3,17-Disubstituted 2-alkylestra-1,3,5(10)-trien-3-ol derivatives: Synthesis, in vitro and in vivo anticancer activity

Article abstract of DOI:10.1021/jm070405v

Estradiol-3,17-O,O-bis-sulfamates inhibit steroid sulfatase (STS), carbonic anhydrase (CA), and, when substituted at C-2, cancer cell proliferation and angiogenesis. C-2 Substitution and 17-sulfamate replacement of the estradiol-3,17-O,O-bis-sulfamates we

Full text of DOI:10.1021/jm070405v

J. Med. Chem. 2007, 50, 4431-4443
4431
3,17-Disubstituted 2-Alkylestra-1,3,5(10)-trien-3-ol Derivatives: Synthesis, In Vitro and In Vivo
Anticancer Activity
Christian Bubert,† Mathew P. Leese,† Mary F. Mahon,‡ Eric Ferrandis,§ Sandra Regis-Lydi,§ Philip G. Kasprzyk,|
Simon P. Newman, Yaik T. Ho, 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, UK, Endocrinology and
Metabolic Medicine and Sterix Ltd., Faculty of Medicine, Imperial College, St. Mary’s Hospital, London W2 1NY, UK, Department of
Chemistry, UniVersity of Bath, BA2 7AY, UK, IPSEN System Biology, 91966 Les Ulis, France, Experimental Oncology Research IPSEN/
Biomeasure, Inc., 27 Maple Street Milford, Massachusetts 01757
ReceiVed April 5, 2007
Estradiol-3,17-O,O-bis-sulfamates inhibit steroid sulfatase (STS), carbonic anhydrase (CA), and, when
substituted at C-2, cancer cell proliferation and angiogenesis. C-2 Substitution and 17-sulfamate replacement
of the estradiol-3,17-O,O-bis-sulfamates were explored with efficient and practical syntheses developed.
Evaluation against human cancer cell lines revealed the 2-methyl derivative 27 (DU145 GI50 ) 0.38 µM)
as the most active novel bis-sulfamate, while 2-ethyl-17-carbamate derivative 52 (GI50 ) 0.22 µM) proved
most active of its series (cf. 2-ethylestradiol-3,17-O,O-bis-sulfamate 4 GI50 ) 0.21 µM). Larger C-2
substituents were deleterious to activity. 2-Methoxy-17-carbamate 50 was studied by X-ray crystallography
and was surprisingly 13-fold weaker as an STS inhibitor compared to parent bis-sulfamate 3. The potential
of 4 as an orally dosed anti-tumor agent is confirmed using breast and prostate cancer xenografts. In the
MDA-MB-231 model, dramatic reduction in tumor growth or regression was observed, with effects sustained
after cessation of treatment. 3-O-Sulfamoylated 2-alkylestradiol-17-O-carbamates and sulfamates have
considerable potential as anticancer agents.
Introduction
Sulfamoylated analogues of 2-methoxyestradiol (2-MeOE2,
1, Figure 1), such as 2-methoxyestradiol-3,17-O,O-bis-sulfamate
(2-MeOE2bisMATE,a 3) and 2-ethylestradiol-3,17-O,O-bis-
sulfamate (2-EtE2bisMATE, 4), have previously been shown
to be potent inhibitors of cancer cell growth1-5 and of steroid
sulfatase (STS, IC50’s of 3 and 4 38 nM and 1 µM, respec-
tively),2,5 a clinical target for the treatment of hormone-
dependent breast cancer.6,7 The inhibition of STS by phenolic
Figure 1. Structures of 2-methoxyestradiol (1), 2-ethylestradiol (2),
2-methoxyestradiol-3,17-O,O-bis-sulfamate (3), 2-ethylestradiol-3,17-
O,O-bis-sulfamate (4), and 2-methylsulfanylestradiol-3,17-O,O-bis-
sulfamate (5).
sulfamates is irreversible and is thought to result from either
the transfer of a sulfamate group to a residue in the active site
of the enzyme or a nucleophilic attack of the sulfamate group
the sulfamate to the zinc atom in the active site of CAII.5,12-14
The uptake of 3 by red blood cells, as a result of its reversible
interaction with CAII (IC50 ) 379 nM),15 and the rapid, almost
complete inhibition of STS combined with the resistance to
deactivating conjugation and metabolism all contribute to the
observed high oral bioavailability (85%) of 2-MeOE2bisMATE
(3), which is much higher than that observed for the parent
2-MeOE2 (1).16,17
As a result of its inhibition of both cancer cell proliferation
and angiogenesis, 2-MeOE2 (1) has generated significant interest
as a potential anticancer agent, and a number of studies have
highlighted possible mechanisms for its cytotoxicity.18,19 The
antiproliferative effects of 1 are independent of the estrogen
receptor status of the cancer cell line and appear to be a result
of its ability to disrupt tubulin polymerization by binding to
the colchicine binding site on tubulin.20 Several cellular
responses, such as Bcl-2 phosphorylation, caspase-3 activation,
down-regulation of hypoxia inducible factor-1, increased ex-
pression of the tumor suppressor protein p53, and G(2)/M cycle
arrest have been reported.20-29 Various analogues of 2MeOE2
(1) have been synthesized and tested for biological activities
and several, such as 2-ethoxyestradiol, 2-((E)-1′-propenyl)-
estradiol, and 2-methoxy-14,15-dehydroestradiol, for example,
on the conserved formylglycine residue of the active site,
generating a dead-end product.8 Metabolic processes similar to
those that metabolize estradiol and other steroidal hormones,
such as conjugation of the 3- or 17-hydroxyl groups to form
glucuronide or sulfate derivatives, or oxidation of the 17-
hydroxyl group by 17â-hydroxysteroid dehydrogenase type II,
rapidly generate less active metabolites of 2-MeOE2 (1).9-11
The sulfamate groups at the 3- and 17-positions are not however
metabolized in the same way, while inhibition of STS prevents
STS-mediated desulfamoylation of the 3-O-sulfamate groups
of 3 and 4. Estrogen 3-O-sulfamates have also been reported to
inhibit carbonic anhydrase II (CAII), which is highly expressed
in red blood cells, by coordination of the monoanionic form of
* To whom correspondence should be addressed. Tel. +44 1225 386639.
Fax +44 1225 386114. E-mail: B.V.L.Potter@bath.ac.uk.
† Department of Pharmacy and Pharmacology & Sterix Ltd., University
of Bath.
‡ Department of Chemistry, University of Bath.
§ Department of System Biology, IPSEN.
| Experimental Oncology Research IPSEN/Biomeasure, Inc.
Endocrinology and Metabolic Medicine and Sterix Ltd., Imperial
College.
a Abbreviations: E2bisMATE, estradiol-3,17-O,O-bis-sulfamate; 2-
MeOE2bisMATE,
2-methoxyestradiol-3,17-O,O-bis-sulfamate;
2-
EtE2bisMATE, 2-ethylestradiol-3,17-O,O-bis-sulfamate.
10.1021/jm070405v CCC: $37.00 © 2007 American Chemical Society
Published on Web 08/15/2007

4432 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Bubert et al.
were found to be more potent than 2-MeOE2 (1) as in vitro
from estrone (6). 2-Methylestradiol (8) was accessed in 47%
yield by reaction of 6 with N,N,N′,N′-tetramethyldiami-
nomethane and paraformaldehyde in a Mannich-type reaction
as described by Patton to give the amine 7,37 which was then
quaternized with iodomethane and the product reduced with
NaBH4 to give 8. Friedel-Crafts alkylation of 6 using tert-
butanol and BF3‚Et2O in chloroform gave 2-tert-butylestrone 9
in high yield (93%),38 which was then reduced with NaBH4 to
give 2-tert-butyl estradiol (10) in an overall yield of 79%.39
Various methods for the syntheses of the 2-n-alkylestradiols 2,
23, and 24 have been reported in the literature, including the
hydrogenation of the corresponding 2-alkenylestradiols that are
available by Wittig reactions of 2-formylestradiol,30 by pal-
ladium-catalyzed cross-couplings of 2-iodoestradiol,40 by a
Claisen rearrangement of estrone allyl ether41 or by alkylation
of ortho-lithiated MOM-protected derivatives of 6.42 Though
all of these methods give good results, we decided to investigate
a different approach that appeared amenable to a multigram
scale, inspired by the synthesis of 2-ethyl-8R-estradiol described
by Gonza´lez et al.43 Friedel-Crafts acylation of the protected
estradiol derivative 1144 with aluminum chloride and acetyl
chloride, propionic anhydride, or butyric anhydride afforded the
2-acylated products 12, 13, and 14 in good yields. These were
then demethylated using aluminum chloride and trimethylam-
monium chloride in DCM to give the desired phenols 15, 16,
and 17 (Scheme 1).45 Catalytic hydrogenation with palladium
on charcoal afforded the 17-O-acetyl protected compounds 18,
19, and 20, which were deprotected with sodium hydroxide in
methanol/water to give the 2-alkylestradiols 2, 23, and 24. In
order to obtain 2-isopropylestradiol (21), the 2-acetyl compound
15 was reacted with 6 equivalents of methylmagnesium bromide
to give the trihydroxy compound 22. The benzylic hydroxyl
group of 22 was removed by catalytic hydrogenation to afford
the target 2-isopropyl estradiol (21) in good yield, as described
by Pert and Ridley.46
inhibitors of tubulin polymerization and cell proliferation.30-34
We previously demonstrated that 2-substituted estrogen
derivatives that are sulfamoylated in the 3-position and in both
the 3- and 17-positions (E2bisMATEs), such as 3 and 4, inhibit
cell cycle progression and cell proliferation of an array of normal
and drug-resistant breast, prostate, and ovarian cancer cells in
vitro.1,2,4 The bis-sulfamates 3 and 4 exhibited mean GI50’s of
87 and 18 nM, respectively, across the NCI 60 cell line screening
panel (cf. 2-MeOE2, 1.3 µM).5,30 The structure-activity relation-
ship of 2-substituted estrogen 3-O-sulfamates and 2-substituted
estrogen 3,17-O,O-bis-sulfamates has been investigated. The
highest antiproliferative activities were observed for 3,17-O,O-
bis-sulfamoylated estradiol derivatives with a methoxy (3), ethyl
(4), or methylsulfanyl (5) group at the 2-position. The corre-
sponding 3-O-mono-sulfamoylated estradiol derivatives were
generally slightly less active in vitro than the bis-sulfamoylated
compounds, though 6.5-150-fold more potent than their parent
estradiol derivatives against the proliferation of MCF7 cells,
while the 17-O-mono-sulfamoylated estradiol derivatives proved
inactive. Analogous results were obtained across the NCI 60-
cell line panel. Alkylation or acylation of the 3-, 17-, or 3,17-
O-sulfamate-nitrogen(s) was not tolerated and caused a dramatic
reduction in antiproliferative activity. These results indicate that
2-substituted estrogen 3-O-sulfamates, although exhibiting
mechanistic similarities with the 2-substituted estradiol deriva-
tives, are not prodrugs of the parent steroids and represent a
novel class of anticancer agent. In addition, the antiangiogenic
effects of 3 and 4 have been shown to be superior to those of
2-MeOE2 (1) with lower concentrations required to inhibit the
growth of human vascular endothelial cells in vitro.35 In vivo
studies have also demonstrated the advantages of the bis-
sulfamoylated compounds over the corresponding 2-substituted
estradiol-3-O-mono-sulfamates and 2-substituted estradiol de-
rivatives, presumably due to a combination of their enhanced
antiproliferative activity and superior ADME properties (see
above).
2MeOE2 (1) is a more potent inhibitor in vitro of MCF7 cell
proliferation than 2-ethylestradiol (2) (respective GI50’s are 2.35
and 10.5 µM). In contrast, in vitro results for the bis-sulfamate
series show that the 2-MeO compound 3 (GI50 ) 0.25 µM),
though nearly 10-fold more active than 1, is less potent than its
2-ethyl analogue 4, which has a GI50 of 0.07 µM and is, in
turn, 100 times more potent than the parent 2-ethylestradiol (2).
In the present study, we set out to determine the effects of
different small alkyl substitutions at the 2-position on antipro-
liferative activity and the effect of bioisosteric replacement of
the 17-O-sulfamate group with a carbamate group. The syntheses
of a series of novel 2-alkylated estradiol bis-sulfamates and a
series of 2-alkylated 3-O-sulfamoylated estradiol 17-carbamates
are described. The new compounds were then evaluated for in
vitro antiproliferative activity, with the most active compound
of the series being further evaluated in vivo in tumor xenograft
models.
Estradiol (25), the series of 2-alkylestradiols 2, 8, 10, 21,
23, 24 and acetates 18, 19, and 20 were sulfamoylated under
standard conditions with sulfamoyl chloride in dimethyl aceta-
mide to give the mono-sulfamate derivatives 32-34 and the
bis-sulfamates 4, 26-31 in good to excellent yields (Scheme
2).47
As part of a continuing SAR program, it was of interest to
explore the replacement of the 17-sulfamate group. We decided
to explore the use of a carbamate as a potential bioisostere for
the sulfamate group, as it seemed likely that a carbamate could
exploit some or all of the key interactions with the binding site
obviously well satisfied by sulfamate. Since one of the reasons
for the success of the bis-sulfamate series is blockade of
metabolic conjugation16 and the sulfamate group per se is
unlikely to be the only, or perhaps optimal, motif at the 17-
position that could imbue good activity in this class of
compound, we undertook to design routes to mixed sulfamate-
carbamate esters. The syntheses of the comprehensive series of
17â-carbamoylated estradiol derivatives 49-56 are thus il-
lustrated in Scheme 3. The sulfamoylated estradiol and 2-meth-
oxyestradiol mono-carbamates 49 and 50 were synthesized in
four steps from the appropriate benzyl ethers 35 and 36, which
were first reacted with trichloroacetyl isocyanate to give the
N-trichloroacetyl carbamates 37 and 38.48 The trichloroacetyl
groups of 37 and 38 were then selectively hydrolyzed with K2-
CO3 in MeOH/THF/H2O to yield the 3-O-benzyl-17â-carbamate
derivatives 39 and 40, respectively, which were then hydroge-
nated to give phenols 41 and 42. Sulfamoylation of 41 and 42
was then carried out as described above to give 49 and 50. A
Chemistry
The syntheses of some 2-substituted estrogen 3,17-O,O-bis-
sulfamates (E2bisMATEs) by sulfamoylation of the correspond-
ing 2-substituted estradiol derivatives have been previously
described.5,13,36 Thus, in order to construct a sensible series of
analogs for SAR purposes, a number of different 2-alkyl
estradiol derivatives were prepared and converted into a series
of 3- and 17-substituted derivatives (Scheme 1). 2-Methylestra-
diol 8 and 2-tert-butylestradiol 10 were obtained in two steps

