-Villiger oxidation ac-
complished with m-chloroperbenzoic acid in buffered
dichloromethane and the product then saponified to give
the desired 2-hydroxy compound 9. Alkylation of this
with potassium carbonate and the appropriate alkyl
iodide in the presence of a catalytic amount of tetra-n-
butylammonium iodide in DMF delivered the protected
2-alkoxy estrones 10 and 11. Simple acidic deprotection
could then be applied to give the desired 2-alkoxy
estrones in good overall yield. 2-Methoxyestrone 1 and
2-ethoxyestrone 12a were thus available for further
manipulation.
The transformation of the 2-alkoxy estrones 1 and 12a
into their respective sulfamates could then be achieved
with either sulfamoyl chloride50 in DMA51 or with 2,6-
di-tertbutyl-4-methylpyridine in dichloromethane52 to
give high yields of the desired 2-substituted estrone-3-
O-sulfamate 3 or 13 (2-substituted EMATE) respectively
as shown in Scheme 2. A selective reduction of the 17-
keto function could then be achieved with sodium
borohydride in THF/methanol to give the 2-substituted
estradiol-3-O-sulfamates (2-substituted E2MATEs) 4
and 14. 2-MeOE2 2 and 2-EtOE2 12b were prepared
from the corresponding estrones 1 and 12a under
analogous conditions. A series of 2-methoxy and 2-ethoxy
estrones, estradiols and their respective 3-O-sulfamate
derivatives was thus available for biological evaluation.
The effect of sulfamate N-substitution on the activity
on 2-MeOEMATE was examined with alkylation (so-
dium hydride/methyl iodide) and acylation (pyridine/
acetic anhydride) of the sulfamate proceeding in a
smooth manner (Scheme 2) to afford the desired sulfa-
mate derivatives 15 and 16 in good yield. The N-
monomethyl sulfamate derivative is not readily acces-
sible by this approach.
a Reagents and conditions: (i) ethylene glycol, p-TsOH, Dean-
Stark, PhMe, quant; (ii) NaH, MOMCl, DMF, 95%; (iii) sec-BuLi,
THF, -78 °C, 1 h then DMF -78 °C to room temperature, 86%;
(iv) mCPBA, Na2HPO4, DCM then NaOH, MeOH, 84%; (v) K2CO3,
TBAI, DMF and MeI (93%) or EtI (89%); (vi) 6 M HCl /THF (R )
Me 90%, R ) Et 92%) vii) NaBH4, THF/IPA (R ) Me 85%, R ) Et
83%).
introduce the 2-acyl group in a selective and high
yielding manner.46 We decided not to use this approach
since we wished to adopt a strategy which would allow
access to a wider range of 2-substituents. Formylation
at the 2-position was used by Cushman et al. in two
different syntheses of 2-MeOE2. The earlier approach
involved an unselective formylation with hexamethyl-
enetetramine, and this gave only 25% of the desired
2-formylE2 which could then be transformed into
2-MeOE2 in 5% overall yield.47 A second approach
proved far superior,22 with a 2-formyl group being
introduced at the 2-position after the ortho-lithiation
method of Pert and Ridley,48 wherein bis-methoxym-
ethyl-protected estradiol undergoes a sterically directed
lithiation at the 2-position. The lithiated intermediate
can then be reacted with a range of electrophiles,49 with
DMF quench, affording the 2-formyl derivative in good
yield and selectivity.48 Given this selectivity and the
scalable nature of the chemistry involved, along with
the opportunity to introduce a range of other functional-
ity, we were attracted to modify this approach to give
direct access to 2-substituted estrones and the corre-
sponding estradiols. Conversion of the 17-carbonyl group
to the 17â-hydroxyl is facile and high yielding, in
contrast to the reverse process.
A protecting group for the 17-keto group of E1 5 was
required to complement the MOM protection required
to direct the ortho-lithiation reaction. A 17-ethylene
dioxolone group appeared ideal since it offered a facile
introduction and deprotection, the latter being realized
in tandem with the acidic MOM cleavage at the 3-O-
position of the steroid if so desired. Reaction of E1 with
ethylene glycol under Dean-Stark conditions delivered
the ketal 6 in quantitative yield (Scheme 1). MOM
protection was then incorporated under standard condi-
tions by addition of chloromethyl methyl ether to the
sodium phenolate of 6 in DMF solution. The desired
MOM/ketal 7 was thus available in a rapid manner and
high 95% yield over two steps. A directed metalation
We were interested to compare the activities of the
2-alkyl sulfanyl estradiols, their 3-O-sulfamate deriva-
tives and the corresponding 2-alkoxy estradiol and

5246 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Leese et al.
Scheme 3a
Scheme 4a
a Reagents and conditions: (i) sec-BuLi, THF, -78 °C, 1 h then
EtI for 29, CF3CH2Br for 35 or CF3CH2I for 38, -78 °C to room
temperature; (ii) 4 M HCl /MeOH; (iii) H2NSO2Cl, DMA, 0 °C to
room temperature; (iv) NaBH4, THF/IPA, 73%.
a Reagents and conditions: (i) sec-BuLi, THF, -78 °C, 1 h then
RSSR, -78 °C to room temperature; (ii) 4 M HCl /MeOH; (iii)
H2NSO2Cl, DBMP, DCM, rt iv) NaBH4, THF/IPA; (v) mCPBA,
DCM, CHCl3.
estradiol-3-O-sulfamates. 2-Ethylsulfanyl estradiol, 2-Et-
SE2, 25, was previously synthesized by Cushman and
co-workers in moderate yield by a nucleophilic substitu-
tion reaction between the sodium salt of ethanethiol and
protected 2-iodo estradiol. Given the success of our
approach to alkoxy estrones, we perceived that the use
of disulfides to quench the lithio-anion of MOM/ketal 7
would provide a rapid, flexible entry to the 2-alkylsul-
fanyl estrogens. As anticipated, disulfides proved to be
excellent electrophiles in these reactions with protected
2-methylsulfanyl and 2-ethylsulfanyl estrones, 17 and
24, being obtained in excellent 91% and 97% yields
respectively as shown in Scheme 3. Acidic deprotection
at the 3- and 17-positions then afforded the desired
2-alkylsulfanyl estrones 18 and 25 which were then
available for transformation into their respective 2-sub-
stituted estradiols 19 and 26, 2-substituted EMATEs
20 and 27 and 2-substituted E2MATEs 21 and 28, as
previously described for the alkoxy series. We were thus
able to compare the effect of transposition of the oxygen
to sulfur at the 2-position.19 The oxidation of 2-MeSE1
18 to the corresponding sulfoxide 22 and sulfone 23 was
achieved by use of the appropriate quantity of mCPBA
in dichloromethane (Scheme 3). The high yields ob-
tained in the synthesis suggest that this approach is
the most efficient and flexible entry to 2-alkylsulfanyl
estrones presently available.
Figure 2. X-ray crystal structure showing of one of the
molecules in the asymmetric unit of 2-ethylestrone 30. El-
lipsoids are represented at 30% probability level.
compounds would thus determine whether an H-bond
acceptor group at the 2-position is necessary for biologi-
cal activity. 2-Methylestrone-3-O-sulfamate 34,
2-MeEMATE, was also synthesized by this method.53
Attempts to introduce a 2,2,2-trifluororoethyl group
at the 2-position by quenching the lithiated MOM/ketal
with 2-bromo-1,1,1-trifluoroethane and 2-iodo-1,1,1-
trifluoroethane proved unsuccessful, with the 2-bromo
MOM/ketal 35 and 2-iodo MOM/ketal 38 being the only
isolated products of the respective reactions (Scheme 4).
The outcome of these reactions is not surprising given
the literature precedents for halogen exchange54 and the
strong deactivating effect the trifluoromethyl group
exerts on the neighboring carbon.55 Deprotection to give
the 2-halo estrones 36 and 39 was accomplished under
acidic conditions in modest overall yield; no attempt was
made to optimize this reaction. 2-Bromoestrone-3-O-
sulfamate 37 (2-BrEMATE) and 2-iodoestrone-3-O-
sulfamate 40 (2-IEMATE) were also derived for evalu-
ation under the standard conditions described above.
X-ray crystal structures of 2-EtE1 30 and 2-MeOE-
MATE 3 were obtained and are presented in Figures 2
and 3, respectively. The two structures confirm that the
integrity of the chiral centers at C-8, -9, -13 and -14 of
the estrane core is maintained. Adjacent 2-EtE1 units
align to allow hydrogen bonding interactions between
the C-3-OH group and the C-17-ketone. Interestingly,
2-Alkyl estrones were also available from this ap-
proach by quenching the lithio-anion of MOM/ketal 7
with the appropriate alkyl iodide. The 2-ethyl MOM/
ketal 29 was obtained in high (84%) yield, allowing,
after the methods described above, access to 2-ethyl
estrone 30, 2-ethyl estradiol 31, 2-EtEMATE 32 and
2-EtE2MATE 33 in a direct manner. Evaluation of these

A-Ring-Substituted Estrogen-3-O-sulfamates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5247
molecules display relatively modest growth inhibitory
effects, with only 2-EtOE1 12a causing more than 50%
growth inhibition at 10 µM concentration. This result
correlates with earlier studies in the estradiol series
where 2-EtOE2 proved to be the most potent of the
2-alkoxy estradiols.22 As had been previously reported,
the 2-substituted estradiol derivatives display far greater
activity than their estrone equivalents, a general trend
across the substituents explored here and clearly il-
lustrated by GI50 values of 2.35 µM and 21.3 µM (Table
1) obtained for 2-MeOE2 2 and 2-MeOE1 1 respectively.
2-MeSE2 19, which had not previously been synthesized
proved to be one of the most active 2-substituted
estradiols displaying similar activity to that of 2-MeOE2
2 (Table 1, 3.96 vs 2.35 µM respectively). The activity
of 2-MeSE2 19 contrasts with that observed for 2-EtSE2
26 in both the present and previous studies22 where the
ethylsulfanyl group proved a poor isostere for the ethoxy
group. It is not clear if this reduced antiproliferative
activity is due to steric factors (the ethylsulfanyl group
is larger than the 2-ethoxy group) or the reduced ability
of sulfur to act as a hydrogen bond acceptor with respect
to oxygen.
A marked increase in growth inhibitory activity was
observed in both the estrone and estradiol series upon
sulfamoylation of the 3-hydroxyl group. Transformation
of the phenolic estrones to their 3-O-sulfamoylated
(EMATE) derivatives, with the exception of the rela-
tively inactive 2-EtS series, produced an increase in
activity ranging from a 2-fold increase from 2-EtOE1
12 to 2-EtOEMATE 13 to a 170-fold increase in activity
from 2-EtE1 30 and 2-EtEMATE 32 (respective GI50s
of 57.6 µM and 0.34 µM). The EMATE derivatives are
also observed to be significantly more active in this
assay than the nonsulfamoylated estradiols, the most
active of which, 2-MeOE2 2, is eight times less active
than the corresponding estrone-3-O-sulfamate, 2-MeOE-
MATE 3, suggesting that the estrogen 3-O-sulfamates
are unlikely to act simply as prodrugs of the corre-
sponding estrogen. This premise is further supported
by the observation that the 2-substituted estrone de-
rivatives display only very modest activity as noted
above. Substitution on the nitrogen in the case of the
N,N-dimethyl 15 and N-acyl 2-MeOEMATEs 16 abol-
ished activity discouraging any further investigation
into modification of the sulfamate group at this time.