Disubstituted 2-Alkylestra-1,3,5(10)-trien-3-ols
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4433
Scheme 1. Syntheses of 2-Alkylestradiol Derivativesa
a Reagents and conditions: (a) N,N,N′,N′-tetramethyldiaminomethane, (CHO)n, PhMe, EtOH, reflux; (b) (1) MeI, Et2O; (2) NaBH4, EtOH; (c) t-BuOH,
BF3‚Et2O, CHCl3; (d) NaBH4, MeOH, 0 °C; (e) AcCl or (RC(O))2O, AlCl3, DCM, 0 °C; (f) AlCl3, NMe3‚HCl, DCM, reflux; (g) 10% Pd/C, H2, MeOH,
THF; (h) 6 equiv MeMgCl, THF, 15; (i) NaOH, MeOH, H2O.
Scheme 2. Synthesis of 2-Substituted Estradiol 3-O-Sulfamates
and 3,17-O,O-Bis-sulfamatesa
chiral centers of the estra-1,3,5(10)-triene core is maintained
through the synthesis and also demonstrates the intramolecular
hydrogen bonding between the sulfamate and carbamate NH2-
groups, with the respective carbamate and sulfamate oxygens
in the crystallographically adjacent molecules of the unit cell
(Figure 3).
Results and Discussion
The sulfamates and carbamates synthesized were evaluated
for their ability to inhibit in vitro the proliferation of DU145
prostate cancer cells, MDA-MB-231 (ER-) human breast cancer
cells, and, in some cases, MCF7 (ER+) breast cancer cells
(Table 1). 2MeOE2 (1); the parent 2-alkyl estradiol derivatives
2, 8, 10, 21, 23, 24; and the bis-sulfamates 3 and 4 were tested
side by side for comparison.
While in the 2-substituted estradiol series 2MeOE2 (1) is
the most potent compound, in the bis-sulfamate series,
2-EtE2bisMATE (4) proved to be the most potent inhibitor of
cancer cell proliferation in vitro in the tested cell lines. Of the
novel bis-sulfamates 27-31, only the 2-methyl substituted
compound 27 displayed high in vitro activity with a GI50 of
0.25 µM in MDA-MB-231 cells, which is comparable to that
obtained for 2-MeOE2bisMATE 3 (0.28 µM). Even a small
increase in substituent size at the 2-position (e.g. 2-n-propyl
28, 2-isopropyl 29) was not well tolerated and resulted in a
significant decrease in antiproliferative activity. The n-butyl and
tert-butyl derivatives 30 and 31 showed only a very modest
activity. An inspection of the 2-alkyl bisMATEs evaluated herein
clearly shows that 2-ethyl substitution is optimal for antipro-
a Reagents and conditions: (a) ClSO2NH2, DMA, 0 °C.
second, more efficient approach to 2-alkylestradiol 17-carbam-
ates was also developed, wherein reaction of the parent estradiol
derivatives 2, 8, 10, 21, 23, and 24 with excess trichloroacetyl
isocyanate was followed by selective hydrolysis of the trichlo-
roacetyl and phenolic carbamate groups in a one-pot procedure
to give carbamates 43-48. Sulfamoylation of phenols 43-48
gave the target sulfamates 51-56 in moderate to good yield.
An X-ray crystal structure of 50 was obtained and is presented
in Figure 2. This structure confirms that the integrity of the

4434 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Bubert et al.
Scheme 3. Syntheses of 17-O-Carbamoylated Estradiol Derivatives and Their Sulfamatesa
a Reagents and conditions: (a) Cl3C(O)NCO, THF; (b) K2CO3, MeOH, THF, H2O; (c) 10% Pd/C, H2, MeOH, THF; (d) (1) Cl3C(O)NCO, THF, 0 °C;
(2) K2CO3, MeOH, THF, H2O; (e) ClSO2NH2, DMA, 0 °C.
Figure 2. X-ray crystal structure of one of the molecules in the asymmetric unit of 50. Ellipsoids are represented at 30% probability. Carbon-
bound hydrogen atoms have been omitted for clarity.
Figure 3. Intermolecular hydrogen bonding viewed perpendicular to the bc plane in the gross structure of 50.
liferative activity and that the activity is proportional to the steric
size of the 2-alkyl group.
emphasizing the poor tolerance of alkyl groups larger than ethyl
for antiproliferative activity in the sulfamate series.
The 2-alkyl-3-hydroxy-17â-acetoxyestra-1,3,5(10)-trienes 18-
20 proved to be devoid of antiproliferative activity (GI50 > 100
µM), while compound 32, the sulfamoylated analogue of 18,
displays fairly good activity with a GI50 of 1.6 µM in the DU145
cell line (cf. 2-MeOE2, 1.22 µM). The 2-n-propyl and 2-n-butyl
analogues 33 and 34 were only modestly active, once again
The findings in the 17â-carbamate series are similar to those
obtained in the bis-sulfamate and 17â-acetate series. The
nonsulfamoylated 17â-carbamates 41-48 displayed very little
or no activity, while most of the corresponding sulfamates, with
the exception of the 2-unsubstituted compound 49, displayed
enhanced antiproliferative activity. The 2-ethyl derivative 52

Disubstituted 2-Alkylestra-1,3,5(10)-trien-3-ols
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4435
Table 1. Antiproliferative Activities of Estradiol Derivatives against
Table 2. Inhibition of Placental STS by 3-O-Sulfamoylated Estradiol
DU145, MDA MB-231, and MCF7 Cancer Cell Lines
Derivatives
STS
IC50 (nM)
GI50a (µM)
compd
R1
R2
38b
18b
40
compd R1
R2
R3
DU145 MDA-MB-23 1 MCF7
3
26
49
50
MeO
H
H
MeO
SO2NH2
SO2NH2
CONH2
CONH2
1
2
MeO
Et
H
H
H
H
1.22
10.3
0.34
0.21
0.94
8.0
0.28
0.21
2.35c
10.5c
0.25c
0.07c
n/d
520
3
MeO SO2NH2 SO2NH2
a IC50 figures are the mean values obtained from experiments performed
4
Et
SO2NH2 SO2NH2
in triplicate, SEM < (7%. b Taken from ref 5.
8
Me
t-Bu
i-Pr
n-Pr
n-Bu
H
H
H
H
H
H
H
H
H
H
H
51
n/d
10
21
23
24
26
27
28
29
30
31
32
33
34
49
50
51
52
53
54
55
56
24
23
n/d
n/d
n/d
n/d
15
16.6
n/d
>100
>100
18.3
0.25
6.4
n/d
10.7
n/d
n/d
n/d
SO2NH2 SO2NH2
SO2NH2 SO2NH2
11.5
0.38
>10c
Me
n/d
n/d
n/d
n/d
n/d
n/d
n/d
n/d
45
n-Pr SO2NH2 SO2NH2
i-Pr SO2NH2 SO2NH2
n-Bu SO2NH2 SO2NH2
t-Bu SO2NH2 SO2NH2 >10
Et
n-Pr SO2NH2 Ac
n-Bu SO2NH2 Ac
H
MeO SO2NH2 CONH2
Me
Et
3.4
1.8
9
SO2NH2 Ac
1.6
61.3
18.4
20.4
21.6
>100
SO2NH2 CONH2 >100
1.34
1.9
0.22
1.15
4.7
0.39
3.5
n/d
0.64
SO2NH2 CONH2
SO2NH2 CONH2
n-Pr SO2NH2 CONH2
i-Pr SO2NH2 CONH2
n-Bu SO2NH2 CONH2
t-Bu SO2NH2 CONH2
21
2.4
15
13
14.5
3.4
>100
17
n/d
n/d
n/d
n/d
Figure 4. Comparison effect of 2EtE2bisMATE (4) using various po
dosing schedules on the MDA-MB-231 xenograft model in female
athymic nude mice. Dosing schedules: start of treatment on day 10;
qd (daily) end of treatment on day 38; MWF (Monday, Wednesday,
Friday) end of treatment on day 36; qwk (once a week) end of treatment
on day 32.
a GI50 figures are the mean values obtained from experiments performed
in triplicate, SEM ) (10%. b n/d ) not determined. c Taken from refs 5,
42.
was the most potent compound tested in this series, with GI50’s
of 0.22 and 0.39 µM in the DU145 and MDA-MB-231 cell
lines, repectively. These results compare well with those
obtained for 3 and 4. However, while 4 displayed a GI50 of just
0.07 µM in the MCF7 cell line, a near 10-fold increase was
observed for 52. The carbamates with a methoxy, methyl, and
isopropyl substituent in 2-position (50, 51, and 54) also showed
good inhibitory effects against cancer cell growth but signifi-
cantly less than 52, while the 2-n-propyl, 2-n-butyl, and 2-tert-
butyl substituted compounds 53, 55, and 56 displayed highly
reduced activity in these assays. It would thus appear that
replacement of the 17-O-sulfamate with a carbamate group is
valid in the generation of 2-substituted estradiol-3-O-sulfamates
with antiproliferative activity, and although such derivatives do
not show uniformly high activity, their SAR mirrors that found
in the estradiol-bis-sulfamate series.
To investigate the effect of carbamoylation of the 17-hydroxy
group on the steroid sulfatase (STS) inhibitory activity of the
estradiol-3-O-sulfamate compounds 49 and 50 were assayed
using the radiometric assay developed by Purohit et al.,49
wherein the STS-mediated conversion of tritiated E2-sulfate to
E2 by placental microsomes is determined (Table 2). The bis-
sulfamates 3 and 26 have been found in previous studies to be
very potent inhibitors of STS, with IC50’s of 38 and 18 nM,
respectively. Replacement of the 17-O-sulfamate group of 26
with a carbamate group results in a slight decrease in sulfatase
inhibitory activity. The IC50 for the carbamate 49 was deter-
mined as 40 nM, a 2-fold increase compared to the correspond-
ing estradiol bis-sulfamate 26. The IC50 for the 2-methoxy
carbamate 50 is, however, surprisingly more than 13-times
higher than that observed for the corresponding parent bis-
sulfamate 3. We cannot rationalize this relative to the results
for 26 vs 49 beyond supposing that differential binding of the
carbamate motif presumably distorts binding of the group at
the distal end relative to a sulfamate. The activity in the 2-alkyl
series was not explored, as it had previously been determined
that 2-EtE2bisMATE (4) is substantially less potent than
2-MeOE2bisMATE (3) as an irreversible inhibitor of STS.5
In Vivo Studies. 2-EtE2bisMATE (4) was selected for in
vivo evaluation of its ability to inhibit tumor growth. These
assessments were performed using xenografts derived from
MDA-MB-231 (ER-) human breast cancer cells in female
athymic nude mice and xenografts derived from DU145 human
prostate cancer cells in male athymic nude mice using various
po dosing schedules. The results for the MDA-MB-231 xe-
nograft model are presented in Figure 4. All three different
dosing schedules [i, 60 mg/kg, qd (once a day), 28d; ii, 90 mg/
kg, MWF (Monday, Wednesday, Friday) × 4; iii, 200 mg/kg,
qwk (once a week) × 4] using an unoptimized THF/PG vehicle
system caused a dramatic reduction in tumor growth (qwk
cohort) or tumor regression over the course of the dosing (qd
and MWF cohorts); furthermore, these effects were sustained
after the cessation of the treatment on day 38. No treatment-
related deaths or significant weight loss were observed in any
of the three cohorts.
The high efficacy demonstrated by 4 in this study underlines
the potential of this agent as an orally dosed antitumor agent.

4436 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Bubert et al.
all three series, although the 2-methyl bis-sulfamate (27) also
displayed promising activity.
In addition to these findings from in vitro assays, the potential
of 2-EtE2bisMATE (4) as an antitumor agent was confirmed
in MDA-MB-231 and DU145 xenograft models. In the MDA-
MB-231 xenografts significant tumor regression was observed
on p.o. dosing in two out of three schedules, while the remaining
schedule still achieved significant growth inhibition. In the
highly aggressive DU145 xenograft 65% growth inhibition
was observed in the higher (40 mg/kg) dosed cohort with no
signs of toxicity. Bis-substituted 2-alkylestradiol sulfamates
show considerable potential as anticancer agents. Further
studies directed toward clinical development, particularly of
2-EtE2bisMATE (4), are in progress.
Experimental Section
Figure 5. Comparison effect of various doses 2-EtE2bisMATE (4)
using a qd × 28 schedule on the DU145 xenograft model in male
athymic nude mice.
Biology. In Vitro Studies: Cell Lines. DU145 (brain metastasis
carcinoma of the prostate), MCF7, and MDA-MB-231 (Metastatic
pleural effusion of breast adenocarcinoma) established human cell
lines were obtained from ATCC Global Bioresource Center. Cells
were maintained in a 5% CO2 humidified atmosphere at 37 °C in
RPMI-1640 medium, supplemented with 10% fetal bovine serum,
penicillin, and streptomycin.
The observed reduction in tumor volume over the treatment
period augers well for tumor suppression over extended dosing
schedules.
The effects of different doses (10, 20, and 40 mg/kg, qd, 28d)
of 4 in the highly aggressive DU145 human prostate cancer
xenograft model in male athymic nude mice are shown in Figure
5. Here, after initiation of treatment, the various dosing cohorts
show little activity over the first 7 days. After 25 days of
treatment (day 35), however, the tumor growth in the 40 mg/
kg treatment group appears to plateau with a 65% inhibition of
tumor growth at this time point (control and vehicle group were
euthanized at this point for ethical reasons). The 10 and 20 mg/
kg groups effected more modest tumor growth, 24% and 27%,
though there is no statistically significant difference between
the inhibition effected by these groups. The activity of 4 in this
highly aggressive xenograft model at nonoptimized doses was
very encouraging.
Antiproliferative Assays. DU145 and MDA-MB-231 cells were
seeded into 96-well microtiter plates (5000 cells/well) and treated
with 10-9-10-4 M of compounds or with vehicle control. At 96 h
post-treatment, live cell counts were determined by WST-1 cell
proliferation assay (Roche, Penzberg, Germany), as per the
manufacturer’s instructions. Viability results were expressed as a
percentage of mean control values resulting in the calculation of
the 50% growth inhibition (GI50). All experiments were performed
in triplicate. The MCF7 antiproliferative assay was carried out as
reported previously.42
The inhibition of STS was assayed by the method of Purohit et
al.49
In Vivo Studies. Studies were carried out following Institutional
Guidelines that comply with the recommendations of the Guide
for Care and Use of Laboratory Animals with respect to restraint,
husbandry, surgical procedures, feed, and fluid regulation.
MDA-MB-231 Study. Mice: Female NCr-nude mice, 6-8
weeks of age, were fed water ad libitum (reverse osmosis, 0.17%
Cl) and an autoclaved standard rodent (NIH31) diet consisting of
18% protein, 5% fat, 5% fiber, 8% ash, and 3% minerals. Mice
were housed in microisolators on a 12-hour light cycle at 22 °C
and 40%-60% humidity.
Conclusion
In the present study, we have synthesized a series of novel
2-alkylestradiol-3,17-O,O-bis-sulfamates and sulfamoylated
2-alkylestradiol-17-O-carbamates. The compounds were evalu-
ated as inhibitors of in vitro cancer cell growth in DU145, MDA-
MB-231, and MCF7 human cancer cell cultures and compared
to the parent estradiol derivatives. In the bis-sulfamate series
the 2-methyl derivative 27 displayed antiproliferative activity
similar to that of 2-ethylestradiol-3,17-O,O-bis-sulfamate (4),
while introduction of larger alkyl-substituents at the C-2 position
proved detrimental to antiproliferative activity.
Carbamoylation of the 17â-hydroxy group of the 2-substituted
estradiols afforded the 2-alkyl-17â-carbamoyloxyestra-1,3,5(10)-
trien-3-ol series of compounds 41-48, which did not substan-
tially inhibit cancer cell proliferation. After sulfamoylation of
the phenolic hydroxy group, a series of 2-alkyl-3-sulfamoyloxy-
17â-carbamoyloxyestra-1,3,5(10)-trien-3-ols (compounds 49-
56) was obtained that showed much improved antiproliferative
activity, with optimal activity obtained for the 2-ethyl-carbamate
52 in the three cell lines assayed. Introduction of an acetate
group in the 17-position led to a reduction of cancer cell growth
inhibition compared to the carbamate and bis-sulfamate analogs.
Bioisosteric replacement of the 17-O-sulfamate group of 4 with
a carbamate group (52) or, with less success, an acetate group
(32) was thus achieved, albeit the bis-sulfamate compound
remains the most active of the series. It was also confirmed
that of the 2-alkyl substituents the 2-ethyl group is optimal in
Tumors: Mice were implanted subcutaneously with 5 × 106
MDA-MB-231 cells in the flank. Tumors were monitored initially
twice weekly, and then daily as the neoplasms reached the desired
size, approximately 50-100 mm3. Estimated tumor volume was
calculated using the formula tumor volume (mm3) ) (w2 × l)/2,
where w ) width and l ) length in mm of the breast carcinoma.
Statistical significance calculated by one-way analysis of variance
for all three treatment groups with respect to control gave P <
0.001.
DU145 Study. Mice: Male NCr-nude mice, 6-8 weeks of age,
were fed water ad libitum (reverse osmosis, 0.17% Cl) and an
autoclaved standard rodent (NIH31) diet consisting of 18% protein,
5% fat, 5% fiber, 8% ash, and 3% minerals. Mice were housed in
microisolators on a 12-hour light cycle at 22 °C and 40%-60%
humidity.
Tumors: Mice were implanted subcutaneously with 5 × 106 cells
in the flank. Tumors were monitored initially twice weekly and
then daily as the neoplasms reached the desired size, approximately
50-100 mm3. Estimated tumor volume was calculated using the
formula tumor volume (mm3) ) (w2 × l)/2, where w ) width and
l ) length in mm of the carcinoma. Statistical significance
calculated by one-way analysis of variance for all three treatment
groups with respect to control gave P < 0.001.

Disubstituted 2-Alkylestra-1,3,5(10)-trien-3-ols
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4437
Chemistry. All chemicals were either purchased from Aldrich
Chemical Co. (Gillingham, UK) or Lancaster Synthesis (More-
combe, UK). E1 (6) was purchased from Sequoia Research Products
(Oxford, UK). All organic solvents of A.R. grade were supplied
by Fisher Scientific (Loughborough, UK). Sulfamoyl chloride was
prepared by an adaptation 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.51 The protected
estradiol 11 was prepared according to the method of Bielman and
Branlant.52 Compound 35 was synthesized from estradiol (25),53
and compound 36 was made according to a procedure from Rao
and Cessac.54 The bis-sulfamates 3, 4, and 26 have been described
previously.5,12,36 Thin-layer chromatography (TLC) was performed
and the residue was purified by flash chromatography using
mixtures of chloroform and acetone (typically between 20:1 and
5:1 by volume) to give the sulfamate or bis-sulfamate derivative.
General Procedure 2. Preparation of Carbamates. Trichlo-
roacetyl isocyanate (8-9 equiv) was added to a solution of the
estradiol (2 mmol) in THF at 0 °C. Stirring was continued for 30
min and water (5 mL) was then added carefully. MeOH (30 mL)
and K2CO3 (2.76 g, 20.0 mmol) were then added, and the mixture
was stirred vigorously at room temperature for 3 h. The mixture
was poured into water (60 mL) and the products were extracted
with EtOAc (3 × 50 mL). The combined organic layers were
washed with brine (50 mL) and dried (Na2SO4). The solvents were
removed under reduced pressure, and the residue was purified by
flash chromatography (eluent CHCl3/acetone typically between 20:1
and 5:1 by volume) to give the carbamate.
on precoated plates (Merck TLC aluminum sheets silica gel 60 F254
,
Art. No. 5554). Product(s) and starting materials(s) were detected
by either viewing under UV light or treating with an ethanolic
solution of phosphomolybdic acid followed by heating. Flash
column chromatography was performed on silica gel (Matrex C60).
IR spectra were determined on a Perkin-Elmer Spectrum RXI FT-
2-Ethylestradiol (2). To a suspension of 18 (1.03 g, 3.0 mmol)
in MeOH (20 mL) was added 5 M NaOH (3 mL). The mixture
was stirred for 2 h at room temperature before being poured into 2
M KHSO4 (50 mL). The product was extracted with EtOAc (3 ×
50 mL), and the combined organic layers were then washed with
brine (50 mL) and dried (Na2SO4). The solvents were removed
under reduced pressure, and the residue was purified by flash
chromatography (CHCl3/acetone 10:1 by volume) to give 2 (865
mg, 96%) as a white solid: mp 168-169 °C (lit.30 mp 165-
1
IR as KBr disks, and peak positions are expressed in cm-1. H
NMR and 13C NMR spectra were recorded with a Varian Mercury
VX 400 NMR spectrometer at 400 and 100.6 MHz, respectively,
and chemical shifts are reported in parts per million (ppm, δ) relative
to tetramethylsilane (TMS) as an internal standard. The following
abbreviations are used to describe resonances in 1H NMR and 13
C
1
166 °C); H NMR (CDCl3) δ 0.77 (s, 3H), 1.22 (t, J ) 7.5 Hz,
NMR spectra: br, broad; s, singlet; d, doublet; t, triplet; q, quartet;
m, multplet; hpt, heptet. Chemical shifts for AB systems were
approximated by taking the middle of each doublet. HPLC analyses
were performed on a Waters Millenium 32 instrument equipped
with a Waters 996 PDA detector. A Waters Radialpack C18 reversed
phase column (8 × 100 mm) was eluted with a methanol/water
gradient at 2 mL/min. Mass spectra were recorded at the Mass
Spectrometry Service Centre, University of Bath or at the EPSRC
National Mass Spectrometry Service Centre, University of Wales
Swansea. FAB-MS were carried out using m-nitrobenzyl alcohol
(NBA) as the matrix. Electrospray (ES) and atmospheric pressure
chemical ionization (APCI) low-resolution mass spectra were
obtained on a Waters Micromass ZQ. Elemental analyses were
performed by the Microanalysis Service, University of Bath.
Melting points were determined using a Stanford Research Systems
OptiMelt MPA100 and are uncorrected.
Crystal Data for 50. Empirical formula: C20H28N2O6S, M )
424.50, T ) 150(2) K, λ ) 0.710 73 Å, monoclinic, space group
P21, a ) 9.0350(1) Å, b ) 25.5210(4) Å, c ) 9.1370(1) Å, â )
100.288(1)°, U ) 2072.96(5) Å3, Z ) 4, Dc ) 1.360 g cm-3, µ )
0.196 mm-1; F(000) ) 904, crystal size 0.35 × 0.25 × 0.10 mm,
unique reflections ) 9299 [R(int) ) 0.0528], observed I >2σ(I) )
6850, data/restraints/parameters: 9299/9/544, R1 ) 0.0416 wR2
) 0.0780 (obsd data), R1 ) 0.0737, wR2 ) 0.0871 (all data), max
peak/hole 0.385 and -0.376 e Å-3, absolute structure parameter
) 0.06(4). Data were collected on a Nonius kappaCCD diffracto-
meter. The asymmetric unit was seen to consist of two independent
molecules. Hydrogen atoms attached to N1 and N3 were located
and refined at a distance of 0.89 Å from the parent atoms. The
gross structure is dominated by extensive hydrogen bonding.
Crystallographic data for 50 have been deposited with the
Cambridge Crystallographic Data Centre as supplementary publica-
tion CCDC 632335. Copies of these 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].
General Procedure 1. Preparation of Sulfamates. The sulfa-
mates were prepared using the method of Okada et al.47 The
estradiol, 17â-carbamoyloxyestra-1,3,5(10)-trien-3-ol, or 17â-ac-
etoxyestra-1,3,5(10)-trien-3-ol (0.35-1.0 mmol) was added to a
stirred solution of sulfamoyl chloride (3 equiv per hydroxyl group)
in anhydrous dimethylacetamide (5 mL) at 0 °C under an
atmosphere of N2. The reaction mixture was stirred for 2 h at 0 °C
and then for another 14 h at room temperature. EtOAc (50 mL)
and water (30 mL) were added. The organic layer was separated,
washed with water (2 × 30 mL) and brine (30 mL), dried (Na2-
SO4), and filtered. The solvent was removed under reduced pressure
3H), 1.24-1.56 (m, 7H), 1.60-1.74 (m, 2H), 1.80-1.88 (m, 1H),
1.92-1.98 (m, 1H), 2.06-2.22 (m, 2H), 2.30-2.38 (m, 1H), 2.60
(q, J ) 7.5 Hz, 2H), 2.72-2.86 (m, 2H), 6.49 (s, 1H), 7.05 (s,
1H); MS (APCI-) m/z 299.2 (100, [M - H]-). Anal. (C20H30O2)
C, H.
2-(N,N-Dimethylamino)methylestrone (7). To a solution of
estrone (6, 6.76 g, 25.0 mmol) in EtOH (30 mL) and toluene (30
mL) were added N,N,N′,N′-tetramethyldiaminomethane (5.0 g, 50
mmol) and paraformaldehyde (750 mg). The mixture was heated
to reflux for 18 h and then cooled to room temperature before being
concentrated under reduced pressure to a volume of ca. 10 mL.
Water (50 mL) was added, and the products were extracted with
Et2O (3 × 60 mL). The combined organic layers were washed with
brine (30 mL) and dried (Na2SO4). The solvents were removed
under reduced pressure to give the crude product, which contained
ca. 10% 4-(N,N-dimethylamino)methylestrone. Recrystallization
from EtOH gave pure 7 (4.83 g, 59%) as a white crystalline solid:
1
mp 174-175 °C (lit.37 mp 169.5-171.5 °C); H NMR (CDCl3) δ
0.89 (s, 3H), 1.32-1.66 (m, 6H), 1.89-2.25 (m, 5H), 2.30 (s, 6H),
2.32-2.40 (m, 1H), 2.48 (dd, J ) 18.8, 8.2 Hz, 1H), 2.80-2.88
(m, 2H), 3.53 (d, J ) 13.7 Hz, 1H), 3.62 (d, J ) 13.7 Hz, 1H),
6.56 (s, 1H), 6.86 (s, 1H), 11.92 (br s, 1H); MS (APCI+) m/z 328.1
(100, [M + H]+). Anal. (C21H29NO2) C, H, N.
2-Methylestradiol (8). To a solution of 7 (3.93 g, 12.0 mmol)
in Et2O (100 mL) was added methyl iodide (3.55 g, 25 mmol) and
the mixture was stirred for 24 h at room temperature. The resulting
white precipitate was collected by filtration, washed with Et2O, and
then suspended in EtOH (50 mL). NaBH4 (1.0 g, 26.3 mmol) was
added at 0 °C and the mixture was stirred for 18 h at room
temperature. The reaction mixture was then poured into water (100
mL), and the products were extracted with EtOAc (3 × 50 mL).
The combined organic layers were washed with water (50 mL) and
brine (50 mL) and dried (Na2SO4). The solvent was removed under
reduced pressure and the residue was purified by flash chroma-
tography (CHCl3/acetone 10:1) to give 8 as a white solid (2.72 g,
79%): mp 180-182 °C (EtOH, crystals contain 1 equiv EtOH)
1
(lit.30 mp 185-186 °C from ether); H NMR (CDCl3) δ 0.82 (s,
3H), 1.18-1.59 (m, 7H), 1.64-1.78 (m, 2H), 1.86-1.94 (m, 1H),
1.98-2.02 (m, 1H), 2.10-2.24 (m, 2H), 2.26 (s, 3H), 2.32-2.40
(m, 1H), 2.76-2.90 (m, 2H), 3.74-3.81 (m, 1H), 5.16 (s, 1H),
6.55 (s, 1H), 7.08 (s, 1H); MS (APCI+) m/z 287.3 (65, [M + H]+),
269.3 (100). Anal. (C19H26O2‚C2H5OH) C, H.
2-tert-Butylestrone (9). To a stirred suspension of estrone (6,
5.41 g, 20.0 mmol) in CHCl3 (100 mL) and tert-butanol (2 mL)
was added BF3‚(Et)2O (10 mL) with a syringe at room temperature.
The resulting orange solution was stirred for 30 min before water