While the exact role of the sulfamate moiety in the high
inhibitory potency observed for these 2-substituted
estrogen sulfamates remains unestablished, the biologi-
cal activity displayed by 15 and 16 clearly suggested
that an unsubstituted sulfamate group (i.e. H2NSO2O)
is crucial for high potency. This is to be contrasted, at
least for the N-acetyl sulfamates, by the excellent
inhibitory activity of N-acetyl EMATE against the
steroid sulfatase mediated conversion of estradiol sul-
fate to estradiol.56,57 The antiproliferative activity of
2-substituted estradiol 3-O-sulfamates (E2MATEs) in
the MCF-7 cell line proved to be equivalent or, in the
case of 2-EtE2MATE 33, slightly enhanced with respect
to their 2-substituted EMATE equivalents.
Figure 3. X-ray crystal structure showing one of the mol-
ecules in the asymmetric unit of 2-methoxyestrone-3-O-sulfa-
mate (2-MeOEMATE) 3. Ellipsoids are represented at 30%
probability level.
Table 1. Antiproliferative Activities of Estrogen Derivatives
against MCF-7 Human Breast Cancer Cellsa
compound
R1
R2
R3
R4 MCF-7 GI50 (µM)
21.3
2.35
1
MeO
MeO
MeO
MeO
MeO
MeO
EtO
EtO
EtO
MeS
MeS
MeS
MeS
MeSO
MeSO2
EtS
EtS
EtS
EtS
Et
H
H
O
OH
O
O
H
O
H
O
O
O
O
H
O
H
O
H
O
O
O
H
O
H
O
H
O
H
O
O
O
O
O
2
3
SO2NH2
SO2NH2
SO2NMe2
SO2NHAc
H
SO2NH2
SO2NH2
H
0.30
0.36
>10
168
9.04
4.84
0.61
33.4
4
OH
O
15
16
12a
13
14
18
19
20
21
22
23
25
26
27
28
30
31
32
33
34
36
37
39
40
O
O
O
OH
O
H
OH
O
OH
O
O
O
OH
O
OH
O
3.96
0.40
0.43
SO2NH2
SO2NH2
H
>10
66
H
H
31.9
23.2
35.2
23.4
57.6
10.5
0.34
0.07
0.26
33
6.95
38
1.59
H
SO2NH2
SO2NH2
H
Et
H
OH
O
Et
SO2NH2
SO2NH2
SO2NH2
H
SO2NH2
H
SO2NH2
Et
OH
O
Me
Br
O
Br
O
I
I
O
O
a GI50 figures are the mean values obtained from experiments
performed in triplicate, SEM ) (7%.
the crystal structure of 2-MeOEMATE reveals that the
NH2 group of the sulfamate is appropriately (but pos-
sibly not optimally) rotated about the 01-S1 vector to
facilitate a H-bond interaction with the oxygen of the
neighboring methoxy group, thus illustrating the H-
bonding potential of the sulfamate group and, perhaps,
the ability of the neighboring methoxy group to stabilize
the conformation of an adjacent sulfamate.
Biology. As a first stage predictor of biological
activity, the compounds were evaluated for their ability
to inhibit the proliferation of MCF-7 cells, a ER+ human
breast cancer cell line. Previous experience had shown
that results from this assay were predictive of the
spectrum of desirable biological properties already
obtained with 2-MeOEMATE. The results of this assay
are presented in Table 1.
Inspection of Table 1 reveals that the steric size of
the 2-substituent is crucial for high activity, with the
larger 2-ethylsulfanyl 27 and 2-ethoxy EMATEs 13
exhibiting significantly lower activity to the homologous
A brief examination of the results obtained for the
2-substituted estrone derivatives shows that all of these

5248 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Leese et al.
Table 2. GI50 Values Obtained from the NCI 55 Cell Panel and Mean Graph Midpoint (MGM) Values
GI50 (µM)
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 (µM)
2
0.7
0.16
0.21
27
0.47
0.065
0.066
20
0.088
8.5
3.2
3.5
<0.01
<0.01
0.32
0.12
0.044
14
0.045
16
1.4
3.3
<0.01
<0.01
0.36
0.017
0.039
12
0.052
11
5.9
-
<0.01
<0.01
0.21
<0.01
0.026
22
0.012
14
2.3
2.9
<0.01
<0.01
0.95
0.6
0.32
19
0.76
18
11
7.3
<0.01
0.04
1.8
0.32
0.19
85
0.08
<0.01
0.02
12
<0.01
2
2.2
1.7
<0.01
<0.01
1.3
0.095
0.11
20.4
0.229
10
5.62
6.5
0.014
0.016
3
4
18
20
26
27
31
32
33
35
22
16
6.9
6.1
<0.01
<0.01
24
13
11
<0.01
<0.01
a Data for compounds 2, 26 and 31 are taken from ref 22.
2-methylsulfanyl and 2-methoxy EMATEs 20 and 4. The
equivalence in activity of the 2-CH3X (where X is either
O, S, or CH2)-substituted EMATEs 4, 21 and 32 in this
assay in conjunction with the previously demonstrated
lack of estrogenicity for 2-MeOEMATE 335 highlights
these molecules as being of particular interest and
would suggest that groups of this size with similar
electronic influence on the aromatic A-ring are optimal
on the grounds of antiproliferative activity and absence
of uterotropic activity. This is supported by the observed
activity of 2-MeEMATE 3453 which is equal to that
observed for the larger but electronically similar
2-EtEMATE 32, and far greater than that obtained for
the sterically similar 2-IEMATE 40 and 2-BrEMATE
37. It would seem that electronic-withdrawing 2-sub-
stituents diminish the antiproliferative activity in the
estrogen-3-O-sulfamate series, with the relative anti-
proliferative activities of 2-IEMATE 40 and 2-BrEMATE
37 reflecting the electron-withdrawing ability of their
respective substituents.
A selection of these compounds was then screened in
the NCI 55 human cancer cell line panel, thus allowing
an evaluation of activity across various cancer pheno-
types.58 Data obtained in eight individual cell lines from
this assessment are presented in Table 2. The cell lines
were selected due to the availability of comparative data
for 2-MeOE2 2, 2-EtE2 31 and 2-EtSE2 26 which are
drawn from the literature.22 The MDA-MB-435 cell line
has been highlighted as a cell line especially sensitive
to 2-MeOE2 2, which generally shows higher activity
in the breast cancer cell lines of the panel.22 The mean
graph midpoint (MGM) data represent the mean con-
centration required to cause 50% growth inhibition in
all the cell lines successfully evaluated and thus indi-
cates 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 per-
formed, in this case 10-4 to 10-8M, thus a numerical
value of 0.01 is maximal and would be obtained for a
compound which causes 50% growth inhibition at
concentrations of 10-8M or less in all 55 cell lines.
Examination of the data displayed for individual cell
lines and the MGM values show that the EMATE
derivatives 3, 4, 20, 32 and 33 (with the exception of
2-EtSEMATE 27) exhibit their antiproliferative effects
at much lower concentrations than 2-MeOE2 2 as, by
extension, do their analogous estradiol derivatives. In
the 2-MeO series both EMATE 3 and E2MATE 4
derivatives are more active than 2-MeOE2 2 in all the
cell lines for which comparative data are available; the
degree of enhancement afforded by introduction of the
sulfamate is clearly illustrated by the MGM values
which indicate that across the panel the mean activity
of the 2-MeO estrogen sulfamates 3 and 4 across the
panel is over 10-fold greater than that of 2-MeOE2 2
(values 3 0.095 µM, 4 0.11 µM and 2 1.3 µM). All data
obtained are consistent with those obtained in our
preliminary MCF-7 screen with, for example, the influ-
ence of the large 2-ethylsulfanyl substituent proving
deleterious for antiproliferative activity (vide supra).
The 2-CH3X (where X is either O, S, or CH2)-substituted
EMATEs 3, 20 and 32 and E2MATEs 4 and 33 all
proved to be highly active. In particular, 2-EtEMATE
32 and 2-EtE2MATE 33 display 50% growth inhibition
at less than 10 nM concentration almost uniformly
across the panel as reflected in the 90- and 80-fold
greater MGM values than 2-MeOE2 2, figures which
are intrinsically limited due to the concentration sur-
veyed being greater than that required for 50% growth
inhibition for these highly active molecules.
The ability of estrone-3-O-sulfamate, EMATE, to act
as an irreversible inhibitor of steroid sulfatase (STS), a
clinical target for the treatment of hormone-dependent
breast cancer, is well documented (vide supra).32 Previ-
ous studies have shown that selected 2-substituted
EMATEs, including 2-MeOEMATE, are potent STS
inhibitors.35;53 The activity of 2-MeO-, 2-EtO-, 2-MeS-
and 2-Et-substituted EMATE derivatives to inhibit STS
activity in placental microsomes was evaluated along-
side EMATE (IC50 22 nM). 2-MeOEMATE 3 (IC50 42
nM) and 2-MeOE2MATE 4 (IC50 34 nM) proved to be
the most active of the substituted EMATEs assayed,
with IC50 values slightly higher than EMATE. Unsur-
prisingly, the N,N-dimethyl sulfamate 15 showed no
inhibition of STS activity at a concentration of 1 µM.
Introduction of the larger 2-EtO substituent caused a
significant reduction in STS inhibition (2-EtOEMATE
IC50 890 nM), indicating that the ethoxy group is
disfavored relative to the methoxy group presumably
due to steric constraints at the site of action. 2-MeSE-
MATE (IC50 120 nM) however proved highly active as
an inhibitor of STS; clearly this group is of a similar
steric size to the methoxy group and can participate in
H-bond acceptor interactions in the binding pocket in
an analogous manner to 2-MeOEMATE. It appears
reasonable to suggest that the relative activities of
2-MeOEMATE and 2-MeSEMATE are due to the re-
spective H-bond acceptor potential of the ether and
thioether groups. The importance of the putative H-
bonding interactions is further illustrated by the relative

A-Ring-Substituted Estrogen-3-O-sulfamates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5249
taxis assays to select compounds with antiangiogenic
activity.59 2-EtE2MATE 33 gave IC50 values of 0.33 µM,
0.06 µM and 0.36 µM in each respective assay, thus
confirming the antiangiogenic properties of this mol-
ecule in agreement with results previously reported for
2-MeOEMATE.60
On the basis of the in vitro activity displayed by
2-EtE2MATE 33, this compound was selected for evalu-
ation by the NCI in the in vivo hollow fiber assay which
entails the intraperitoneal (ip) and subcutaneous (sc)
implanting of hollow polyvinylidene fluoride fibers in
an animal, each of which contains a culture of one of
twelve human tumor cell lines.61 Each tumor line is thus
examined in the ip and sc environment. The vehicle
animals are then treated in an ip manner at two dose
levels (100 and 150 mg/kg, 25% and 37.5% respectively
of the MTD) on a daily basis for 4 days, and after a
further day the fibers are collected for analysis and the
analyzed compound awarded a score of 2 if a 50%
reduction in viable cell mass compared to the vehicle
control samples is observed. In this assay 2-EtE2MATE
33 obtained an ip score of 10 and an sc score of 8 (i.e.
50% growth inhibition was observed in 5 of the ip
implanted fibers and four of the sc implanted fibers, the
maximum possible score is 96), a result which demon-
strates the good in vivo activity of this class of molecules
and the ability of the compound to be transported from
the peritoneum to peripheral tissue.