4438 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Bubert et al.
(50 mL) was added carefully. The organic layer was separated,
washed with saturated aqueous NaHCO3 (50 mL) and brine (30
mL), and dried (Na2SO4). The solvent was removed under reduced
pressure, the solid residue was suspended in MeOH (50 mL), and
the mixture was heated to reflux for 5 min. The product 9 (6.07 g,
93%), a white solid, was collected by filtration after cooling to
room temperature, washed with MeOH, and dried under high
vacuum: mp 242-245 °C (lit.38 mp 242-245 °C); 1H NMR
(CDCl3) δ 0.83 (s, 3H), 1.40 (s, 9H), 1.42-1.70 (m, 6H), 1.93-
2.30 (m, 5H), 2.40-2.55 (m, 2H), 2.75-2.88 (m, 2H), 4.94 (s,
1H, OH), 6.44 (s, 1H), 7.19 (s, 1H); MS (APCI-) m/z 325.4 (100,
[M - H]-). Anal. (C22H30O2) C, H.
× 50 mL). The combined organic layers were washed with water
(50 mL) and brine (50 mL), dried (MgSO4), and the solvent was
removed under reduced pressure. The residue was crystallized from
MeOH to give the 14 (7.85 g, 78%) as fine colorless needles: mp
1
145-147 °C; H NMR (CDCl3) δ 0.81 (s, 3H), 0.94 (t, J ) 7.4
Hz, 3H), 1.20-1.58 (m, 7H), 1.62-1.76 (m, H), 1.84-1.91 (m,
1H), 2.05 (s, 3H), 2.12-2.25 (m, 2H), 2.30-2.39 (m, 1H), 2.83-
2.98 (m, 4H), 3.84 (s, 3H), 4.66 (dd, J ) 9.0, 7.8 Hz, 1H), 6.64 (s,
1H), 7.61 (s, 1H); MS (ES+) m/z 399.4 (100, [M + H]+). Anal.
(C25H34O4) C, H.
17â-Acetoxy-2-acetylestra-1,3,5(10)-trien-3-ol (15). Trimethy-
lammonium hydrochloride (15.77 g, 165 mmol) was added to a
suspension of AlCl3 (44.0 g, 0.33 mmol) in DCM (100 mL) at
0 °C. The resulting mixture was stirred for 30 min at room
temperature before a solution of 12 (11.12 g, 30 mmol) in DCM
(100 mL) was added slowly. The mixture was brought to reflux
for 2 h and then, after cooling to room temperature, poured onto
crushed ice. The organic layer was separated, washed with water
(50 mL) and brine (50 mL), dried (MgSO4), and the solvent was
removed under reduced pressure. The residue was crystallized from
EtOAc/hexane to give 15 (9.30 g, 87%) as light yellow needles:
mp 198-200 °C (lit.44 mp 202-204 °C); 1H NMR (CDCl3) δ 0.83
(s, 3H), 1.20-1.60 (m, 7H), 1.70-1.79 (m, 1H), 1.84-1.94 (m,
2H), 2.05 (s, 3H), 2.13-2.31 (m, 3H), 2.58 (s, 3H), 2.76-2.92
(m, 2H), 4.68 (dd, J ) 9.0, 7.8 Hz, 1H), 6.67 (s, 1H), 7.58 (2, 1H),
12.02 (s, 1H, OH); MS (ES+) m/z 357.5 (100, [M + H]+). Anal.
(C22H28O4) C, H.
2-tert-Butylestradiol (10). To a solution of 9 (2.47 g, 7.56 mmol)
in MeOH (25 mL) and THF (25 mL) cooled to 0 °C was added
NaBH4 (378 mg, 10.0 mmol). The resulting solution was stirred at
this temperature for 2 h and then treated with water (50 mL) and
EtOAc (100 mL). The organic layer was separated washed with
water (50 mL) and brine (50 mL) and dried (Na2SO4). The solvents
were removed under reduced pressure, and the residue was purified
by flash chromatography (CHCl3/acetone 10:1) to give 10 as a white
solid (2.11 g, 85%): mp 174-176 °C (lit.39 mp 174-176 °C); 1H
NMR (CDCl3) δ 0.78 (s, 3H), 1.14-1.36 (m, 4H), 1.38 (s, 9H),
1.41-1.58 (m, 4H), 1.65-1.72 (m, 1H), 1.82-1.87 (m, 1H), 1.92-
1.98 (m, 1H), 2.06-2.22 (m, 2H), 2.30-2.38 (m, 1H), 2.68-2.84
(m, 2H), 3.74-3.77 (m, 1H), 4.86 (s, 1H, OH), 6.42 (s, 1H), 7.18
(s, 1H); MS (APCI-) m/z 327.5 (100, [M - H]-); HRMS (ES+)
m/z calcd for C22H33O2 [M + H]+ 329.2475, found 329.2473.
17â-Acetoxy-2-Acetyl-3-methoxyestra-1,3,5(10)-triene (12).
Acetyl chloride (4.0 mL, 3.62 g, 46.1 mmol) was added dropwise
to a cooled (0 °C) suspension of AlCl3 (6.40 g, 48.0 mmol) in DCM
(50 mL), and then a solution of 11 (7.90 g, 24.0 mmol) in DCM
(25 mL) was added slowly and stirring was continued for 15 min
at this temperature. The mixture was poured onto crushed ice,
chloroform (100 mL) and water (100 mL) were added, and the
resultant mixture was stirred vigorously for 10 min. The organic
layer was separated, the aqueous layer was extracted with CHCl3
(2 × 50 mL), and the combined organic layers were dried (Na2-
SO4). The solvents were removed under reduced pressure and the
residue was crystallized from i-Pr2O to give 12 (3.78 g, 85%) as a
white crystalline solid: mp 187-189 °C (lit.44 mp 192-194 °C);
1H NMR (CDCl3) δ 0.80 (s, 3H), 1.20-1.58 (m, 7H), 1.66-1.86
(m, 1H), 1.83-1.92 (m, 2H), 2.05 (s, 3H), 2.12-2.26 (m, 2H),
2.32-2.40 (m, 1H), 2.57 (s, 3H), 2.83-2.91 (m, 2H), 3.86 (3H,
s), 4.66 (dd, J ) 9.4, 7.8 Hz, 1H), 6.65 (s, 1H), 7.67 (s, 1H); MS
(APCI+) m/z 371.4 (100, [M + H]+). Anal. (C23H30O4) C, H.
17â-Acetoxy-3-methoxy-2-propionylestra-1,3,5(10)-triene (13).
Propionic anhydride (6.50 g, 50 mmol) was added dropwise to a
cooled (0 °C) suspension of AlCl3 (8.0 g, 60 mmol) in DCM (100
mL). To this was added over a period of 10 min a solution of 11
(8.21 g, 25 mmol) in DCM (50 mL) and stirring was continued for
30 min, at which time the reaction mixture was poured onto crushed
ice and allowed to warm to room temperature. The organic layer
was separated and the aqueous layer was extracted with DCM (2
× 50 mL). The combined organic layers were washed with water
(50 mL) and brine (50 mL), dried (MgSO4), and the solvents were
removed under reduced pressure. The resulting solid was recrystal-
lized from MeOH to give 13 (7.306 g, 76%) as fine colorless
needles: mp 160-161 °C; 1H NMR (CDCl3) δ 0.81 (s, 3H), 1.13
(t, J ) 7.4 Hz, 3H), 1.20-1.60 (m, 1H), 1.84-1.92 (m, 2H), 2.05
(s, 3H), 2.13-2.26 (m, 2H), 2.31-2.39 (m, 1H), 2.84 (m, 2H),
2.92-3.01 (m, 2H), 3.85 (s, 3H), 4.66 (dd, J ) 9.0, 7.8 Hz, 1H),
6.64 (s, 1H), 7.64 (s, 1H); MS (APCI+) m/z 385.5 (100, [M +
H]+). Anal. (C24H32O4) C, H.
17â-Acetoxy-2-propionylestra-1,3,5(10)-trien-3-ol (16). Tri-
methylammonium hydrochloride (7.17 g, 75 mmol) was added to
a suspension of AlCl3 (20.0 g, 150 mmol) in DCM (100 mL) at
0 °C. The resulting mixture was stirred for 30 min at room
temperature before a solution of 13 (5.77 g, 30 mmol) in DCM
(100 mL) was added slowly. The mixture was brought to reflux
for 2 h and then, after cooling to room temperature, poured onto
crushed ice. The organic layer was separated, washed with water
(50 mL) and brine (50 mL), dried (MgSO4), and the solvent was
removed under reduced pressure. The residue was crystallized from
EtOAc/hexane to give 16 (4.39 g, 79%) as light yellow needles:
1
mp 167-168 °C; H NMR (CDCl3) δ 0.87 (s, 3H), 1.27 (t, J )
7.4 Hz, 3H), 1.28-1.62 (m, 7H), 1.74-1.82 (m, 1H), 1.90-1.97
(m, 2H), 2.10 (s, 3H), 2.19-2.36 (m, 3H), 2.80-2.96 (m, 2H),
3.00-3.11 (m, 2H), 4.47 (dd, J ) 9.0, 7.8 Hz, 1H), 6.72 (s, 1H),
7.68 (2, 1H), 12.19 (s, 1H, OH); MS (APCI+) m/z 371.6 (100, [M
+ H]+). Anal. (C23H30O4) C, H.
17â-Acetoxy-2-butyrylestra-1,3,5(10)-trien-3-ol (17). Trim-
ethylammonium hydrochloride (8.60 g, 90 mmol) was added to a
suspension of AlCl3 (24.0 g, 180 mmol) in DCM (100 mL) at
0 °C. The resulting mixture was stirred for 30 min at room
temperature before a solution of 14 (7.17 g, 18.0 mmol) in DCM
(50 mL) was added slowly. The mixture was heated to reflux for
2 h and then, after cooling to room temperature, poured onto crushed
ice. The organic layer was separated, washed with water (50 mL)
and brine (50 mL), dried (MgSO4), and the solvent was removed
under reduced pressure. The residue was crystallized from MeOH
to give 17 (5.81 g, 84%) as light yellow needles: mp 112-113
1
°C; H NMR (CDCl3) δ 0.82 (s, 3H), 1.00 (t, J ) 7.4 Hz, 3H),
1.20-1.58 (m, 7H), 1.70-1.81 (m, 3H), 1.84-1.92 (m, 2H), 2.05
(s, 3H), 2.12-2.32 (m, 3H), 2.79-2.95 (m, 2H), 4.68 (dd, J )
9.0, 8.2 Hz, 1H), 6.66 (s, 1H), 7.61 (2, 1H), 12.17 (s, 1H, OH);
MS (ES-) m/z 383.3 (100, [M - H]-). Anal. (C24H32O4) C, H.
17â-Acetoxy-2-ethylestra-1,3,5(10)-trien-3-ol (18). Pd/C (250
mg, 10% Pd) was added to a solution of 15 (1.78 g, 5.0 mmol) in
MeOH (20 mL) and THF (20 mL). The mixture was stirred under
a H2 atmosphere for 24 h before the catalyst was filtered off, and
the solvents were removed under reduced pressure. The residue
was crystallized from CHCl3/hexane to give 18 (1.473 g, 86%) as
fine colorless needles: mp 175-177 °C; 1H NMR (CDCl3) δ 0.82,
1.20 (t, J ) 7.4 Hz, 3H), 1.22-1.60 (m, 7H), 1.68-1.78 (m, 1H),
1.82-1.90 (m, 2H), 2.07 (s, 3), 2.13-2.34 (m, 3H), 2.59 (q, J )
7.4 Hz, 2H), 2.70-2.86 (m, 2H), 4.68 (dd, J ) 9.0, 7.8 Hz, 1H),
17â-Acetoxy-2-butyryl-3-methoxyestra-1,3,5(10)-triene (14).
Butyric anhydride (9.5 g, 60 mmol) was added dropwise to a cooled
(0 °C) suspension of AlCl3 (8.0 g 60 mmol) in DCM (100 mL).
To this was added over a period of 10 min a solution of 11 (8.21
g, 25 mmol) in DCM (50 mL) and stirring was continued for 30
min, at which time the reaction mixture was poured onto crushed
ice and allowed to warm to room temperature. The organic layer
was separated and the aqueous layer was extracted with DCM (2