A further assessment of the ability of 2-MeOE2MATE
4 and 2-EtE2MATE 33 to inhibit tumor growth in vivo
was performed using xenografts derived from MDA-MB-
435 (ER-) human breast cancer cells transplanted
into female mice. The oral administration of either
2-MeOE2MATE or 2-EtE2MATE significantly reduced
the growth of tumors derived from MDA-MB-435 cancer
cells (Figure 4). A 20 mg/kg dose, administered orally
for 28 days, of 2-MeOE2MATE reduced tumor growth
by 33% (p < 0.05) while 2-EtE2MATE reduced tumor
growth by 40% (p < 0.01) compared with the growth of
tumors in untreated animals. Four weeks after the
cessation of dosing the tumor volumes in mice that had
received 2-EtE2MATE remained significantly smaller
(45% reduction, p < 0.01) than those for the control
group. The difference in tumor volumes for the
2-MeOE2MATE treated group was not significant at
this time point. From this study, therefore, it would
appear that the in vivo efficacy of 2-EtE2MATE is
greater than that of 2-MeOE2MATE and that both of
these molecules are orally bioavailable.
Figure 4. In vivo effects of 2-MeOE2MATE or 2-EtE2MATE
on the growth of MDA-MB-435 tumor xenografts in nude mice.
Vehicle (THF/propylene glycol) or compounds were adminis-
tered orally (20 mg/kg) 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) (a, p < 0.05 vs controls; b, p < 0.01 vs
controls, Student’s t test).
inactivity of 2-EtEMATE and 2-EtE2MATE with respect
to 2-MeOEMATE (IC50 values of 1.0 µM, 1.8 µM and 42
nM). The inability of the ethyl group to act as an H-bond
acceptor results in the greater than 25-fold increase in
STS IC50 value observed for 2-EtEMATE. These results
are in agreement with a previous study in which the
excellent STS inhibition (IC50 100 pM) displayed by
2-difluoromethyl EMATE was ascribed to the H-bond
interactions between the difluoromethyl group and
H-bond donor residues of the active site.53 Thus, in
addition to their direct antiproliferative effect against
tumors, these compounds should act against hormone-
dependent tumors by inhibiting the STS mediated
conversion of estradiol sulfate to estradiol, thereby
causing a reduction in the circulatory estrogens required
to stimulate their growth. The SAR of the 2-substituted
EMATEs as inhibitors of STS clearly contrasts with the
SAR for antiproliferative effects discussed above (see
Figure 5).
2-EtE2MATE 33 was also evaluated using the NCI
antiangiogenesis resource, which assesses HUVEC
growth inhibition, and uses cord formation and chemo-
Figure 5. In vitro SAR of 2-RX-substituted estradiol-3-O-sulfamates as (a) antiproliferative agents and (b) irreversible inhibitors
of steroid sulfatase.

5250 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Leese et al.
University of Bath.UK. FAB-MS were carried out using
m-nitrobenzyl alcohol (NBA) as the matrix. Elemental analyses
were performed by the Microanalysis Service, University of
Bath. Melting points were determined using a Reichert-Jung
Thermo Galen Kofler block and are uncorrected.
The X-ray crystallographic studies of 2-EtE1 and 2-MeOE-
MATE were carried out on a Nonius Kappa CCD diffractome-
ter with area detector. Crystallographic data for 2-EtE1 and
2-MeOEMATE have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publications
CCDC 260114 and 260115, respectively. Copies of the data can
be obtained free of charge on application to CCDC, 12 Union
Road, Cambridge CB2 1EZ, UK [fax(+44) 1223 336033,
e-mail: deposit@ccdc.cam.ac.uk].
Crystal Data for 2-EtE1: C20 H26 O2, M ) 298.41, λ )
0.71073 Å, monoclinic, space group P21, a ) 12.0690(3), b )
11.1390(3), c ) 12.7150(4)Å, â ) 90.702(1)°, U ) 1709.23(8)
Å3, Z ) 4, Dc ) 1.160 Mg/m3, µ ) 0.073 mm-1, F(000) ) 648,
crystal size 0.20 × 0.13 × 0.13 mm, unique reflections ) 7656
[R(int) ) 0.0779], observed I > 2σ(I) ) 5186, data/restraints/
parameters ) 7656/1/403, R1 ) 0.0496 wR2 ) 0.1026 (obs.
data), R1 ) 0.0897 wR2 ) 0.1174 (all data), max peak/hole
0.179 and -0.177 eÅ-3. Absolute structure parameter )
0.6(11).
Thus, in summary, in the present study a flexible
practical approach to 2-substituted estrone derivatives,
2-substituted estrone-3-O-sulfamates (EMATEs) and
2-substituted estradiol-3-O-sulfamates (E2MATEs) has
been developed. Of the molecules synthesized the 2-sub-
stituted estrogen-3-O-sulfamates exhibited high anti-
proliferative activity in vitro against a wide range of
tumor cells. The structure activity relationship for
antiproliferative activity obtained for these molecules
is summarized in Figure 5. These 3-O-sulfamate deriva-
tives are significantly more active in vitro than the
closely related nonsulfamoylated 2-substituted estradiol
derivatives and exhibit a contrasting SAR. Substitution
at the 2-position for the 3-O-sulfamates series is opti-
mally a group of the formula CH3X (X is either O, S, or
CH2) which appear ideal on both steric and electronic
grounds. Substitution of the sulfamate moiety leads to
a reduction in activity while the oxidation state of C-17,
of key importance in the estradiol series, has little effect
on in vitro activity. Further assessment of one of these
compounds, 2-EtE2MATE 33, has also demonstrated
antiangiogenic activity as well as in vivo antitumor
activity. Mechanistic studies have shown that these
molecules inhibit the taxol-promoted polymerization of
tubulin, and, additionally, they also trigger BCL-2
phosphorylation and upregulation of p53 and induce
apoptosis.36,37 We have also previously shown that
2-MeOEMATE inhibits the growth of NMU-induced
tumors in rats.36 When considered alongside our previ-
ous observations that 2-substituted EMATEs are ad-
ditionally nonestrogenic irreversible inhibitors of steroid
sulfatase,35 itself a clinical target for the treatment of
hormone-dependent breast cancer, these molecules are
clearly highlighted as a promising class of potential
therapeutic agents which have multitargeted antitumor
activities. Additionally, the sequestration of such 3-O-
sulfamate derivatives into red blood cells by binding to
CAII provides a route to minimize metabolism and
maximize the oral bioavailability of such compounds.
We are actively investigating the effects of further
modification of the 2-substituted estrogen-3-O-sulfa-
mates with a view to producing new classes of potent
anticancer agents.
Crystal Data for 2-MeOEMATE: C19 H25 N O5 S, M )
379.46, λ ) 0.71073 Å, orthorhombic, space group P212121,
a ) 7.6190(1), b ) 15.2780(2), c ) 15.4400(2)Å, U )
1797.26(4) Å3, Z ) 4, Dc ) 1.402 Mg/m3, µ ) 0.211 mm-1
,
F(000) ) 808, crystal size 0.20 × 0.15 × 0.15 mm, unique
reflections ) 4104 [R(int) ) 0.0558], observed I > 2σ(I) ) 3433,
data/restraints/parameters ) 4104/2/246, R1 ) 0.0352 wR2
) 0.0798 (obs. data), R1 ) 0.0491 wR2 ) 0.0860 (all data),
max peak/hole 0.164 and -0.252 eÅ-3. Absolute structure
parameter ) 0.02(6).
Biology. Cell proliferation was analyzed using the Cell
Titer 96 Aqueous One Solution Cell Proliferation Assay
(Promega, WI). In brief, MCF-7 cells were seeded in a 96-well
multiplate at a density of 4000 cells/well (0.05 mL) in MEM
with 10% FBS and supplements. Four to five hours after
seeding, medium alone or medium containing the test com-
pounds (0.002-20.0 µM, 0.05 mL) was added. After an
additional 96 h of culture, cells were incubated for 2h at 37
°C with MTS (0.02 mL/well, 3-(4,3-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium re-
agent). The absorbance of the colored formazan product was
recorded at 492 nm. Data were expressed as percentage
inhibition of MCF-7 proliferation, and the GI50 values were
obtained from these data. STS inhibition was assayed by the
method of Purohit et al.63 The ability of 2-MeOE2MATE and
2-EtE2MATE 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 mm3. 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 tumors was
measured at weekly intervals, from which tumor volumes were
calculated using the formula (l × w2/2). Monitoring of tumor
volumes continued for a further 28 day period after the end of
drug administration.
Experimental Section
Materials and Methods. Chemistry. All chemicals were
either purchased from Aldrich Chemical Co. (Gillingham, UK),
Fluka (Gillingham, UK) or Lancaster Synthesis (Morecambe,
UK). Organic solvents of A. R. grade were supplied by Fisher
Scientific (Loughborough, UK) and used as supplied. Anhy-
drous N,N-dimethylformamide (DMF) and N,N-dimethylac-
etamide (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 adaption of the method of Appel and Berger50
and was stored in the refrigerator under positive pressure of
N2 as a solution in toluene as described by Woo et al.62 An
appropriate volume of this solution was freshly concentrated
in vacuo immediately before use. E1 was purchased from
Sequoia Research Products (Oxford, UK). Flash column chro-
matography was performed on silica gel (MatrexC60).
17,17-Ethylenedioxy estrone 6. A suspension of estrone
5 (12.5 g, 46.2 mmol), toluene (150 mL), ethylene glycol (14
mL) and tosic acid (120 mg) was refluxed for 14 h under Dean-
Stark conditions. The resultant pale pink solution was poured
onto saturated sodium bicarbonate solution (150 mL) and
diluted with ethyl acetate (250 mL). The organic layer was
separated, the aqueous layer extracted with a further aliquot
of ethyl acetate (100 mL) and the combined organics were
washed with water (150 mL) and brine (150 mL), dried and
evaporated to yield crude dioxolone 6 (15 g, 103%) as an off-
white crystalline solid mp 183-184 °C [lit. 180-181 °C64]
1H NMR and 13C NMR spectra were recorded in chloroform
solution (unless otherwise indicated) with a JMN-GX 400 NMR
spectrometer at 400 and 100 MHz, respectively. Chemical
shifts are reported in parts per million (ppm, δ) relative to
tetramethylsilane (TMS) as an internal standard. Mass spectra
were recorded at the Mass Spectrometry Service Center,

A-Ring-Substituted Estrogen-3-O-sulfamates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5251
which was used without further purification (1H NMR shows
after which time the reaction was allowed to come to 0 °C over
2 h. Ammonium chloride (50 mL, saturated) quench was
followed by dilution in ethyl acetate (500 mL). The organic
layer was separated, washed with sodium thiosulfate (100 mL,
10% aqueous), water (3 × 100 mL) and brine (200 mL), dried
and evaporated. The desired product 29 was isolated by
column chromatography (hexane/ethyl acetate gradient 10:0
purity >95%).
3-O-Methoxymethyl-17,17-ethylenedioxyestrone. So-
dium hydride (3.48 g, 87 mmol) was added in a portionwise
manner to a stirred 0 °C solution of protected estrone 6 (18.2
g, 58 mmol) in dimethylformamide (250 mL). Methyl chlorom-
ethyl ether (8.81 mL, 2 equiv) was then introduced in a
dropwise manner after evolution of H2 had ceased. On comple-
tion of addition, the external cooling was removed and the
reaction was left to stir at room temperature for 16 h.
Ammonia (100 mL, 2 M) was then added to destroy any
residual MOMCl before extraction of the mixture into ethyl
acetate (500 mL). The aqueous layer was then separated, and
the organic layer was washed with brine (5 × 200 mL), then
dried and evaporated. The resultant pale yellow oil was
purified by column chromatography (4:1 hexane/ethyl acetate)
to give compound 7 as a colorless oil which on standing gave
a white solid (19.8 g, 95%): mp 62-63 °C, Rf 0.55 (3:1 hexane/
1
to 9:1) as a colorless oil (36.5 g, 84%) which showed H NMR
δ 7.08 (1H, s), 6.78 (1H, s), 5.16 (2H, s), 3.86-3.98 (4H, m),
3.48 (3H, s), 2.78-2.98 (2H, m), 2.61 (2H, q, J 7.4), 1.22-2.44
(13H, m), 1.21 (3H, t, J 7.4) and 0.88 (3H, s); m/z [FAB+] 386.2
(100%, M+); HRMS [FAB+] 386.24571, calculated 386.24571.
2-Bromoestrone 36. Estrone MOM/ketal 7 (3.58 g, 10
mmol) in THF (70 mL) was reacted with sec-BuLi (23 mL, 30
mmol) as described for the synthesis of 8 and then quenched
with 1,1,1-trifluoro-2-bromoethane (5 g, 30 mmol). After
workup the resulting was partially purified by chromatography
(9:1 hexane/ethyl acetate) to give a mixture (2.7 g) of starting
material and 2-bromo compound 35 which showed significant
resonances at δH (CDCl3) 7.43 (1H, s), 6.86 (1H, s), 5.20 (2H,
s, OCH2O), 3.86-3.98 (4H, m), 3.51 (3H, s), 2.76-2.84 (2H,
m), and 0.89 (3H, s); m/z [FAB+] 437.1 (M+). A sample of this
mixture (1 g) was treated with methanolic HCl (made from
AcCl and MeOH [5.64 mL and 14.4 mL respectively see above])
for 1 h at room temperature. The white powder obtained from
standard workup was purified by column chromatography (3%
ethyl acetate in chloroform) to give pure 2-bromoestrone 36
(620 mg, 48% over two steps) mp 211-14 °C [lit 194-195 °C65].
1H NMR δ 7.34 (1H, s), 6.76 (1H, s), 5.37 (1H, s), 2.78-2.86
(2H, m), 1.36-2.56 (13H, m) and 0.91 (3H, s); m/z [FAB+]
350.1 (M+ -H, 81Br, 95%) and 348.1 (M+ -H, 79Br, 100%); Anal.
(C18H21BrO2) C, H.
1
ethyl acetate). H NMR δ 7.21 (1H, d, J 8.6), 6.82 (1H, dd, J
8.6 and 2.7), 6.77 (1H, d, J 2.7), 5.13 (2H, s), 3.89-3.98 (4H,
m), 3.46 (3H, s), 2.83 (2H, m), 2.27 (2H, m), 1.25-2.05 (11H,
m) and 0.88 (3H, s, CH3); m/z [EI] 358.2 (100%, M+); HRMS
[EI] 358.214410 C22H30O4 requires 358.21441.
2-Formyl-3-O-methoxymethyl-17,17-ethylenedioxyestro-
ne 8. A well stirred solution of protected estrone 7 (24.3 g,
67.9 mmol) in THF (400 mL) was cooled to -78 °C and then
treated with sec-butyllithium (156 mL, 203 mmol) in a drop-
wise manner over 1 h. Cooling was maintained for a further 2
h prior to introduction of dimethylformamide (21 mL, 272
mmol) over 10 min after which time the reaction was allowed
to come to 0 °C over 1 h. The reaction was then quenched with
aqueous ammonium chloride (20 mL, saturated) and then
partitioned between ethyl acetate (500 mL) and water (200
mL). The organic layer was separated, washed with water (3
× 100 mL) and brine (200 mL), dried and evaporated. The
desired product 8 was isolated by column chromatography
(gradient hexane to 10% ethyl acetate in hexane) as a colorless
oil (22.5 g, 86%) which solidified on standing to give a cream
2-Iodoestrone 39. Estrone MOM/ketal 7 (2.86 g, 8 mmol)
in THF (50 mL) was reacted with sec-BuLi (18 mL, 23.5 mmol)
as described for the synthesis of 8 and then quenched with
1,1,1-trifluoro-2-iodoethane (5 g, 23.8 mmol). After standard
workup, the crude product was partially purified by chroma-
tography (14:1 hexane/ethyl acetate) to give a mixture (2 g) of
starting material 7 and 2-iodinated compound 38 as a pale
1
solid mp 108-110 °C. H NMR δ 10.42 (1H, s), 7.77 (1H, s),
1
6.91 (1H, s), 5.26 (2H, s), 3.89-3.98 (4H, m), 3.51 (3H, s), 2.80-
2.94 (2H, m), 1.30-2.46 (13H, m), and 0.87 (3H, s); m/z [EI+]
386.2 (100%, M+); HRMS [EI] 386.20932, calculated 386.20932.
yellow oil which showed H NMR δ 7.65 (1H, s), 6.78 (1H, s),
5.19 (2H, s), 3.88-3.98 (4H, m), 3.50 (3H, s), 2.78-2.86 (2H,
m), and 0.87 (3H, s); m/z [FAB+] 437.1 (M+). A sample of this
mixture (1.95 g) was treated with methanolic HCl (generated
from acetyl chloride (11.2 mL) and methanol (28.2 mL)) and
then sonicated for 8 min. The resultant orange solution was
then stirred for 0.5 h prior to addition of water (50 mL) and
neutralization with NaHCO3. The solution was then extracted
with ethyl acetate (2 × 100 mL), the organic layers were
washed with water (100 mL) and then brine (100 mL), dried
and evaporated to give a pale yellow solid. 2-Iodo estrone 39
was isolated by column chromatography (3% ethyl acetate in
chloroform) as a white crystalline solid (1.2 g, 38% over two
2-Methylsulfanyl-3-O-methoxymethylene-17,17-ethyl-
enedioxyestrone 17. Protected estrone 7 (20 g, 55.8 mmol)
in THF (400 mL) was reacted with sec-butyllithium (129 mL,
167 mmol) as described for the synthesis of 8. The resulting
anion was quenched with dimethyl disulfide (20 mL, 223
mmol) over a period of 5 min. After a further 2 h, the reaction
was subjected to the standard workup to yield a pale yellow
oil which was purified by chromatography (hexane/ethyl
acetate gradient 100:0 to 85:15). The desired sulfide 17 (20.5
1
g, 91%), a clear colorless oil, showed H NMR δ 7.16 (1H, s),
1
steps) mp 195 °C (dec) [lit. 200-205 °C43] which showed H
6.81 (1H, s), 5.21 (2H, s), 3.86-3.98 (4H, m) 3.51 (3H, s), 2.80-
2.88 (2H, m), 2.43 (3H, s), 1.25-2.38 (13H, m) and 0.88 (3H,
s); m/z [FAB+] 404.1 (M+); HRMS [FAB+] 404.2004, calculated
404.2021.
NMR δ 7.52 (1H, s, ArH), 6.74 (1H, s, ArH), 5.18 (1H, s, OH),
2.80-2.88 (2H, m, 6-CH2), 1.30-2.56 (13H, m) and 0.91 (3H,
s, 18-CH3); m/z [APCI-] 395.3 (M+ - H, 100%).
2-Ethylsulfanyl-3-O-methoxymethylene-17,17-ethyl-
enedioxyestrone 24. Protected estrone 7 (5 g, 13.9 mmol) in
THF (100 mL) was reacted with sec-butyllithium (32.2 mL,
41.8 mmol) as described for the synthesis of 8. The resulting
anion was quenched with diethyl disulfide (9.1 mL, 70 mmol)
over a period of 5 min. Standard workup yielded a very pale
yellow purified by chromatography (hexane/ethyl acetate
gradient 100:0 to 85:15) to give the desired sulfide 24 (5.3 g,
12.6 mmol, 97%) as a clear colorless oil which showed 1H NMR
δ 7.24 (1H, s), 6.82 (1H, s), 5.21 (2H, s), 3.86-3.98 (4H, m),
3.51 (3H, s), 2.79-2.94 (4H, m) 1.20-2.36 (16H, m [including
1.30 (3H, t)) and 0.88 (3H, s); m/z [FAB+] 418.1 (M+); HRMS
[FAB+] 418.1275, calculated 418.2178.
2-Hydroxy-3-O-methoxymethyl-17,17-ethylenedioxy-
estrone 9. A room-temperature solution of 2-formyl compound
8 (17.3 g, 44.7 mmol) in dichloromethane (150 mL) and
chloroform (150 mL) was treated with sodium hydrogenphos-
phate (22.2 g, 157 mmol) and then, in a dropwise manner, a
solution of mCPBA (15.4 g, 80 mmol) in dichloromethane (150
mL). The reaction was stirred for 1 h and then filtered to
remove solid residues. The filtrate was then washed with ice-
water (400 mL), and the aqueous washings were extracted
with dichloromethane (100 mL). The combined organic layers
were washed with and saturated sodium bicarbonate (200 mL)
then brine (200 mL) prior to evaporation. The resultant white
solid was dissolved in degassed methanol (400 mL), treated
with sodium hydroxide solution (81 mL, 81 mmol, 1 M) and
then stirred for 2 h. The reaction was then carefully neutral-
ized with 2 M HCl, and the methanol was then removed by
evaporation. The mixture was then extracted into ethyl acetate
(400 mL), the aqueous layer separated and the organic layer
2-Ethyl-3-O-methoxymethyl-17,17-ethylenedioxyestro-
ne 29. Protected estrone 7 (40 g, 112 mmol) in THF (400 mL)
was reacted with sec-butyllithium (258 mL, 336 mmol) as
described for the synthesis of 8. The resulting anion was
quenched with ethyl iodide (23.7 mL, 336 mmol) over 10 min

5252 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Leese et al.
washed with water (200 mL) and brine (200 mL), dried and
evaporated. The desired product 9 was isolated by column
chromatography (5:1 hexane/ethyl acetate) to give a colorless
oil which solidified on standing to give a white crystalline solid
(13.8 g, 84%) mp 88-89 °C which showed 1H NMR δ 6.90 (1H,
s), 6.78 (1H, s), 5.80 (1H, br), 5.15 (2H, s), 3.88-3.98 (4H, m),
3.51 (3H, s), 2.70-2.82 (2H, m), 1.20-2.28 (13H, m) and 0.87
(3H, s); m/z [EI] 374.3 (100%, M+); HRMS [EI] 374.20932,
calculated 374.20932; Anal. (C22H30O5) C, H.
then washed with water (2 × 50 mL) and brine (100 mL) before
drying (MgSO4) and evaporating. Purification by gradient
elution with hexane/ethyl acetate mixtures (100:0 to 80:20) on
silica gel gave 18 as a white crystalline solid (1.06 g, 90%) mp
153-5 °C which showed: 1H NMR δ 7.40 (1H, s), 6.73 (1H, s),
6.49 (1H, s), 2.82-2.91 (2H, m), 2.34-2.56 (2H, m), 2.30 (3H,
s) 1.36-2.26 (11H, m) and 0.91 (3H, s); m/z [FAB+] 316.2
(100%, M+); HRMS [FAB+] 316.149702 calculated 316.14970;
Anal. (C19H24O2S) C, H.
2-Ethylsulfanylestrone 25. Protected estrone 24 (1.8 g,
4.3 mmol) was reacted with 4 M methanolic HCl as described
for the synthesis of 18. The crude product was crystallized
(ethanol) to give white crystals of 25 (1.23 g, 87%) mp 139-
141 °C which showed: 1H NMR δ 7.36 (1H, s), 6.73 (1H, s),
6.57 (1H, s), 2.82-91 (2H, m), 2.66 (2H, q, J 7.4), 1.36-2.55
(13H, m), 1.21 (3H, t, J 7.4) and 0.91 (3H, s); m/z [FAB+] 330.2
(100%, M+), HRMS [FAB+] 330.165352, calculated 330.16535;
Anal. (C20H26O2S) C, H.