Disubstituted 2-Alkylestra-1,3,5(10)-trien-3-ols
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4439
5.01 (s, 1H), 6.50 (s, 1H), 7.04 (s, 1H); MS (APCI-) m/z 341.5
(100, [M - H]-). Anal. (C22H30O3) C, H.
2.24-2.36 (m, 1H), 2.49-2.56 (m, 2H), 2.70-2.84 (m, 2H), 3.70-
3.78 (m, 1H), 4.96 (s, 1H), 6.45 (s, 1H), 7.03 (s, 1H); MS (APCI-)
m/z 313.6 (100, [M - H]-); HRMS (ES+) calcd for C21H31O2 [M
+ H]+ 315.2330, found 315.2318.
17â-Acetoxy-2-propylestra-1,3,5(10)-trien-3-ol (19). Pd/C (250
mg, 10%Pd) was added to a solution of 16 (4.11 g, 11.1 mmol) in
MeOH (60 mL) and THF (20 mL). The mixture was stirred under
a H2 atmosphere for 24 h before the catalyst was filtered off, and
the solvents were removed under reduced pressure. The residue
was crystallized from CHCl3/hexane to give 19 (3.54 g, 89%) as
fine colorless needles: mp 167-169 °C; 1H NMR (CDCl3) δ 0.82
(s, 3H), 0.97 (t, J ) 7.4 Hz, 3H), 1.20-1.60 (m, 10H) 1.80-1.90
(m, 2H), 2.06 (s, 3H), 2.13-2.33 (m, 3H), 2.48-2.60 (m, 2H),
2.70-2.86 (m, 2H), 4.68 (dd, J ) 9.0, 8.2 Hz, 1H), 4.78 (s, 1H),
6.49 (s, 1H), 7.01 (s, 1H); MS (APCI+) m/z 357.6 (15, [M + H]+),
297.5 (100, [M - OAc]+; HRMS (ES+) calcd for C23H33O3 [M +
H]+ 357.2435, found 357.2429.
2-n-Butylestradiol (24). To a suspension of 20 (2.96 g, 8.0
mmol) in MeOH (40 mL) was added 5 M NaOH (5 mL). The
mixture was stirred for 2 h at room temperature before being poured
into 2 M KHSO4 (50 mL). The product was extracted with EtOAc
(3 × 50 mL), the combined organic layers were washed with brine
(50 mL), dried (Na2SO4), and the solvents were removed under
reduced pressure. The residue was purified by flash chromatography
on silica (CHCl3/acetone 10:1) to give 24 (2.33 g, 89%) as a white
solid: mp 138-141 °C (lit.30 mp 142-143 °C); 1H NMR (CDCl3)
δ 0.77 (s, 3H), 0.93 (t, J ) 7.3 Hz, 3H), 1.14-1.73 (m, 13H),
1.80-1.84 (m, 1H), 1.91-1.97 (m, 1H), 2.04-2.21 (m, 2H), 2.29-
2.36 (m, 1H), 2.48-2.60 (m, 2H), 2.70-2.85 (m, 2H), 3.68-3.78
(m, 1H), 4.70 (s, 1H), 6.49 (s, 1H), 7.02 (s, 1H); MS (ES-) m/z
327.5 (100, [M - H]-). Anal. (C22H32O4) C, H.
17â-Acetoxy-2-butylestra-1,3,5(10)-trien-3-ol (20). Pd/C (300
mg, 10% Pd) was added to a solution of 17 (4.72 g, 12.2 mmol) in
MeOH (30 mL) and THF (30 mL). The mixture was stirred under
a H2 atmosphere for 24 h before the catalyst was filtered off, and
the solvents were removed under reduced pressure. The residue
was crystallized from MeOH to give 20 (3.66 g, 81%) as fine
2-Methyl-3,17â-disulfamoyloxyestra-1,3,5(10)-triene (27). Ac-
cording to general procedure 1, compound 8 (143 mg, 0.50 mmol)
was reacted with sulfamoyl chloride (6 equiv) in DMA (5 mL) to
give 27 (196 mg, 88%) as a white solid. Recrystallization from
1
colorless needles: mp 161-162 °C; H NMR (CDCl3) δ 0.82 (s,
1
acetone/hexane gave fine colorless needles: mp 155-158 °C; H
3H), 0.93 (t, J ) 7.3 Hz, 3H), 1.20-1.60 (m, 11H), 1.69-1.78
(m, 1H), 1.81-1.90 (m, 2H), 2.06 (s, 3H), 2.14-2.31 (m, 3H),
2.38-2.60 (m, 2H), 2.70-2.83 (m, 2H), 4.68 (dd, J ) 9.0, 7.8 Hz,
1H), 4.75 (s, 1H), 6.49 (s, 1H), 7.00 (s, 1H); MS (ES-) m/z 369.6
(100, [M - H]-); HRMS (ES-) calcd for C24H33O3 [M - H]-
369.2435, found 369.2428.
NMR (DMSO-d6) δ 0.77 (s, 3H), 1.16-1.44 (m, 6H), 1.63-1.75
(m, 2H), 1.79-1.86 (m, 1H), 1.90-2.00 (m, 1H), 2.10-2.21 (m,
2H), 4.34 (dd, J ) 8.6, 8.2 Hz, 1H), 6.98 (s, 1H), 7.20 (s, 1H),
7.39 (s, 2H, OSO2NH2), 7.92 (s, 2H); MS (APCI-) m/z 443.5 (100,
[M - H]-), 212.2 (88%). Anal. (C19H28N2O6S2) C, H, N.
2-n-Propyl-3,17â-disulfamoyloxyestra-1,3,5(10)-triene (28).
According to general procedure 1, compound 23 (157 mg, 0.50
mmol) was reacted with sulfamoyl chloride (6 equiv) in DMA (5
mL) to give 28 (189 mg, 80%) as a white solid: mp 182-185 °C;
1H NMR (DMSO-d6) δ 0.77 (s, 3H), 0.89 (t, J ) 7.4 Hz, 3H),
1.20-1.45 (m, 6H), 1.48-1.60 (m, 2H), 1.63 (m, 2H), 1.80-1.86
(m, 1H), 1.88-2.00 (m, 1H, 2.10-2.26 (m, 2H), 2.29-2.40 (m,
1H), 2.53-2.62 (m, 2H), 2.74-2.82 (m, 2H), 4.34 (dd, J ) 8.6,
8.1 Hz,), 7.00 (s, 1H), 7.19 (s, 1H), 7.41 (s, 2H), 7.95 (s, 2H); MS
(APCI-) m/z 471.5 (100, [M - H]-). Anal. (C21H32N2O6S2) C, H,
N.
2-Isopropylestradiol (21). Pd/C (250 mg, 10% Pd) was added
to a solution of 22 (2.63 g, 7.96 mmol) in MeOH. The mixture
was stirred under a H2 atmosphere for 24 h at room temperature
before the catalyst was filtered off and the solvent was removed
under reduced pressure. The solid residue was recrystallized from
CHCl3/hexane to give 21 as fine colorless needles (2.23 g, 89%):
mp 149-152 °C (lit.46 mp 103-105 °C); 1H NMR (CDCl3) δ 0.78
(s, 3H), 1.23 (d, J ) 6.7 Hz, 3H), 1.25 (d, J ) 6.7 Hz, 3H), 1.26-
1.58 (m, 7H), 1.60 (s, 1H), 1.65-1.74 (m, 1H), 1.82-1.88 (m,
1H), 1.93-1.99 (m, 1H), 2.07-2.23 (m, 2H), 2.31-2.40 (m, 1H),
2.70-2.86 (m, 2H), 3.18 (hpt, J ) 6.7 Hz, 1H), 3.72-3.80 (m,
1H), 5.04 (s, 1H), 6.48 (s, 1H), 7.12 (s, 1H); MS (APCI-) m/z
313.5 (100, [M - H]-); HRMS (ES-) calcd for C21H29O2 [M -
H]- 313.2173, found 313.2182.
2-Isopropyl-3,17â-disulfamoyloxyestra-1,3,5(10)-triene (29).
According to general procedure 1, compound 21 (143 mg, 0.50
mmol) was reacted with sulfamoyl chloride (6 equiv) in DMA (5
mL) to give 29 (201 mg, 85%) as a white solid: mp 193-195 °C;
1H NMR (DMSO-d6) δ 0.78 (s, 3H), 1.14 (d, J ) 7.0 Hz, 3H),
1.16 (d, J ) 7.0 Hz, 3H), 1.20-1.45 (m, 6H), 1.62-1.76 (m, 2H),
1.79-1.86 (m, 1H), 1.92-2.00 (m, 1H), 2.10-2.26 (m, 2H), 2.34-
2.44 (m, 1H), 2.74-2.82 (m, 2H), 3.29 (hpt, J ) 7.0 Hz, 1H), 4.34
(dd, J ) 8.6, 8.2 Hz, 1H), 7.00 (s, 1H), 7.25 (s, 1H), 7.40 (s, 2H),
7.96 (s, 2H); MS (APCI-) m/z 471.6 (100, [M - H]-). Anal.
(C21H32N2O6S2) C, H, N.
2-n-Butyl-3,17â-disulfamoyloxyestra-1,3,5(10)-triene (30). Ac-
cording to general procedure 1, compound 24 (143 mg, 0.50 mmol)
was reacted with sulfamoyl chloride (6 equiv) in DMA (5 mL) to
give 30 (189 mg, 78%) as a white solid: mp 148-150 °C; 1H NMR
(DMSO-d6) δ 0.77 (s, 3H), 0.89 (t, J ) 7.4 Hz, 3H), 1.18-1.55
(m, 10H), 1.62-1.75 (m, 2H), 1.77-1.85 (m, 1H), 1.90-2.00 (m,
1H), 2.11-2.25 (m, 2H), 2.30-2.42 (m, 1H), 2.53-2.65 (m, 2H),
2.70-2.82 (m, 2H), 4.34 (dd, J ) 8.6, 8.2 Hz,), 7.00 (s, 1H), 7.19
(s, 1H), 7.41 (s, 2H), 7.95 (s, 2H); MS (ES-) m/z 485.5 (100, [M
- H]-). Anal. (C22H34N2O6S2) C, H, N.
2-(1-Hydroxy-1-methylethyl)estradiol (22). A solution of 15
(3.56 g, 10.0 mmol) in THF (20 mL) was added slowly to a stirred
solution of 3 M methylmagnesium chloride in THF (20 mL, 60
mmol). The mixture was stirred for 1 h at room temperature. Water
(5 mL) was added carefully and the mixture was poured into
saturated NH4Cl solution (70 mL). The product was extracted with
EtOAc (2 × 50 mL), and the combined organic layers were washed
with water (50 mL) and brine (50 mL) and dried (Na2SO4). The
solvents were removed under reduced pressure, and the solid white
residue was recrystallized from EtOAc/hexane to give 22 as fine
colorless needles (2.98 g, 90%): mp 155-158 °C (lit.46 mp 160-
1
162 °C); H NMR (CDCl3) δ 0.76 (s, 3H), 1.12-1.51 (m, 7H),
1.66 (s, 3H), 1.68 (s, 3H), 1.69-1.73 (m, 1H), 1.80-1.89 (m, 1H),
1.90-1.98 (m, 1H), 2.05 2.19 (m, 2H), 2.23-2.32 (m, 1H), 2.50
(s, 1H), 2.75-2.85 (m, 2H), 3.66-3.75 (m, 1H), 6.60 (s, 1H), 6.98
(s, 1H), 8.70 (s, 1H); MS (APCI-) m/z 329.4 (100, [M - H]-);
HRMS (ES-) calcd for C21H29O3 [M - H]- 329.2122, found
329.2129.
2-tert-Butyl-3,17â-disulfamoyloxyestra-1,3,5(10)-triene (31).
According to general procedure 1, compound 10 (329 mg, 1.00
mmol) was reacted with sulfamoyl chloride (3 equiv) in DMA (3
mL) to give 31 (260 mg, 53%) as a white solid, which was then
recrystallized from ethyl acetate/hexane: mp 218-219 °C; 1H NMR
(DMSO-d6) δ 0.78 (s, 3H), 1.32 (s, 9H), 1.15-2.40 (m, 13H), 2.72-
2.84 (m, 2H), 4.33 (dd, J ) 8.4, 8.2 Hz, 1H), 7.22 (s, 1H), 7.24 (s,
1H), 7.42 (s, 2H) and 8.15 (s, 2H); MS (APCI-) m/z 471.6 (100,
[M - H]-). Anal. (C22H34N2O6S2) C, H, N.
2-n-Propylestradiol (23). To a suspension of 19 (2.85 g, 8.0
mmol) in MeOH (40 mL) was added 5 M NaOH (5 mL). The
mixture was stirred for 2 h at room temperature before being poured
into 2 M KHSO4 (50 mL). The product was extracted with EtOAc
(3 × 50 mL), and the combined organic layers were washed with
brine (50 mL), dried (Na2SO4), and the solvents were removed
under reduced pressure. The residue was purified by flash chro-
matography (CHCl3/acetone 10:1) to give 23 (2.29 g, 91%) as a
1
white solid: mp 131-133 °C (lit.30 mp 135-136 °C); H NMR
(CDCl3) δ 0.77 (s, 3H), 0.97 (t, J ) 7.4 Hz, 3H), 1.14-1.73 (m,
11H), 1.80-1.88 (m, 1H), 1.92-1.97 (m, 1H), 2.05-2.20 (m, 2H),
17â-Carbamoyloxy-2-ethylestra-1,3,5(10)-trien-3-ol (32). Ac-
cording to general procedure 1, compound 18 (171 mg, 0.50 mmol)