2-Ethylestrone 30. A solution of methanolic HCl was
generated by cautious addition of acetyl chloride (122 mL) to
ice cold methanol (324 mL) and then poured onto 29 (21 g,
54.3 mmol). Sonication was applied for 5 min to aid solution
and after 10 min (400 mL) water was added to the reaction.
The mixture was cooled in an ice bath causing precipitation
of the product as a white powder which was collected by
filtration and then crystallized from methanol to give 30 white
crystals (14.7 g over three crops, 91%) mp 208-210 °C. 1H
NMR δ 7.03 (1H, s), 6.51 (1H, s), 4.92 (1H, s), 2.78-2.86 (2H,
m), 2.59 (2H, q, J 7.4), 1.30-2.54 (13H, m), 1.21 (3H, t, J 7.4)
and 0.91 (3H, s); m/z [ES-] 297.1 (100%, M+ - H); Anal.
(C20H30O2.EtOAc) C, H.
2-Methoxy-3-O-methoxymethyl-17,17-ethylenedioxy-
estrone 10. A solution of 2-hydroxy compound 9 (14 g, 37.4
mmol) in anhydrous DMF (300 mL) was treated with potas-
sium carbonate (63 g, 455 mmol) and then stirred for 10 min
prior to addition of methyl iodide (22.7 mL, 411 mmol) then
tetrabutylammonium iodide (100 mg). The reaction was stirred
at room temperature for 72 h, then filtered through a sinter
to remove the potassium carbonate. The residues were washed
with ethyl acetate (400 mL), and then combined filtrate and
washings were then poured onto water (200 mL). The organic
layer was separated and washed with a further five aliquots
of water (100 mL) and then brine (200 mL), dried and
evaporated to give 10 as a pale yellow solid (13.5 g, 93%) which
was used without further purification. 1H NMR δ 6.86 (1H,
s), 6.84 (1H, s), 5.18 (2H, s), 3.88-3.98 (4H, m), 3.51 (3H, s),
2.76-2.83 (2H, m), 1.24-2.36 (13H, m) and 0.89 (3H, s); m/z
[EI] 388.3 (100%, M+); HRMS [EI] 388.22497, calculated
388.22497.
2-Ethoxy-3-O-methoxymethyl-17,17-ethylenedioxyestro-
ne 11. A solution of 9 (2.0 g, 5.34 mmol) in DMF (80 mL) was
treated with potassium carbonate (7.38 g, 53.4 mmol), and then
after 10 minutes, ethyl iodide (4.70 mL, 58.7 mmol) and tetra-
n-butylammonium iodide (120 mg). After 36 h stirring, the
reaction was poured onto brine (150 mL) and extracted with
ethyl acetate (200 mL). The organic layer was then separated
and washed exhaustively with brine (5 × 100 mL), dried and
evaporated to give 11 as a colorless oil (1.91 g, 89%) which
was used without further purification. 1H NMR δ 6.88 (1H,
s), 6.87 (1H, s), 5.20 (2H, s), 4.11 (2H, q, J 7.0), 3.92-4.02 (4H,
m), 3.55 (3H, s), 2.80-2.86 (2H, m), 1.30-2.36 (16H, m
including 1.46 (3H, t, J 7.0)) and 0.93 (3H, s); m/z [FAB+] 402.1
(100%, M+); HRMS [FAB+] 402.24062, calculated 402.24062.
2-Methoxyestrone 1. A solution of 10 (2.12 g, 5.4 mmol)
in THF (50 mL) was treated with hydrochloric acid (20 mL, 6
M). After 1 h stirring, the reaction was poured onto brine (50
mL) and extracted into ethyl acetate (150 mL). The organic
layer was separated, washed with water (50 mL), sodium
hydrogen carbonate (50 mL), water (50 mL) and brine (50 mL),
then dried and evaporated to give an off-white solid. Crystal-
lization from methanol gave 2-methoxyestrone 1 as white
crystals (1.45 g, 90%) mp 189-191 °C [lit. 190s192 °C45] which
showed 1H NMR δ 6.79 (1H, s), 6.65 (1H, s), 5.50 (1H, br),
3.86 (3H, s), 2.76-2.84 (2H, m), 1.35-2.55 (13H, m) and 0.92
(3H, s); m/z [FAB+] 300.2 (100%, M+); HRMS [FAB+] 300.17254,
calculated 300.17254; Anal. (C19H24O3) C, H.
2-Methylsulfoxyestrone 22. A solution of 2-methylsulfan-
ylestrone 18 (632 mg, 2 mmol) in dichloromethane (40 mL) at
10 °C was treated with mCPBA (652 mg, 3.4 mmol) in a
portionwise manner over 2 min. After 15 min, the reaction was
diluted with dichloromethane (50 mL) and then washed with
brine, sodium hydrogen carbonate solution, water (50 mL each)
and brine (2 × 50 mL), dried and evaporated to give an off
white solid which was crystallized from ethanol to give 22 as
1
a white crystalline solid (420 mg, 63%) mp 250 °C (dec); H
NMR δ 10.0 (1H, s), 6.95 (1H,s), 6.70 (1H, s), 2.80-2.96 (2H,
m), 2.95 (3H, s), 1.40-2.56 (13H, m) and 0.91 (3H, s,); m/z
[ES-] 331.1 (M+ - H, 35%) and 316.5 (M+ - OH, 100%); Anal.
(C19H24O3S) C, H.
2-Methylsulfonylestrone 23. A solution of 2-methyl-
sulfanylestrone 18 (632 mg, 2 mmol) in dichloromethane (40
mL) was treated with mCPBA (1.34 g, 7 mmol) at room
temperature. After 1 h, TLC analysis showed no sign of
starting material or intermediate sulfoxide. The reaction was
filtered, washed with saturated sodium hydrogen carbonate
solution (2 × 50 mL), water (50 mL) then brine (50 mL), dried
and evaporated to give a white solid. The product 23 was
isolated by crystallization from ethanol as colorless prisms (553
mg, 78%) mp 265-266 °C which showed 1H NMR δ 8.59 (1H,
s), 7.54 (1H, s), 6.78 (1H, s), 3.10 (3H, s), 2.84-3.00 (2H, m),
2-Ethoxyestrone 12a. Protected 2-ethoxyestrone 11 (1.91
g, 4.75 mmol) was dissolved in THF (50 mL) and then treated
with hydrochloric acid (20 mL, 6 M) as described for the
synthesis of 1. The crude product was purified by column
chromatography (4:1 hexane/ethyl acetate) to give 12a as a
white crystalline solid (1.38 g, 92%) mp 145-147 °C [lit. 140-
1.40-2.56 (13H, m) and 0.92 (3H, s); m/z [ES-] 347.2 (M+
H); Anal. (C19H24O4S) C, H.
-
2-Methoxyestradiol 2. A room-temperature solution of
2-methoxyestrone 1 (900 mg, 3 mmol) in THF (5 mL) and
isopropyl alcohol (25 mL) was treated with sodium borohydride
(111 mg, 3 mmol) in a portionwise manner. The reaction was
stirred for 16 h then treated with saturated ammonium
chloride solution (10 mL) and then partitioned between ethyl
acetate (50 mL) and water (30 mL). The organic layer was
separated, washed with water (2 × 25 mL) and brine (50 mL),
dried and evaporated to give a white solid. Recrystallization
from chloroform gave the pure estradiol 2 as white crystals
(770 mg, 85%) mp 189-190 °C [lit. 188-190 °C40] which
1
142 °C24] which showed H NMR δ 6.77 (1H, s), 6.65 (1H, s),
5.55 (1H, br), 4.08 (2H, m), 2.76-2.84 (2H, m), 1.38-2.55 (16H,
m including 1.43 (3H, t, J 7.0)) and 0.91 (3H, s); m/z [FAB+]
314.2 (100%, M+); HRMS [FAB+] 314.18819, calculated
314.18819.
2-Methylsulfanylestrone 18. A 4 M solution of methanolic
HCl was prepared by cautious addition of acetyl chloride (6.1
mL) to ice cold methanol (15.6 mL). After 5 min of stirring,
this solution was poured onto protected estrone 17 (1.5 g, 3.72
mmol). Sonication was applied to speed the dissolution of the
protected estrone, a pink color emerged in the reaction mixture
after 5 min and, after a further 10 minutes, ethyl acetate (100
mL) and then sufficient sodium hydrogen carbonate to neu-
tralize the reaction mixture was added. The organic layer was
1
showed H NMR δ (d6-DMSO) 8.57 (1H, s), 6.76 (1H, s), 6.43
(1H, s), 4.48 (1H, d, J 4.9) 3.70 (3H, s), 3.45-3.55 (1H, m),
2.60-2.68 (2H, m), 1.05-2.35 (13H, m) and 0.67 (3H, s); Anal.
(C19H26O3) C, H.
2-Ethoxyestradiol 12b. A solution of 2-ethoxyestrone 12a
(237 mg, 0.75 mmol) in THF (6 mL) and isopropyl alcohol (6

A-Ring-Substituted Estrogen-3-O-sulfamates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5253
mL) was cooled to 0 °C then treated with sodium borohydride
(114 mg, 3.02 mmol). The reaction was stirred for 14 h then
brought to pH 6 by addition of glacial acetic acid. Solvents were
then removed by evaporation and the desired product 12b
isolated by column chromatography (3:1 hexane/ethyl acetate)
as a white crystalline solid (197 mg, 83%) which showed mp
reacted with sulfamoyl chloride (5.4 mL, 0.7 M, 3.77 mmol)
as described for the synthesis of 3. The desired product 13 was
isolated by column chromatography (3:2 hexane/ethyl acetate)
as a white solid (282 mg, 57%), a sample was crystallized from
ethyl acetate/hexane to give as white needles mp 179-181 °C
1
which showed H NMR δ 7.30 (1H, s), 6.92 (1H, s), 5.20 (2H,
1
153-155 °C [lit. 154-155 °C22] H NMR δ 6.78 (1H, s), 6.64
s), 4.11 (2H, m), 2.78-2.86 (2H, m), 1.12-2.55 (16H, m
including 1.43 (3H, t, J 7.0)), and 0.91 (3H, s); m/z [FAB+] 395.2
(100%, M+); HRMS [FAB+] 393.16099, calculated 393.16099;
Anal. (C20H27NO5S) C, H, N.
(1H, s), 5.58 (1H, s), 4.10 (2H, m), 3.72 (1H, dd, J 8.6 and 8.2),
2.72-2.80 (2H, m), 1.16-2.30 (16H, m including 1.42 (3H, t,
J 7.0)), and 0.78 (3H, s); m/z [FAB+] 316.2 (100%, M+); HRMS
[FAB+] 316.20385, calculated 316.20385.
2-Methylsulfanylestrone-3-O-sulfamate 20. A stirred
solution of 2-methylsulfanylestrone 19 (400 mg, 1.26 mmol)
and 2,6-di-tert-butyl-4-methyl pyridine (776 mg, 3.78 mmol)
in dichloromethane (20 mL) was reacted with sulfamoyl
chloride (5.4 mL, 3.78 mmol, 0.7 M in toluene) as described
for the synthesis of 3. The desired sulfamate 20 was purified
by column chromatography (chloroform/ethyl acetate 9:1) to
give a white crystalline solid (358 mg, 72%) mp 184-185 °C
2-Methylsulfanylestradiol 19. Sodium borohydride (37
mg) was added to a solution of 2-methylsulfanylestrone 18 (316
mg, 1 mmol) in THF/IPA (8 mL each) at 0 °C. After 14 h,
stirring saturated ammonium chloride (5 mL) was added to
quench the reaction and the mixture was then extracted with
ethyl acetate (50 mL). The organic layers were washed with
water (3 × 20 mL) and brine (2 × 20 mL), dried (MgSO4) and
evaporated to give a white solid which was crystallized from
ethanol to give 19 white crystals (296 mg, 83%) mp 163-164
°C which showed: 1H NMR δ 7.40 (1H, s), 6.71 (1H, s), 6.47
(1H, s), 3.73 (1H, dd, J 8.4 and 8.4), 2.80-2.86 (2H, m), 1.14-
2.36 (16H, m including 2.29 (3H, s)) and 0.91 (3H, s); m/z
[FAB+] 318.2 (100%, M+); HRMS [FAB+] 318.16535 calculated
316.16535; Anal. (C19H26O2S) C, H.