4440 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Bubert et al.
was reacted with sulfamoyl chloride (3 equiv) in DMA (5 mL) to
J ) 8.6 Hz, 1H), 7.34-7.48 (m, 5H); MS (FAB+) m/z 405.2.0
(35, [M]+), 91.0 (100); HRMS (FAB+) cald for C26H31NO3 [M]+
405.2304, found 405.2296.
give 32 (196 mg, 93%) as a white solid, which was recrystallized
1
from CHCl3/hexane to give fine needles: mp 212-213 °C; H
NMR (CDCl3) δ 0.81 (s, 3H), 1.20 (t, J ) 7.4 Hz, 3H), 1.22-1.60
(m, 7H), 1.68-1.79 (m, 1H), 1.84-1.92 (m, 2H), 2.05 (s, 3H),
2.15-2.33 (m, 3H), 2.68 (q, J ) 7.3 Hz, 2H), 2.79-2.86 (m, 2H),
4.68 (dd, J ) 8.7, 7.8 Hz, 1H), 4.98 (s, 2H), 7.07 (s, 1H), 7.17 (s,
1H); MS (APCI-) m/z 420.5 (100, [M - H]-). Anal. (C22H31NO5)
C, H, N.
3-Benzyloxy-17â-carbamoyloxy-2-methoxyestra-1,3,5(10)-
triene (40). A solution of K2CO3 (414 mg, 3.0 mmol) in water (10
mL) was added to a solution of 38 (407 mg, 0.70 mmol) in THF
(10 mL) and MeOH (10 mL) as described for the synthesis of 39.
The crude product was crystallized from DCM/hexane to give 40
1
(262 mg, 86%) as colorless plates: mp 180-181 °C; H NMR
17â-Carbamoyloxy-2-n-propylestra-1,3,5(10)-trien-3-ol (33).
According to general procedure 1, compound 19 (178 mg, 0.50
mmol) was reacted with sulfamoyl chloride (3 equiv) in DMA (5
mL) to give 33 (194 mg, 89%) as a white solid, which was
recrystallized from CHCl3/hexane: mp 202-206 °C; 1H NMR
(CDCl3) δ 0.81 (s, 3H), 0.95 (t, J ) 7.4 Hz, 3H), 1.20-1.66 (m,
9H), 1.69-1.78 (m, 1H), 1.84-1.91 (m, 2H), 2.05 (s, 3H), 2.14-
2.34 (m, 3H), 2.56-2.64 (m, 2H), 2.78-2.86 (m, 2H), 4.68 (dd, J
) 9.1, 7.8 Hz, 1H), 4.96 (br s, 2H), 7.07 (s, 1H), 7.15 (s, 1H); MS
(ES-) m/z 434.5 (100, [M - H]-). Anal. (C23H33NO5S) C, H, N.
17â-Acetoxy-2-n-butyl-3-sulfamoyloxyestra-1,3,5(10)-triene (34).
According to general procedure 1, compound 20 (185 mg, 0.50
mmol) was reacted with sulfamoyl chloride (3 equiv) in DMA (5
mL) to give 34 (201 mg, 89%) as a white solid: mp 175-177 °C;
1H NMR (CDCl3) δ 0.81 (s, 3H), 0.92 (t, J ) 7.4 Hz, 3H), 1.20-
1.61 (m, 11H), 1.68-1.77 (m, 1H), 1.83-1.91 (m, 2H), 2.05 (s,
3H), 2.15-2.32 (m, 3H), 2.61-2.68 (m, 2H), 2.79-2.85 (m, 2H),
4.68 (dd, J ) 9.0, 7.8 Hz, 1H), 5.09 (br s, 2H), 7.06 (s, 1H), 7.14
(s, 1H); MS (ES-) m/z 448.6 (100, [M - H]-). Anal. (C24H35-
NO5S) C, H, N.
(CDCl3) δ 0.82 (s, 3H, H-18), 1.25-1.64 (m, 7H), 1.68-1.78 (m,
1H), 1.82-1.96 (m, 2H) 2.16-2.32 (m, 3H), 2.64-2.81 (m, 2H),
3.87 (s, 3H), 4.58 (br s, 2H) 4.63 (dd, J ) 9.4, 8.2 Hz, 1H), 5.11
(s, 2H), 6.62 (s, 1H), 6.84 (s, 1H), 7.27-7.46 (m, 5H); MS (FAB+)
m/z 435.0 (90, [M]+), 90.9 (100); HRMS (FAB+) calcd for C27H33-
NO4 [M]+ 435.2410, found 435.2404.
17â-Carbamoyloxyestra-1,3,5(10)-trien-3-ol (41). Pd/C (50 mg,
10% Pd) was added to a solution of 39 (405 mg, 1.0 mmol) in
MeOH (10 mL) and THF (10 mL). The mixture was stirred under
a H2 atmosphere for 18 h at room temperature, the catalyst was
filtered off, and the solvents were removed under reduced pressure.
The residue was crystallized from EtOAc/hexane to give 41 (271
mg, 86%) as fine needles: mp 280-284 °C (dec); 1H NMR
(DMSO-d6) δ 0.76 (s, 3H), 1.18-21.52 (m, 7H), 1.60-1.82 (m,
3H), 2.00-2.15 (m, 2H), 2.18-2.30 (m, 1H), 2.64-2.78 (m, 2H),
4.56 (dd, J ) 9.0, 8.2 Hz, 1H), 6.37 (br s, 2H), 6.42 (d, J ) 2.7
Hz, 1H), 6.49 (dd, J ) 8.6, 2.7 Hz, 1H), 7.03 (d, J ) 8.6 Hz, 1H),
8.99 (s, 1H); MS (FAB+) m/z 315.0 (100, [M]+), 255.0 (50);
HRMS (FAB+) calcd for C19H25NO3 [M]+ 315.1834, found
315.1830. Anal. (C19H25NO3) C, H, N.
17â-Carbamoyloxy-2-methoxyestra-1,3,5(10)-trien-3-ol (42).
Pd/C (50 mg, 10%) was added to a solution of 40 (218 mg, 0.50
mmol) in MeOH (10 mL) and THF (10 mL). The mixture was
stirred under a H2 atmosphere for 18 h at room temperature, the
catalyst was filtered off, and the solvents were removed under
reduced pressure. The residue was precipitated from EtOAc with
hexane to give 42 (173 mg, 100%) as a white powder: mp 235-
238 °C; 1H NMR (DMSO-d6) δ 0.77 (s, 3H), 1.28-1.50 (m, 7H),
1.59-1.70 (m,1H), 1.72-1.81 (m, 2H), 1.99-2.16 (m, 2H), 2.22-
2.31 (m, 1H), 2.55-2.68 (m, 2H), 3.70 (s, 3H), 4.45 (dd, J ) 9.0,
7.8 Hz, 1H), 6.40 (bs, 2H), 6.43 (s, 1H), 6.75 (s, 1H), 8.60 (s, 1H);
MS (FAB+) m/z 345.2 (100, [M]+); HRMS (FAB+) calcd for
C20H27NO4 [M]+ 345.1940, found 345.1943. Anal. (C20H27NO4)
C, H, N.
3-Benzyloxy-17â-[N-(2,2,2-trichloroacetyl)]carbamoyloxyestra-
1,3,5(10)-triene (37). Trichloroacetyl isocyanate (0.50 mL, 4.20
mmol) was added to a solution of 35 (906 mg, 2.50 mmol) in THF
(20 mL). The solution was stirred for 15 min at room temperature
and then treated with water (0.5 mL) to destroy the excess of
trichloroacetyl isocyanate. EtOAc (50 mL) and more water (30 mL)
were added, the organic layer was separated and dried (Na2SO4),
and the solvents were removed under reduced pressure. The residue
was purified by flash chromatography (EtOAc/hexane 1:3) followed
by precipitation from a solution in DCM by addition of hexane to
give 37 (1.32 g, 96%) as a white solid: mp 177-179 °C; 1H NMR
(CDCl3) δ 0.91 (s, 3H), 1.29-2.00 (m, 10H), 2.20-2.38 (m, 3H),
2.79-2.90 (m, 2H), 4.81 (dd, J ) 9.0, 7.8 Hz, 1H), 5.31 (s, 2H),
6.73 (d, J ) 2.3 Hz, 1H), 6.79 (dd, J ) 8.6, 2.3 Hz, 1H), 7.20 (d,
J ) 8.6 Hz, 1H), 7.30-7.45 (m, 5H), 8.32 (s, 1H, NH); MS (FAB+)
m/z 549.1 (15, [M]+), 345.0 (25), 90.9 (100); HRMS (FAB+) calcd
for C28H30Cl3NO4 [M]+ 549.1240, found 549.1207.
17â-Carbamoyloxy-2-methylestra-1,3,5(10)-trien-3-ol (43). Ac-
cording to general procedure 2, compound 8 (573 mg, 2.0 mmol)
was reacted with trichloroacetyl isocyanate (1.0 mL, 8.4 mmol) in
THF (10 mL). After partial hydrolysis, workup, and purification,
43 (455 mg, 69%) was obtained as a white solid: mp >230 °C
3-Benzyloxy-17â-[N-(2,2,2-trichloroacetyl)]carbamoyloxy-2-
methoxyestra-1,3,5(10)-triene (38). Trichloroacetyl isocyanate
(0.20 mL, 1.68 mmol) was reacted with 36 (393 mg, 1.00 mmol)
in THF (20 mL), as described for the synthesis of 37. The crude
product was purified by flash chromatography (EtOAc/hexane 1:5)
1
(dec); H NMR (DMSO-d6) δ 0.75 (s, 3H), 1.15-1.38 (m, 6H),
1.40-1.51 (m, 1H), 1.58-1.68 (m, 1H), 1.72-1.80 (m, 2H), 1.98-
2.12 (m, 5H), 2.18-2.26 (m, 1H), 2.58-2.73 (m, 2H), 4.45 (dd, J
) 8.9, 8.1 Hz, 1H), 6.40 (br s, 2H), 6.42 (s, 1H), 6.92 (s, 1H), 8.87
(s, 1H); MS (APCI+) m/z 330.5 (17, [M + H]+), 269.4 (100). Anal.
(C20H27NO3) C, H, N.
1
to give 38 (534 mg, 92%) as a white solid: mp 193-195 °C; H
NMR (CDCl3) δ 0.91 (s, 3H), 1.26-1.28 (m, 6H), 1.65-1.91 (m,
3H), 1.95-2.02 (m, 1H), 2.20-2.40 (m, 3H), 2.68-2.84 (m, 2H),
3.88 (s, 3H, OCH3), 4.82 (dd, J ) 9.0, 7,8 Hz, 1H), 5.11 (s, 2H),
6.63 (s, 1H), 6.84 (s, 1H), 7.27-7.47 (m, 5H), 8.31 (s, 1H); MS
(FAB+) m/z 579.1 (100, [M]+), 375.2 (66), 73 (58); HRMS
(FAB+) calcd for C29H32Cl3NO5 [M]+ 579.1346, found 579.1323.
Anal. (C29H32Cl3NO5) C, H, N.
17â-Carbamoyloxy-2-ethylestra-1,3,5(10)-trien-3-ol (44). Ac-
cording to general procedure 2, compound 2 (601 mg, 2.0 mmol)
was reacted with trichloroacetyl isocyanate (1.0 mL, 8.4 mmol) in
THF (10 mL). After partial hydrolysis, workup, and purification,
44 (577 mg, 84%) was obtained as a white solid: mp 243-
245 °C; 1H NMR (CDCl3) δ 0.80 (s, 3H), 1.20 (t, J ) 7.4 Hz, 3H),
1.21-1.74 (m, 9H), 1.80-1.95 (m, 2H), 2.10-2.36 (m, 2H), 2.68
(q, J ) 7.4 Hz, 2H), 2.60-2.72 (m, 2H), 4.49-4.62 (m, 3H), 4.90
(s, 2H), 7.07 (s, 1H), 7.17 (s, 1H); MS (FAB+) m/z 343.1 (100,
[M]+); HRMS (FAB+) m/z calcd for C21H29NO3 [M]+ 343.2147,
found 343.2147. Anal. (C21H29NO3) C, H, N.
3-Benzyloxy-17â-carbamoyloxyestra-1,3,5(10)-triene (39). A
solution of K2CO3 (414 mg, 3.0 mmol) in water (10 mL) was added
to a solution of 37 (1.10 g, 2.0 mmol) in THF (20 mL) and MeOH
(20 mL). The mixture was stirred for 3 h at room temperature,
EtOAc (60 mL) and water (60 mL) were added, the organic layer
was separated, dried (Na2SO4), and the solvents were removed under
reduced pressure. The residue was crystallized from DCM/hexane
to give 39 (745 mg, 92%) as colorless needles: mp 161-162 °C;
1H NMR (CDCl3) δ 0.86 (s, 3H), 1.26-1.86 (m, 7H), 1.73-1.82
(m, 1H), 1.88-1.98 (m, 2H), 2.22-2.38 (m, 3H), 2.82-2.96 (m,
2H), 4.67 (dd, J ) 9.1, 7.7 Hz, 1H), 4.73 (bs, 2H), 5.07 (s, 2H),
6.76 (d, J ) 2.7 Hz, 1H), 6.82 (dd, J ) 8.6, 2.7 Hz, 1H), 7.24 (d,
17â-Carbamoyloxy-2-n-propylestra-1,3,5(10)-trien-3-ol (45).
According to general procedure 2, compound 23 (629 mg, 2.0
mmol) was reacted with trichloroacetyl isocyanate (1.0 mL, 8.4
mmol) in THF (10 mL). After partial hydrolysis, workup, and
purification, 45 (579 mg, 81%) was obtained as a white solid: mp
1
225-229 °C; H NMR (CDCl3) δ 0.80 (s, 3H), 0.97 (t, J ) 7.4

Disubstituted 2-Alkylestra-1,3,5(10)-trien-3-ols
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4441
Hz, 3H), 1.20-1.48 (m, 6H), 1.54-1.64 (m, 3H), 1.68-1.74 (m,
1H), 1.82-1.86 (m, 1H), 1.88-1.92 (m, 1H), 2.14-2.23 (m, 2H),
2.26-2.31 (m, 1H), 2.49-2.56 (m, 2H), 2.75-2.83 (m, 2H), 4.59-
4.63 (m, 3H), 4.67 (s, 1H), 6.49 (s, 1H), 7.01 (s, 1H); MS (ES-)
m/z 356.6 (100, [M - H]-); HRMS (EI) calcd for C22H31NO3 [M]+
357.2298, found 357.2293. Anal. (C22H31NO3) C, H, N.
(m, 1H), 2.72-2.81 (m, 2H), 4.47 (dd, J ) 8.6, 8.2 Hz, 1H), 6.40
(br s, 2H), 6.98 (s, 1H), 7.19 (s, 1H), 7.93 (s, 2H); MS (ES-) m/z
407.7 (100, [M - H]-); HRMS (ES-) m/z calcd for C20H27N2O5S
[M - H]- 407.1646, found 407.1643. Anal. (C20H28N2O5S) C, H,
N.
17â-Carbamoyloxy-2-ethyl-3-sulfamoyloxyestra-1,3,5(10)-
triene (52). According to general procedure 1, compound 44 (344
mg, 1.0 mmol) was reacted with sulfamoyl chloride (3 equiv) in
DMA (5 mL) to give 52 (254 mg, 60%) as a white solid: mp 220-
223 °C; 1H NMR (DMSO-d6) δ 0.77 (s, 3H), 1.12 (t, J ) 7.4 Hz,
3H), 1.20-1.54 (m, 7H), 1.60-1.72 (m, 1H), 1.74-1.85 (m, 2H),
2.00-2.12 (m, 1H), 2.16-2.23 (m, 1H), 2.26-2.38 (m, 1H), 2.61
(q, J ) 7.4 Hz, 2H), 2.74-2.82 m, 2H), 4.47 (dd, J ) 9.0, 8.2 Hz,
1H), 6.43 (br s, 2H), 7.00 (s, 1H), 7.20 (s, 1H), 8.32 (s, 2H);
MS (APCI-) m/z 421.5 (100, [M - H]-); HRMS (FAB+) m/z
calcd for C21H30N2O5S [M]+ 422.1875, found 422.1872. Anal.
(C21H30N2O5S) C, H, N.
17â-Carbamoyloxy-2-n-propyl-3-sulfamoyloxyestra-1,3,5(10)-
triene (53). According to general procedure 1, compound 45 (179
mg, 0.50 mmol) was reacted with sulfamoyl chloride (3 equiv) in
DMA (5 mL) to give 53 (129 mg, 59%) as a white solid: mp 171-
173 °C; 1H NMR (CDCl3) δ 0.80 (s, 3H), 0.95 (t, J ) 7.4 Hz, 3H),
1.20-1.78 (m, 10H), 1.82-1.94 (m, 2H), 2.13-2.32 (m, 3H),
2.58-2.64 (m, 2H), 2.79-2.85 (m, 2H), 4.56-4.68 (m, 3H), 5.12
(s, 2H), 7.08 (s, 1H), 7.15 (s, 1H); HRMS (ES-) m/z calcd for
C22H31N2O5S [M - H]- 435.1959, found 435.1959. Anal.
(C22H32N2O5S) C, H, N.
17â-Carbamoyloxy-2-isopropylestra-1,3,5(10)-trien-3-ol (46).
According to general procedure 2, compound 21 (629 mg, 2.0
mmol) was reacted with trichloroacetyl isocyanate (1.0 mL, 8.4
mmol) in THF (10 mL). After partial hydrolysis, workup, and
purification, 46 (509 mg, 71%) was obtained as a white solid: mp
1
221-222 °C; H NMR (CDCl3) δ 0.81 (s, 3H), 1.23 (d, J ) 6.7
Hz, 3H), 1.25 (d, J ) 6.7 Hz, 3H), 1.28-1.63 (m, 7H), 1.67-1.76
(m, 1H), 1.80-1.92 (m, 2H), 2.14-2.26 (m, 2H), 2.28-2.36 (m,
1H), 2.70-2.84 (m, 2H), 3.18 (hpt, J ) 6.7 Hz, 1H), 4.63 (dd, J )
8.7, 8.1 Hz, 1H), 4.74 (s, 2H), 5.27 (s, 1H), 6.50 (s, 1H), 7.10 (s,
1H); MS (APCI-) m/z 356.5 (12, [M - H]-), 313.4 (50), 295.4
(100), 187.1 (95). Anal. (C22H31NO3) C, H, N.
2-n-Butyl-17â-carbamoyloxyestra-1,3,5(10)-trien-3-ol (47). Ac-
cording to general procedure 2, compound 24 (657 mg, 2.0 mmol)
was reacted with trichloroacetyl isocyanate (1.0 mL, 8.4 mmol) in
THF (10 mL). After partial hydrolysis, workup, and purification,
47 (587 mg, 79%) was obtained as a white solid: mp 171-
174 °C; 1H NMR (CDCl3) δ 0.80 (s, 3H), 0.93 (t, J ) 7.4 Hz, 3H),
1.20-1.62 (m, 11H), 1.66-176 (m, H), 1.80-1.92 (m, 2H), 2.11-
2.32 (m, 3H), 2.50-2.61 (m, 2H), 2.70-2.83 (m, 2H), 4.62 (dd, J
) 9.0 Hz, 7.8 Hz, 1H), 4.66 (br s, 2H), 4.92 (s, 1H), 6.50 (s, 1H),
7.01 (s, 1H); MS (ES-) m/z 370.7 (100, [M - H]-); HRMS (EI)
calcd for C23H33NO3 [M]+ 371.2455, found 371.2452. Anal.
(C23H33NO3) C, H, N.
17â-Carbamoyloxy-2-isopropyl-3-sulfamoyloxyestra-1,3,5-
(10)-triene (54). According to general procedure 1, compound 46
(179 mg, 0.50 mmol) was reacted with sulfamoyl chloride (3 equiv)
in DMA (5 mL) to give 54 (142 mg, 65%) as a white solid: mp
2-tert-Butyl-17â-carbamoyloxyestra-1,3,5(10)-trien-3-ol (48).
According to general procedure 2, compound 10 (657 mg, 2.0
mmol) was reacted with trichloroacetyl isocyanate (1.0 mL, 8.4
mmol) in THF (10 mL). After partial hydrolysis, workup, and
purification, 48 (539 mg, 80%) was obtained as a white solid: mp
1
202-205 °C; H NMR (DMSO-d6) δ 0.78 (s, 3H), 1.13 (d, J )
6.5 Hz, 3H), 1.16 (d, J ) 6.5 Hz, 3H) 1.20-1.51 (m, 7H), 1.61-
1.72 (m, 1H), 1.74-1.86 (m, 2H), 2.01-2.13 (m, 1H), 2.15-2.25
(m, 1H), 2.31-2.42 (m, 1H),2.70-2.84 (m, 2H), 3.29 (hpt, J )
6.5 Hz, 1H), 4.47 (dd, J ) 8.6, 8.2 Hz, 1H), 6.40 (br s, 2H), 6.99
(s, 1H), 7.24 (s, 1H), 7.96 (s, 2H); MS (ES-) m/z 435.5 (100, [M
- H]-); HRMS (ES-) m/z calcd for C22H31N2O5S [M - H]-
435.1959, found 435.1959. Anal. (C22H32N2O5S) C, H, N.
2-n-Butyl-17â-carbamoyloxy-3-sulfamoyloxyestra-1,3,5(10)-
triene (55). According to general procedure 1, compound 47 (186
mg, 0.50 mmol) was reacted with sulfamoyl chloride (3 equiv) in
DMA (5 mL) to give 55 (137 mg, 61%) as a white solid: mp 162-
165 °C; 1H NMR (CDCl3) δ 0.79 (s, 3H), 0.92 (t, J ) 7.4 Hz, 3H),
1.20-1.62 (m, 11H), 1.66-1.75 (m, 1H), 1.82-1.94 (m, 2H),
2.12-2.32 (m, 3H), 2.60-2.67 (m, 2H), 2.78-2.84 (m, 2H), 4.60
(dd, J ) 8.6, 8.2 Hz, 1H), 4.73 (br s, 2H), 5.35 (s, 2H), 7.07 (s,
1H), 7.14 (s, 1H); HRMS (ES+) m/z calcd for C23H38N3O5S
[M+NH4]+ 468.2527, found 468.2527. Anal. (C23H34N2O5S) C, H,
N.
1
>250 °C (dec); H NMR (DMSO-d6) δ 0.76 (s, 3H), 1.14-1.51
(m, 16H), 1.58-1.70 (m, 1H), 1.72-1.82 (m, 2H), 1.99-2.15 (m,
1H),2.18-2.25 (m, 1H), 2.56-2.74 (m, 2H), 4.45 (dd, J ) 8.6 Hz,
8.2 Hz, 1H), 6.40 (br s, 2H), 6.43 (s, 1H), 7.00 (s, 1H), 8.93 (s,
1H); MS (APCI-) m/z 370.0 (100, [M - H]-). Anal. (C23H33NO3)
C, H, N.
17â-Carbamoyloxy-3-sulfamoyloxyestra-1,3,5(10)-triene (49).
According to general procedure 1, compound 41 (157 mg, 0.50
mmol) was reacted with sulfamoyl chloride (3 equiv) in DMA (5
mL) to give 49 (193 mg, 98%) as a white solid: mp 206-210 °C;
1H NMR (DMSO-d6) δ 0.77 (s, 3H), 1.20-1.55 (m, 7H), 1.62-
1.71 (m, 1H), 1.75-1.88 (m, 2H), 2.00-2.12 (m, 1H), 2.16-2.36
(m, 2H), 2.78-2.86 (m, 2H), 4.47 (dd, J ) 9.0, 7.8 Hz, 1H), 6.40
(bs, 2H), 6.96 (d, J ) 2.3 Hz, 1H), 7.00 (dd, J ) 8.6, 2.3 Hz, 1H),
7.34 (d, J ) 8.6 Hz, 1H), 7.90 (s, 1H); MS (FAB-) m/z 393.2 (100,
[M - H]-); HRMS (FAB+) calcd for C19H26N2O5S [M]+ 394.1562,
found 394.1555.
17â-Carbamoyloxy-2-methoxy-3-sulfamoyloxyestra-1,3,5(10)-
triene (50). According to general procedure 1, compound 42 (120
mg, 0.35 mmol) was reacted with sulfamoyl chloride (3 equiv) in
DMA (5 mL) to give 50 (130 mg, 88%) as a white solid, which
was recrystallized from acetone/cyclohexane to give colorless
monoclinic crystals: mp 201-204 °C; 1H NMR (DMSO-d6) δ 0.78
(s, 3H), 1.30-1.52 (m, 7H), 1.61-1.70 (m, 1H), 1.76-1.84 (m,
2H), 2.00-2.12 (m, 1H), 2.16-2.24 (m, 1H), 2.33-2.40 (m, 1H),
2.70-2.76 (m, 2H), 3.76 (s, 3H), 4.47 (dd, J ) 9.0, 7.8 Hz, 1H),
6.40 (bs, 2H), 6.98 (s, 2H), 7.83 (s, 2H); MS (FAB+) m/z 424.1
(100, [M]+); HRMS (FAB+) calcd for C20H28N2O6S [M]+ 424.1668,
found 424.1666. Anal. (C20H28N2O6S) C, H, N.
2-tert-Butyl-17â-carbamoyloxy-3-sulfamoyloxyestra-1,3,5(10)-
triene (56). According to general procedure 1, compound 48 (186
mg, 0.50 mmol) was reacted with sulfamoyl chloride (3 equiv) in
DMA (5 mL) to give 56 (178 mg, 79%) as a white solid: mp 217-
1
220 °C; H NMR (CDCl3) δ 0.78 (s, 3H), 1.20-1.52 (m, 16H),
1.60-1.70 (m, 1H), 1.75-1.86 (m, 2H), 2.00-2.12 (m, 1H), 2.14-
2.22 (m, 1H), 2.26-2.36 (m, 1H), 2.72-2.80 (m, 1H), 4.47 (dd, J
) 8.6, 8.2 Hz, 1H), 6.40 (br s, 2H), 7.22 (s, 1H), 7.23 (s, 1H), 8.14
(s, 2H); MS (APCI-) m/z 449.6 (100, [M - H]-). Anal.
(C23H34N2O5S) C, H, N.
Acknowledgment. This work was supported by Sterix Ltd.,
a member of the Ipsen group. We thank Ms A. Smith for tech-
nical support and Dr. L. W. L. Woo for useful discussions during
the course of this work.
17â-Carbamoyloxy-2-methyl-3-sulfamoyloxyestra-1,3,5(10)-
triene (51). According to general procedure 1, compound 43 (165
mg, 0.50 mmol) was reacted with sulfamoyl chloride (3 equiv) in
DMA (5 mL) to give 53 (114 mg, 56%) as a white solid: mp 209-
211 °C; 1H NMR (DMSO-d6) δ 0.77 (s, 3H), 1.13 (d, J ) 6.5 Hz,
3H), 1.20-1.53 (m, 8H), 1.60-.170 (m, 1H), 1.72-1.85 (m, 2H),
2.00-2.11 (m, 1H), 2.12-2.20 (m, 1H), 2.22 (s, 3H), 2.26-2.35
Supporting Information Available: Microanalysis data for 2,
7-9, 12-18, 24, 27-34, 38, 41-48, and 50-56; 13C NMR data
(all compounds); IR data for 37-42, 49, and 50; and crystal-
lographic data for compound 50. This material is available free of
charge via the Internet at http://pubs.acs.org.