2-Ethylsulfanylestradiol 26. Sodium borohydride (37 mg)
was added to a room-temperature solution of 2-ethylsulfa-
nylestrone 25 (330 mg, 1 mmol) in THF/isopropyl alcohol (8
mL each). After 14 h stirring, saturated ammonium chloride
(5 mL) was added to quench the reaction and the mixture was
then extracted with ethyl acetate (50 mL). The organic layers
were washed with water (3 × 20 mL) and brine (2 × 20 mL),
dried (MgSO4) and evaporated to give a white solid which was
crystallized from ethanol/hexane to give 2-ethylsulfanylestra-
diol 26 as white crystals (305 mg, 92%) mp 165-166 °C [lit.
166-167 °C22] which showed 1H NMR δ 7.37 (1H, s), 6.72 (1H,
s), 6.56 (1H, s), 3.73 (1H, m), 2.80-86 (2H, m), 2.69 (2H, qd, J
7.4 and 1.5), 1.20-2.46 (16H, m including 1.23 (3H, td, J 7.4
and 1.5) and 0.79 (3H, s); m/z [FAB+] 332.1 (100%, M+), HRMS
[FAB+] 332.18100, calculated 332.18100; Anal. (C20H28O2S) C,
H.
2-Ethylestradiol 31. 2-Ethylestrone 30 (1.19 g, 4 mmol)
was dissolved in THF (5 mL) and isopropyl alcohol (20 mL),
cooled to 0 °C and treated with sodium borohydride (151 mg,
4 mmol) in a portionwise manner. Monitoring of the reaction
by TLC showed no residual starting material remained after
2 h, at which stage ammonium chloride (10 mL) was added to
quench excess borohydride. The reaction was then extracted
with ethyl acetate (50 mL), and the organic layers were washed
with water and brine, dried and evaporated to give the product
as a white powder which was recrystallized from ethyl acetate/
hexane to give 31 as a white crystalline solid (1.03 g, 86%)
mp 168-169 °C [lit. 165-166 °C22] which showed 1H NMR
(DMSO) δ 8.80 (1H, s), 6.90 (1H, s), 6.40 (1H, s), 4.47 (1H, d,
J 4.7), 3.47-3.54 (1H, m), 2.58-2.72 (2H, m), 2.45 (2H, q, J
7.4), 1.00-2.28 (16H, m including 1.05 (3H, t, J 7.4)), and 0.66
(3H, s); Anal. (C20H28O2) C, H.
2-Methoxyestrone-3-O-sulfamate 3. To a solution of
2-methoxyestrone 1 (300 mg, 1 mmol) and 2,6-di-tert-butyl-4-
methyl pyridine (616 mg, 3 mmol) in dichloromethane (16 mL)
was added sulfamoyl chloride (4.2 mL, 0.7M, 2.94 mmol). The
reaction was left to stir overnight, the mixture then poured
onto water (25 mL) and the organic layer was then separated
and washed with water (3 × 10 mL), dried and evaporated.
The desired product 3 was isolated by column chromatography
(3:2 hexane/ethyl acetate) as a white crystalline solid mp 183-
185 °C (321 mg, 84%) [lit. 183-186 °C52] which showed 1H
NMR δ 7.04 (1H, s), 6.91 (1H, s), 5.03 (2H, br), 3.87 (3H, s),
2.82-2.88 (2H, m, 6-CH2), 1.36-2.55 (13H, m), and 0.92 (3H,
s, CH3); m/z [FAB+] 379.2 (100%, M+); HRMS [FAB+]
379.14534, calculated 379.14534; Anal. (C19H25NO5S) C, H, N.
1
which showed H NMR (CD3OD) δ 7.23 (1H,s), 7.12 (1H, s),
4.88 (2H, br s), 2.82-2.90 (2H, m), 1.38-2.54 (16H, m
including 2.41 (3H, s)) and 0.91 (3H, s); m/z [FAB+] 395.2 (M+,
55%), 255.2 (17%); HRMS [FAB-] 394.1 (100%); HRMS [FAB+]
395.12250, calculated 395.12250; Anal. (C19H25NS2O4‚H2O) C,
H, N.
2-Ethylsulfanylestrone-3-O-sulfamate 27. A stirred so-
lution of 2-ethylsulfanylestrone 25 (300 mg, 0.91 mmol) and
2,6-di-tert-butyl-4-methyl pyridine (615 mg, 3 mmol) in dichlo-
romethane (20 mL) was treated with sulfamoyl chloride (4.28
mL, 3 mmol, 0.7 M in toluene) as described for the synthesis
of 3. Column chromatography (chloroform: ethyl acetate 9:1)
gave the desired sulfamate 27 (300 mg, 81%) as a white solid,
a sample of this material was crystallized from ethyl acetate/
hexane to give white crystals mp 179-181 °C, which showed
1H NMR δ 7.34 (1H, s), 7.16 (1H, s), 5.13 (2H, br s), 2.86-2.98
(4H, m), 1.40-2.57 (13H, m), 1.29 (3H, t, J 7.2) and 0.92 (3H,
s); m/z [FAB+] 409.2 (100%, M+), HRMS [FAB+] found
409.13815, calculated 409.13815; Anal. (C20H27NO4S2‚H2O) C,
H, N.
2-Ethylestrone-3-O-sulfamate 32. 2-Ethylestrone 30 (600
mg, 2 mmol) was added to a 0 °C solution of sulfamoyl chloride
(3 mmol) in DMA (1.5 mL). After 16 h stirring, ethyl acetate
(25 mL) and then water (25 mL) were added, and the organic
layer was separated and then washed with water (4 × 25 mL)
and brine (25 mL), dried and evaporated to give a white solid.
Column chromatography (chloroform to 10% acetone in chlo-
roform) gave the desired sulfamate 32 (660 mg, 87%) as a
white crystalline solid mp 161-163 °C which showed 1H NMR
δ 7.18 (1H, s), 7.10 (1H, s), 5.23 (2H, s), 2.85-2.90 (2H, m,),
2.70 (2H, q, J 7.6), 1.32-2.56 (H, m), 1.21 (3H, t, J 7.6) and
0.93 (3H, s); m/z [APCI-] 376.2 (100%, M+ - H); Anal. (C20H27-
NO4S) C, H, N.
2-Bromoestrone-3-O-sulfamate 37. An ice cold solution
of sulfamoyl chloride (1 mmol) in dimethylacetamide (1.5 mL)
was reacted with 2-bromoestrone 36 (150 mg, 0.43 mmol) as
described for the synthesis of 32. The product 37, a white
powder (168 mg, 91%), was recrystallized from acetone/hexane
to give colorless needles mp 225-6 °C which showed 1H NMR
δ 7.52 (1H, s), 7.24 (1H, s), 4.99 (2H, s), 2.84-2.94 (2H, m),
1.36-2.58 (13H, m) and 0.92 (3H, s); m/z (AP-) 428.2 (M+
-
H, 81Br, 30%) and 426.2 (M+ -H, 79Br, 40%); Anal. (C18H22-
BrSO4N) C, H, N.
2-Iodoestrone-3-O-sulfamate 40. An ice cold solution of
sulfamoyl chloride (0.5 mmol) in dimethylacetamide (1.5 mL)
was reacted with 2-iodoestrone 39 (120 mg, 0.30 mmol) as
described for the synthesis of 32. The desired product 40, a
white powder (123 mg, 85%), was recrystallized from acetone/
hexane to give white needles mp 225-226 °C which showed
1H NMR δ 7.72 (1H, s), 7.23 (1H, s), 5.01 (2H, s), 2.86-2.96
(2H, m), 1.36-2.58 (13H, m) and 0.91 (3H, s); m/z (AP-) 474.2
(M+ - H, 70%) and 395.2 (100%); Anal. (C18H22ISO4N) C, H,
N.
2-Ethoxyestrone-3-O-sulfamate 13. A solution of 2-ethoxy-
estrone 12 (395 mg, 1.26 mmol), 2,6-di-tert-butyl-4-methyl
pyridine (820 mg, 4 mmol) in dichloromethane (20 mL) was
2-Methoxyestrone-3-O-(N,N-dimethyl)sulfamate 15. To
a solution of 3 (136 mg, 0.36 mmol) in DMF (5 mL) was added
sodium hydride (32 mg, 0.79 mmol) and then, after 15 min,

5254 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Leese et al.
methyl iodide (67 µL, 1.08 mmol). The reaction was stirred at
room temperature for 16 h then poured onto brine (10 mL)
and extracted with ethyl acetate (10 mL). The organic layer
was then washed with brine (4 × 15 mL), dried and evapo-
rated. The product was isolated by column chromatography
(3:2 hexane/ethyl acetate) as a white solid (86 mg, 59%), a
sample of which was recrystallized from ethyl acetate/hexane
(1H, s), 5.16 (2H, br s), 3.71 (1H, dd, J 8.6 and 7.7), 2.92 (2H,
q, J 7.4), 2.78-2.88 (2H, m), 1.10-2.36 (17H, m including 1.26
(3H, t, J 7.4)) and 0.76 (3H, s); m/z [ES-] 410.2 (M+ - H, 50%)
316.5 (100%); Anal. (C20H29NO4S2‚H2O) C, H, N.
2-Ethylestradiol-3-O-sulfamate 33. A solution of 32 (300
mg, 0.8 mmol) in THF (5 mL) and isopropyl alcohol (20 mL)
was treated with sodium borohydride (60 mg, 1.58 mmol) as
described for the synthesis of 4. The crude product, a white
solid, was recrystallized from ethyl acetate/hexane to give 33
as a fine white powder (220 mg, 73%): mp 193-195 °C which
showed 1H NMR (DMSO) δ 7.94 (2H, s), 7.20 (1H, s), 6.99 (1H,
s), 4.52 (1H, d, J 5.0), 3.45-3.55 (1H, m), 2.72-2.82 (2H, m),
2.63 (2H, q, J 7.4), 1.05-2.40 (H, m including 1.12 (3H, t, J
7.4)) and 0.67 (3H, s); m/z [APCI-] 379.2 (100%, M+ - H); Anal.
(C20H29NO4S) C, H, N.
1
to give 15 as fine white needles mp 218-220 °C. H NMR δ
7.06 (1H, s), 6.88 (1H, s), 3.85 (3H, s), 2.98 (6H, s), 2.78-2.86
(2H, m), 1.23-2.60 (15H, m), and 0.92 (3H, s); m/z [FAB+]
407.2 (100%, M+); HRMS [FAB+] 407.17664, calculated
407.17664; Anal. (C21H29NO5S) C, H, N.
2-Methoxyestrone-3-O-(N-acetyl)sulfamate 16. A solu-
tion of 3 (379 mg, 1 mmol) in pyridine (10 mL) at room
temperature was treated with acetic anhydride (10 mL). After
2 h, the reaction was treated with ice (20 g) then chloroform
(20 mL), causing precipitation of the product in the aqueous
layer. The product 16, a white powder mp 175-177 °C (329
Acknowledgment. This work was supported by
Sterix Ltd. as a member of the IPSEN group. We thank
Dr. G. Sohal and D. Bennetto for technical support and
Drs. L.W.L. Woo and B. LeBlond for useful discussions
during the course of this work.
1
mg, 78%), was collected by filtration. H NMR δ 7.15 (1H, s),
6.84 (1H, s), 3.81 (3H, s), 3.10 (1H, br), 2.78-2.86 (2H, m),
1.32-2.64 (16H, m including 1.96 (3H, s)), and 0.91 (3H, s);
m/z [FAB+] 421.1 (100%, M+); HRMS [FAB+] 421.15591,
calculated 421.15591.
2-Methoxyestradiol-3-O-sulfamate 4. A solution of 3 (1.2
g, 3,16 mmol) in THF (8 mL) and isopropyl alcohol (16 mL)
was added sodium borohydride (239 mg, 6.32 mmol) in a
portionwise manner. The reaction was stirred at room tem-
perature for 3 h then quenched with ammonium chloride
solution. The mixture was then extracted with ethyl acetate
(2 × 50 mL), and the organic layers were washed with water
(50 mL) and brine (50 mL), dried and evaporated. The desired
product was isolated by chromatography (chloroform/ethyl
acetate gradient 8:1 to 4:1) to give the desired product as an
off-white solid. Crystallization from ethyl acetate/hexane gave
4 as colorless crystals (1.02 g, 85%) mp 187-188 °C. 1H NMR
δ 7.80 (2H, s), 6.96 (1H, s), 6.95 (1H, s), 4.52 (1H, s), 3.75 (3H,
s), 3.48-3.56 (1H, m), 2.68-2.76 (2H, m), 1.08-2.40 (13H, m),
and 0.67 (3H, s); m/z [ES-] 380.2 (M+ - H, 100%); Anal.
(C19H27NO5S) C, H, N.
2-Ethoxyestradiol-3-O-sulfamate 14. 2-Ethoxyestrone-3-
O-sulfamate 13 (100 mg, 0.25 mmol) was dissolved in THF (3
mL) and methanol (3 mL), and the solution was cooled to 0 °C
and then treated with sodium borohydride (32 mg, 3.3 eq) in
the same manner as described for the synthesis of 4. Column
chromatography (3:2 hexane/ethyl acetate) gave the desired
product 14 as a white foam (78 mg, 78%). A sample of this
material was recrystallized from chloroform/hexane to give
colorless prisms mp 103-105 °C which showed 1H NMR δ 7.01
(1H, s), 6.92 (1H, s), 5.36 (2H, s), 4.10 (2H, m), 3.70 (1H, dd,
J 8.6 and 8.6), 2.73-2.83 (2H, m), 1.12-2.30 (16H, m including
1.43 (3H, t, J 7.0)), and 0.75 (3H, s); m/z [FAB+] 395.2 (100%,
M+); HRMS [FAB+] 395.17665, calculated 393.17665. Anal.
(C20H29NO5S‚H2O) C, H, N.
Supporting Information Available: Microanalysis and
13C NMR data. This material is available free of charge via
the Internet at http://pubs.acs.org.
References
(1) Fotsis, T.; Zhang, Y. M.; Pepper, M. S.; Adlercreutz, H.; Mon-
tesano, R.; Nawroth, P. P.; Schweigerer, L. The Endogenous
Estrogen Metabolite 2-Methoxyoestradiol Inhibits Angiogenesis
and Suppresses Tumor-Growth. Nature 1994, 368, 237-239.
(2) Martucci, C.; Fishman, J. Direction of estradiol metabolism as
a control of its hormonal action: uterotropic activity of estradiol
metabolites. Endocrinology 1977, 101, 1709-1715.
(3) Liu, Z. J.; Zhu, B. T. Concentration-dependent mitogenic and
antiproliferative actions of 2-methoxyestradiol in estrogen recep-
tor-positive human breast cancer cells. J. Steroid Biochem. Mol.
Biol. 2004, 88, 265-275.
(4) Seegers, J. C.; Aveling, M. L.; Vanaswegen, C. H.; Cross, M.;
Koch, F.; Joubert, W. S. The Cyto-Toxic Effects of Estradiol-17-
Beta, Catecholestradiols and Methoxyestradiols on Dividing
MCF-7 and Hela-Cells. J. Steroid Biochem. 1989, 32, 797-809.
(5) Klauber, N.; Parangi, S.; Flynn, E.; Hamel, E.; D′Amato, R. J.
Inhibition of angiogenesis and breast cancer in mice by the
microtubule inhibitors 2-methoxyestradiol and taxol. Cancer Res.
1997, 57, 81-86.
(6) Attalla, H.; Makela, T. P.; Adlercreutz, H.; Andersson, L. C.
2-Methoxyestradiol arrests cells in mitosis without depolymer-
izing tubulin. Biochem. Biophys. Res. Commun. 1996, 228, 467-
473.
(7) Nakagawa-Yagi, Y.; Ogane, N.; Inoki, Y.; Kitoh, N. The endog-
enous estrogen metabolite 2-methoxyestradiol induces apoptotic
neuronal cell death in vitro. Life Sci. 1996, 58, 1461-1467.
(8) Mukhopadhyay, T. and Roth, J. A. Induction of apoptosis in
human lung cancer cells after wild- type p53 activation by
methoxyestradiol. Oncogene 1997, 14, 379-384.
(9) Schumacher, G.; Kataoka, M.; Roth, J. A.; Mukhopadhyay, T.
Potent antitumor activity of 2-methoxyestradiol in human
pancreatic cancer cell lines. Clin. Cancer Res. 1999, 5, 493-499.
(10) D’Amato, R. J.; Lin, C. M.; Flynn, E.; Folkman, J.; Hamel, E.
2-Methoxyestradiol, an endogenous mammalian metabolite,
inhibits tubulin polymerization by interacting at the colchicine
site. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 3964-3968.
(11) Attalla, H.; Westberg, J. A.; Andersson, L. C.; Adlercreutz, H.;
Makela, T. P. 2-methoxyestradiol-induced phosphorylation of
Bcl-2: Uncoupling from JNK/SAPK activation. Biochem. Bio-
phys. Res. Commun. 1998, 247, 616-619.
2-Methylsulfanyl-3-O-sulfamoylestradiol 21. 2-Methyl-
sulfanylestrone-3-O-sulfamate 20 (150 mg, 0.38 mmol) was
dissolved in THF (4 mL) and methanol (4 mL), and the solution
was cooled to 0 °C and then treated with sodium borohydride
(20 mg, 0.53 mmol). After 15 min, no starting material was
present by TLC, and the reaction was brought to pH 6 by
dropwise addition of glacial acetic acid. Solvent was then
removed by evaporation, and the desired product 21 was
crystallized from ethanol to give colorless crystals mp 179-
180 °C (113 mg, 75%) which showed 1H NMR (CDCl3 with CD3-
OD) δ 7.21 (1H,s), 7.10 (1H, s), 6.05 (2H, br), 3.67 (1H, dd, J
8.5,8.4), 2.76-2.84 (2H, m), 2.42 (3H, s), 1.10-2.32 (13H, m)
and 0.74 (3H, s); m/z [APCI+] 398.4 (80%, M+ + H); Anal.
(C19H27NS2O4) C, H, N.
(12) Jordan, M. A.; Wilson, L. Microtubules and actin filaments:
dynamic targets for cancer chemotherapy. Curr. Opin. Cell. Biol.
1998, 19, 123-130.
(13) Hughes, R. A.; Harris, T.; Altmann, E.; McAllister, D.; Vlahos,
R.; Robertson, A.; Cushman, M.; Wang, Z.; Stewart, A. G.
2-Methoxyestradiol and analogs as novel anti-proliferative
agents: analysis of three-dimensional quantitative structure-
activity relationships for DNA synthesis inhibition and estrogen
receptor binding. Mol. Pharmacol. 2002, 61, 1053-1069.
(14) Stewart, A. G.; Harris, T.; Guida, E.; Vlahos, R.; Koutsoubos,
V.; Hughes, R. A.; Robertson, A. The estradiol metabolite,
2-methoxyestradiol inhibits proliferation of human cultured
airway smooth muscle. Am. J. Respir. Crit. Care 1999, 159,
A531.
2-Ethylsulfanylestradiol-3-O-sulfamate 28. A room-tem-
perature solution of 27 (150 mg, 0.37 mmol) in THF (2 mL)
and methanol (5 mL) was treated with sodium borohydride
(38 mg, 1 mmol) as described for the synthesis of 4. The crude
product, a white solid was purified by crystallization from ethyl
acetate/hexane to give 28 as colorless needles (124 mg, 82%)
1
mp 110-111 °C which showed H NMR δ 7.33 (1H, s), 7.13

A-Ring-Substituted Estrogen-3-O-sulfamates
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16 5255
(15) Mabjeesh, N. J.; Escuin, D.; LaVallee, T. M.; Pribluda, V. S.;
Swartz, G. M.; Johnson, M. S.; Willard, M. T.; Zhong, H.; Simons,
J. W.; Giannakakou, P. 2ME2 inhibits tumor growth and
angiogenesis by disrupting microtubules and dysregulating HIF.
Cancer Cell 2003, 3, 363-375.
(16) Josefsson, E.; Tarkowski, A. Suppression of type II collagen-
induced arthritis by the endogenous estrogen metabolite 2-meth-
oxyestradiol. Arthritis Rheum. 1997, 40, 154-163.
(17) Nishigaki, I.; Sasaguri, Y.; Yagi, K. Antiproliferative effect of
2-methoxyestradiol on cultured smooth-muscle cells from rabbit
aorta. Atherosclerosis 1995, 113, 167-170.
(18) Dubey, R. K.; Gillespie, D. G.; Jackson, E. K.; Keller, P. J. 17â-
estradiol, its metabolites, and progesterone inhibit cardiac
fibroblast growth. Hypertension 1998, 31, 522-528.
(19) Leese, M. P.; Newman, S. P.; Purohit, A.; Reed, M. J.; Potter, B.
V. L. 2-Alkylsulfanyl estrogen derivatives: synthesis of a novel
class of multi-targeted anti-tumor agents. Bioorg. Med. Chem.
Lett. 2004, 14, 3135-3138.
(20) Cushman, M.; Mohanakrishnan, A. K.; Hollingshead, M.; Hamel,
E. The effect of exchanging various substituents at the 2-position
of 2-methoxyestradiol on cytotoxicity in human cancer cell
cultures and inhibition of tubulin polymerization. J. Med. Chem.
2002, 45, 4748-4754.
(21) Cushman, M.; He, H. M.; Katzenellenbogen, J. A.; Varma, R.
K.; Hamel, E.; Lin, C. M.; Ram, S.; Sachdeva, Y. P. Synthesis of
analogues of 2-methoxyestradiol with enhanced inhibitory effects
on tubulin polymerization and cancer cell growth. J. Med. Chem.
1997, 40, 2323-2334.
(22) Cushman, M.; He, H. M.; Katzenellenbogen, J. A.; Lin, C. M.;
Hamel, E. Synthesis, antitubulin and antimitotic activity, and
cytotoxicity of analogues of 2-methoxyestradiol, an endogenous
mammalian metabolite of estradiol that inhibits tubulin polym-
erization by binding to the colchicine binding site. J. Med. Chem.
1995, 38, 2041-2049.
(23) Brueggemeier, R. W.; Bhat, A. S.; Lovely, C. J.; Coughenour, H.
D.; Joomprabutra, S.; Weitzel, D. H.; Vandre, D. D.; Yusuf, F.;
Burak, W. E. 2-Methoxymethylestradiol: a new 2-methoxy
estrogen analog that exhibits antiproliferative activity and alters
tubulin dynamics. J. Steroid Biochem. Mol. Biol. 2001, 78, 145-
156.