4442 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Bubert et al.
(20) 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.
(21) Hamel, E.; Lin, C. M.; Flynn, E.; D’Amato, R. J. Interactions of
2-methoxyestradiol, an endogenous mammalian metabolite, with
unpolymerized tubulin and with tubulin polymers. Biochemistry 1996,
35, 1304-1310.
(22) Qadan, L. R.; Perez-Stable, C. M.; Anderson, C.; D’Ippolito, G.;
Herron, A.; Howard, G. A.; Roos, B. A. 2-Methoxyestradiol induces
G2/M arrest and apoptosis in prostate cancer. Biochem. Biophys. Res.
Commun. 2001, 285, 1259-1266.
(23) Kumar, A. P.; Garcia, G. E.; Slaga, T. J. 2-Methoxyestradiol blocks
cell-cycle progression at G(2)/M phase and inhibits growth of human
prostate cancer cells. Mol. Carcinog. 2001, 31, 111-124.
(24) Lin, H. L.; Liu, T. Y.; Wu, C. W.; Chi, C. W. 2-Methoxyestradiol-
induced caspase-3 activation and apoptosis occurs through G(2)/M
arrest dependent and independent pathways in gastric carcinoma cells.
Cancer 2001, 92, 500-509.
(25) Yue, T. L.; Wang, X. K.; Louden, C. S.; Gupta, S.; Pillarisetti, K.;
Gu, J. L.; Hart, T. K.; Lysko, P. G.; Feuerstein, G. Z. 2-Methox-
yestradiol, an endogenous estrogen metabolite, induces apoptosis in
endothelial cells and inhibits angiogenesis: Possible role for stress-
activated protein kinase signaling pathway and Fas expression. Mol.
Pharmacol. 1997, 51, 951-962.
(26) 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. Biophys. Res.
Commun. 1998, 247, 616-619.
(27) Huang, P.; Feng, L.; Oldham, E. A.; Keating, M. J.; Plunkett, W.
Superoxide dismutase as a target for the selective killing of cancer
cells. Nature 2000, 407, 390-395.
(28) Qanungo, S.; Basu, A.; Das, M.; Haldar, S. 2-Methoxyestradiol
induces mitochondria dependent apoptotic signaling in pancreatic
cancer cells. Oncogene 2002, 21, 4149-4157.
References
(1) Day, J. M.; Newman, S. P.; Comninos, A.; Solomon, C.; Purohit,
A.; Leese, M. P.; Potter, B. V. L.; Reed, M. J. The effects of
2-substituted oestrogen sulphamates on the growth of prostate and
ovarian cancer cells. J. Steroid Biochem. Mol. Biol. 2003, 84, 317-
325.
(2) Raobaikady, B.; Purohit, A.; Chander, S. K.; Woo, L. W. L.; Leese,
M. P.; Potter, B. V. L.; Reed, M. J. Inhibition of MCF-7 breast cancer
cell proliferation and in vivo steroid sulphatase activity by 2-meth-
oxyoestradiol-bis-sulphamate. J. Steroid Biochem. Mol. Biol. 2003,
84, 351-358.
(3) Raobaikady, B.; Reed, M. J.; Leese, M. P.; Potter, B. V. L.; Purohit,
A. Inhibition of MDA-MB-231 cell cycle progression and cell
proliferation by C-2-substituted oestradiol mono- and bis-3-O-
sulphamates. Int. J. Cancer 2005, 117, 150-159.
(4) Suzuki, R. N.; Newman, S. P.; Purohit, A.; Leese, M. P.; Potter, B.
V. L.; Reed, M. J. Growth inhibition of multi-drug-resistant breast
cancer cells by 2-methoxyoestradiol-bis-sulphamate and 2-ethy-
loestradiol-bis-sulphamate. J. Steroid Biochem. Mol. Biol. 2003, 84,
269-278.
(5) Leese, M. P.; Leblond, B.; Smith, A.; Newman, S. P.; Di Fiore, A.;
Di Santo, R.; De Simone, G.; Supuran, C. T.; Purohit, A.; Reed, M.
J.; Potter, B. V. L. 2-Substituted estradiol bis-sulfamates, multitar-
geted antitumor agents: Synthesis, in vitro SAR, protein crystal-
lography, and in vivo activity. J. Med. Chem. 2006, 49, 7683-7696.
(6) Purohit, A.; Woo, L. W. L.; Barrow, D.; Hejaz, H. A. M.; Nicholson,
R. I.; Potter, B. V. L.; Reed, M. J. Non-steroidal and steroidal
sulfamates: New drugs for cancer therapy. Mol. Cell. Endocrinol.
2001, 171, 129-135.
(7) Reed, M. J.; Purohit, A.; Woo, L. W. L.; Newman, S. P.; Potter, B.
V. L. Steroid sulfatase: Molecular biology, regulation, and inhibition.
Endocr. ReV. 2005, 26, 171-202.
(8) Woo, L. W. L.; Purohit, A.; Malini, B.; Reed, M. J.; Potter, B. V. L.
Potent active site-directed inhibition of steroid sulphatase by tricyclic
coumarin-based sulphamates. Chem. Biol. 2000, 7, 773-791.
(9) Martucci, C. P. Metabolic fate of catechol estrogens. In Catechol
Estrogens; Merriam, G. R., Lipsett, M. B., Eds.; Raven Press: New
York, 1983; pp 115-121.
(29) 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.
(10) Ball, P.; Hoppen, H. O.; Knuppen, R. Metabolism of oestradiol-17
beta and 2-hydroxyoestradiol-17 beta. Hoppe-Seyler’s Z. Physiol.
Chem. 1974, 355, 1451-1462.
(30) Cushman, M.; He, H. M.; Katzenellenbogen, J. A.; Lin, C. M.; Hamel,
E. Synthesis, antitubulin and antimitotic activity, and cytotoxicity
of analogs of 2-methoxyestradiol, an endogenous mammalian
metabolite of estradiol that inhibits tubulin polymerization by binding
to the colchicine binding-site. J. Med. Chem. 1995, 38, 2041-2049.
(31) Cushman, M.; He, H. M.; Katzenellenbogen, J. A.; Varma, R. K.;
Hamel, E.; Lin, C. M.; Ram, S.; Sachdeva, Y. P. Synthesis of analogs
of 2-methoxyestradiol with enhanced inhibitory effects on tubulin
polymerization and cancer cell growth. J. Med. Chem. 1997, 40,
2323-2334.
(32) 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.
(11) Ball, P.; Stubenrauch, G.; Knuppen, R. Metabolism of 2-methoxy-
oestrone and 4-methoxyoestrone in man in vivo. J. Steroid Biochem.
Mol. Biol. 1977, 8, 989-993.
(12) Winum, J. Y.; Vullo, D.; Casini, A.; Montero, J. L.; Scozzafava, A.;
Supuran, C. T. Carbonic anhydrase inhibitors. Inhibition of cytosolic
isozymes I and II and transmembrane, tumor-associated isozyme IX
with sulfamates including EMATE also acting as steroid sulfatase
inhibitors. J. Med. Chem. 2003, 46, 2197-2204.
(13) Winum, J. Y.; Vullo, D.; Casini, A.; Montero, J. L.; Scozzafava, A.;
Supuran, C. T. Carbonic anhydrase inhibitors: Inhibition of trans-
membrane, tumor-associated isozyme IX, and cytosolic isozymes I
and II with aliphatic sulfamates. J. Med. Chem. 2003, 46, 5471-
5477.
(14) 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.
(15) Ho, Y. T.; Purohit, A.; Vicker, N.; Newman, S. P.; Robinson, J. J.;
Leese, M. P.; Ganeshapillai, D.; Woo, L. W. L.; Potter, B. V. L.;
Reed, M. J. Inhibition of carbonic anhydrase II by steroidal and non-
steroidal sulphamates. Biochem. Biophys. Res. Commun. 2003, 305,
909-914.
(16) Ireson, C. R.; Chander, S. K.; Purohit, A.; Perera, S.; Newman, S.
P.; Parish, D.; Leese, M. P.; Smith, A. C.; Potter, B. V. L.; Reed, R.
J. Pharmacokinetics and efficacy of 2-methoxyoestradiol and 2-meth-
oxyoestradiol-bis-sulphamate in ViVo in rodents. Br. J. Cancer 2004,
90, 932-937.
(17) Newman, S. P.; Ireson, C. R.; Tutill, H. J.; Day, J. M.; Parsons, M.
F. C.; Leese, M. P.; Potter, B. V. L.; Reed, M. J.; Purohit, A. The
role of 17â-hydroxysteroid dehydrogenases in modulating the activity
of 2-methoxyestradiol in breast cancer cells. Cancer Res. 2006, 66,
324-330.
(18) Seegers, J. C.; Aveling, M. L.; Vanaswegen, C. H.; Cross, M.; Koch,
F.; Joubert, W. S. The cytotoxic effects of estradiol-17â, catec-
holestradiols and methoxyestradiols on dividing MCF-7 and HeLa-
Cells. J. Steroid Biochem. Mol. Biol. 1989, 32, 797-809.
(19) Lottering, M. L.; Haag, M.; Seegers, J. C. Effects of 17-beta-estradiol
metabolites on cell cycle events in MCF-7 cells. Cancer Res. 1992,
52, 5926-5932.
(33) Rao, P. N.; Cessac, J. W.; Tinley, T. L.; Mooberry, S. L. Synthesis
and antimitotic activity of novel 2-methoxyestradiol analogs. Steroids
2002, 67, 1079-1089.
(34) Tinley, T. L.; Leal, R. M.; Randall-Hlubek, D. A.; Cessac, J. W.;
Wilkens, L. R.; Rao, P. N.; Mooberry, S. L. Novel 2-methoxyestradiol
analogues with antitumor activity. Cancer Res. 2003, 63, 1538-1549.
(35) Ho, Y. T.; Newman, S. P.; Purohit, A.; Leese, M. P.; Potter, B. V.
L.; Reed, M. J. The effects of 2-methoxy oestrogens and their
sulphamoylated derivatives in conjunction with TNF-alpha on
endothelial and fibroblast cell growth, morphology and apoptosis. J.
Steroid Biochem. Mol. Biol. 2003, 86, 189-196.
(36) Ro¨mer, J.; Steinbach, J.; Kasch, H.; Scheller, D. Herstellung und
Charakterisierung der Sulfamate von Estra-3,17ê-diolen. Schnelle
Umsetzung von 16R-Fluorestradiol zum 16R-Fluorestradiol-3,17â-
disulfamat. J. Prakt. Chem./Chem. Ztg. 1999, 341, 574-583.
(37) Patton, T. L. Estrogens. II. The synthesis of 2-dialkylaminomethyl-
estrogens. J. Org. Chem. 1960, 25, 2148-2152.
(38) Lunn, L. H. W.; Farkas, E. The adamantyl carbonium ion as a
dehydrogenating agent, its reactions with estrone. Tetrahedron 1968,
24, 6773-6776.
(39) Miller, C. P.; Jirkovsky, I.; Hayhurst, D. A.; Adelman, S. J. In vitro
antioxidant effects of estrogens with a hindered 3-OH function on
the copper-induced oxidation of low density lipoprotein. Steroids
1996, 61, 305-308.

Disubstituted 2-Alkylestra-1,3,5(10)-trien-3-ols
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18 4443
(40) 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.
(41) Patton, T. L. Estrogens. IV. The synthesis of 2- and 4-alkylestrones.
J. Org. Chem. 1962, 27, 910-914.
(48) Scho¨necker, B.; Tresselt, D.; Draffehn, J.; Ponsold, K.; Engelhardt,
G.; Zeigan, D.; Schneider, G.; Weiszvincze, I.; Dombi, G. Steroide.
LIII. 1H-NMR-Untersuchungen. Konfigurationszuordnung von 16-
substituierten 17-Hydroxy-Steroiden. J. Prakt. Chem. 1977, 319,
419-431.
(49) Purohit, A.; Williams, G. J.; Howarth, N. M.; Potter, B. V. L.; Reed,
M. J. Inactivation of steroid sulfatase by an active-site-directed inhi-
bitor, estrone-3-O-sulfamate. Biochemistry 1995, 34, 11508-11514.
(50) Appel, R.; Berger, G. U¨ ber das Hydrazodisulfid. Chem. Ber. 1958,
91, 1339-1341.
(51) 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 inhibit estrone
sulphatase by a different mechanism. J. Steroid Biochem. Mol. Biol.
1996, 57, 79-88.
(42) Leese, M. P.; Hejaz, H. A. M.; Mahon, M. F.; Newman, S. P.; Purohit,
A.; Reed, M. J.; Potter, B. V. L. A-ring-substituted estrogen-3-O-
sulfamates: Potent multitargeted anticancer agents. J. Med. Chem.
2005, 48, 5243-5256.
(43) Gonzalez, F. B.; Neef, G.; Eder, U.; Wiechert, R.; Schillinger, E.;
Nishino, Y. Synthesis and pharmacological evaluation of 8R-estradiol
derivatives. Steroids 1982, 40, 171-187.
(44) Nambara, T.; Honma, S.; Akiyama, S. Studies on steroid conjugates.
III. New syntheses of 2-methoxyestrogens. Chem. Pharm. Bull. 1970,
18, 474-480.
(52) Biellman, J. F.; Branlant, G. Synthesis and circular-dichroism of
substituted benzoquinones. Bull. Soc. Chim. Fr. 1973, 2086-2090.
(53) Prokai, L.; Oon, S. M.; Prokai-Tatrai, K.; Abboud, K. A.; Simpkins,
J. W. Synthesis and biological evaluation of 17 beta-alkoxyestra-
1,3,5(10)-trienes as potential neuroprotectants against oxidative stress.
J. Med. Chem. 2001, 44, 110-114.
(45) Kemperman, G. J.; Roeters, T. A.; Hilberink, P. W. Cleavage of
aromatic methyl ethers by chloroaluminate ionic liquid reagents. Eur.
J. Org. Chem. 2003, 1681-1686.
(46) Pert, D. J.; Ridley, D. D. Preparation of 2-isopropyloestradiol. Aust.
J. Chem. 1988, 41, 1145-1148.
(54) Rao, P. N.; Cessac, J. W. A new, practical synthesis of 2-methox-
yestradiols. Steroids 2002, 67, 1065-1070.
(47) Okada, M.; Iwashita, S.; Koizumi, N. Efficient general method for
sulfamoylation of a hydroxyl group. Tetrahedron Lett. 2000, 41,
7047-7051.
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