(36) Purohit, A.; Hejaz, H. A. M.; Walden, L.; MacCarthy-Morrogh,
L.; Packham, G.; Potter, B. V. L.; Reed, M. J. The effect of
2-methoxyoestrone-3-O-sulphamate on the growth of breast
cancer cells and induced mammary tumours. Int. J. Cancer 2000,
85, 584-589.
(37) MacCarthy-Morrogh, L.; Townsend, P. A.; Purohit, A.; Hejaz,
H. A. M.; Potter, B. V. L.; Reed, M. J.; Packham, G. Differential
effects of estrone and estrone-3-O-sulfamate derivatives on
mitotic arrest, apoptosis, and microtubule assembly in human
breast cancer cells. Cancer Res. 2000, 60, 5441-5450.
(38) Elger, W.; Schwarz, S.; Hedden, A. M.; Reddersen, G.; Schneider,
B. Sulfamates of various estrogens are prodrugs with increased
systemic and reduced hepatic estrogenicity at oral application.
J. Steroid Biochem. Mol. Biol. 1995, 55, 395-403.
(39) Abbate, F.; Winum, J. Y.; Potter, B. V. L.; Casini, A.; Montero,
J. L.; Scozzafava, A.; Supuran, C. T. Carbonic anhydrase
inhibitors: X-ray crystallographic structure of the adduct of
human isozyme II with EMATE, a dual inhibitor of carbonic
anhydrases and steroid sulfatase. Bioorg. Med. Chem. Lett. 2004,
14, 231-234.
(40) Fishman, J. Synthesis of 2-methoxyestrogens. J. Am. Chem. Soc.
1958, 80, 1213-1216.
(41) Rao, P. N. and Burdett Jr, J. E. A novel, 2-step synthesis of
2-methoxyestradiol. Synthesis 1977, 168-169.
(42) Numazawa, M. and Ogura, Y. Efficient synthesis of 2-methoxy-
estrogen and 4-methoxy-estrogen. J. Chem. Soc. Chem. Com-
mun. 1983, 533-534.
(43) Nambara, T.; Akiyama, S.; Honma, S. C-2 Hydroxylation of
2-deuterioestrogen in the rat. Lack of “NIH Shift”. Chem. Pharm.
Bull. 1971, 19, 1727.
(44) LeBras, J.; Rager, M. N.; Besace, Y.; Amouri, H.; Vaissermann,
J. Activation and regioselective ortho-functionalization of the A-
ring of beta-estradiol promoted by ′′Cp*Ir′′: An efficient orga-
nometallic procedure for the synthesis of 2- methoxyestradiol.
Organometallics 1997, 16, 1765-1771.
(45) Nambara, T.; Honma, S.; Akiyama, S. Studies on steroid
conjugates. III. New syntheses of 2-methoxyestrogens. Chem.
Pharm. Bull. 1970, 18, 474-480.
(46) Rao, P. N.; Cessac, J. W. A new, practical synthesis of 2-meth-
oxyestradiols. Steroids 2002, 67, 1065-1070.
(47) He, H. M.; Cushman, M. A versatile synthesis of 2-methox-
yestradiol, an endogenous metabolite of estradiol which inhibits
tubulin polymerization by binding to the colchicine binding site.
Bioorg. Med. Chem. Lett. 1994, 4, 1725-1728.
(48) Pert, D. J.; Ridley, D. D. Formylation of estrogens. Aust. J. Chem.
1989, 42, 405-419.
(49) Pert, D. J.; Ridley, D. D. An alternative route to 2-bromoestra-
diols and 2-iodoestradiols from estradiol. Aust. J. Chem. 1987,
40, 303-309.
(50) Appel, R.; Berger, G. Uber das hydrazidosulfamid (On hydra-
zidosulfamide). Chem. Ber. 1958, 91, 1339-1341.
(24) Edsall, A. B.; Mohanakrishnan, A. K.; Yang, D. L.; Fanwick, P.
E.; Hamel, E.; Hanson, A. D.; Agoston, G. E.; Cushman, M.
Effects of altering the electronics of 2-methoxyestradiol on cell
proliferation, on cytotoxicity in human cancer cell cultures, and
on tubulin polymerization. J. Med. Chem. 2004, 47, 5126-5139.
(25) Lovely, C. J.; Gilbert, N. E.; Liberto, M. M.; Sharp, D. W.; Lin,
Y. C.; Brueggemeier, R. W. 2-(Hydroxyalkyl)estradiols: Synthe-
sis and biological evaluation. J. Med. Chem. 1996, 39, 1917-
1923.
(26) Lovely, C. J. and Brueggemeier, R. W. Synthesis of 2-substituted
hydroxyalkyl and aminoalkyl estradiols. Tetrahedron Lett. 1994,
35, 8735-8738.
(51) Okada, M.; Iwashita, S.; Koizumi, N. Efficient general method
for sulfamoylation of a hydroxyl group. Tetrahedron Lett. 2000,
41, 7047-7051.
(27) Verdier-Pinard, P.; Wang, Z.; Mohanakrishnan, A. K.; Cushman,
M.; Hamel, E. A steroid derivative with paclitaxel-like effects
on tubulin polymerization. Mol. Pharmacol. 2000, 57, 568-575.
(28) Wang, Z.; Yang, D.; Mohanakrishnan, A. K.; Fanwick, P. E.;
Nampoothiri, P.; Hamel, E.; Cushman, M. Synthesis of B-ring
homologated estradiol analogues that modulate tubulin polym-
erization and microtubule stability. J. Med. Chem. 2000, 43,
2419-2429.
(29) Rao, P. N.; Cessac, J. W.; Tinley, T. L.; Mooberry, S. L. Synthesis
and anti-mitotic activity of novel 2-methoxyestradiol analogs.
Steroids 2002, 67, 1079-1089.
(30) Tinley, T. L.; Leal, R. M.; Randall-Hlubek, D. A.; Cessac, J. W.;
Wilkens, L. R.; Rao, P. N.; Mooberry, S. L. Novel 2-methoxy-
estradiol analogues with antitumor activity. Cancer Res. 2003,
63, 1538-1549.
(31) Dahut, W. L.; Lakhani, N. J.; Kohn, E. C.; Gulley, J.; Arlen, P.
M.; Raggio, M.; Wright, J.; Hussain, M. M.; Kotz, H.; Figg, W.
D. A phase I study of 2-methoxyestradiol (2ME2) in patients with
solid tumors. Proc. Am. Soc. Clin. Onc. 2003, 22, 208.
(32) Reed, M. J.; Purohit, A.; Newman, S. P.; Potter, B. V. L. Steroid
sulfatase: Molecular biology, regulation and inhibition. Endocr.
Rev. 2005, 26, 171-202.
(33) Purohit, A.; Williams, G. J.; Roberts, C. J.; Potter, B. V. L.; Reed,
M. J. In Vivo inhibition of estrone sulfatase and dehydroepi-
androsterone sulfatase by Estrone-3-O-Sulfamate. Int. J. Cancer
1995, 63, 106-111.
(34) Purohit, A.; Woo, L. W. L.; Chander, S. K.; Newman, S. P.; Ireson,
C.; Ho, Y.; Grasso, A.; Leese, M. P.; Potter, B. V. L.; Reed, M. J.
Steroid sulphatase inhibitors for breast cancer therapy. J.
Steroid Biochem. Mol. Biol. 2003, 86, 423-432.
(35) Purohit, A.; Vernon, K. A.; Hummelinck, A. E. W.; Woo, L. W.
L.; Hejaz, H. A. M.; Potter, B. V. L.; Reed, M. J. The development
of A-ring modified analogues of oestrone-3-O-sulphamate as
potent steroid sulphatase inhibitors with reduced oestrogenicity.
J. Steroid Biochem. Mol. Biol. 1998, 64, 269-275.
(52) Schwarz, S.; Thieme, I.; Richter, M.; Undeutsch, B.; Henkel, H.;
Elger, W. Synthesis of estrogen sulfamates: Compounds with a
novel endocrinological profile. Steroids 1996, 61, 710-717.
(53) Reed, J. E.; Woo, L. W. L.; Robinson, J. J.; Leblond, B.; Leese,
M. P.; Purohit, A.; Reed, M. J.; Potter, B. V. L. 2-Difluoro-
methyloestrone 3-O-sulphamate, a highly potent steroid sul-
phatase inhibitor. Biochem. Biophys. Res. Commun. 2004, 317,
169-175.
(54) Vaidyanathan, G.; Shankar, S.; Zalutsky, M. R. Synthesis of ring-
and side-chain-substituted m-iodobenzylguanidine analogues.
Bioconjugate Chem. 2001, 12, 786-797.
(55) Fuchigami, T.; Yamamoto, K.; Nakagawa, Y. Electrolytic reac-
tions of fluoro organic-compounds 7. Anodic methoxylation and
acetoxylation of 2,2,2-trifluoroethyl sulfides - Preparation of
highly useful trifluoromethylated building blocks. J. Org. Chem.
1991, 56, 137-142.
(56) Howarth, N. M.; Purohit, A.; Reed, M. J.; Potter, B. V. L. Estrone
sulfamates - Potent inhibitors of estrone sulfatase with thera-
peutic potential. J. Med. Chem. 1994, 37, 219-221.
(57) Woo, L. W. L.; Purohit, A.; Reed, M. J.; Potter, B. V. L. Oestrone
3-O-(N-acetyl)sulphamate, a potential molecular probe of the
active site of oestrone sulphatase. Bioorg. Med. Chem. Lett. 1997,
7, 3075-3080.
(58) Boyd, M. R.; Paull, K. D. Some practical consideration and
applications of the National Cancer Institute in vitro anticancer
drug discovery screen. Drug Dev. Res. 1995, 34, 91-109.
(59) see http://dtp.nci.nih.gov/aa-resources/aa_index.html for details.
(60) Newman, S. P.; Leese, M. P.; Purohit, A.; James, D. R.; Rennie,
C. E.; Potter, B. V. L.; Reed, M. J. Inhibition of in vitro
angiogenesis by 2-methoxy- and 2-ethyl-estrogen sulfamates. Int.
J. Cancer 2004, 109, 533-540.
(61) Hollingshead, M.; Alley, M. C.; Camalier, R. F.; Abbott, B. J.;
Mayo, J. G.; Malspeis, L.; Grever, M. R. In vivo cultivation of
tumor cells in hollow fibres. Life Sci. 1995, 57, 131-141.

5256 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 16
Leese et al.
(62) Woo, L. W. L.; Lightowler, M.; Purohit, A.; Reed, M. J.; Potter,
B. V. L. Heteroatom-substituted analogues of the active-site
directed inhibitor estra-1,3,5(10)-trien-17-one-3-sulphamate in-
hibit estrone sulphatase by a different mechanism. J. Steroid
Biochem. Mol. Biol. 1996, 57, 79-88.
(63) Purohit, A.; Williams, G. J.; Howarth, N. M.; Potter, B. V. L.;
Reed, M. J. Inactivation of steroid sulfatase by an active site
directed inhibitor, estrone-3-O-sulfamate. Biochemistry 1995, 34,
11508-11514.
(64) Rao, P. N.; Wang, Z. New Synthesis of ∆(6)-estrogens. Steroids
1997, 62, 487-490.
(65) Page, P. C. B.; Hussain, F.; Maggs, J. L.; Morgan, P.; Park, B.
K. Efficient regioselective A-ring functionalisation of estrogens.
Tetrahedron 1990, 46, 2059-2068.
JM050066A