Synthesis and evaluation of analogues of estrone-3-O-sulfamate as potent steroid sulfatase inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.03.007

Estrone sulfamate (EMATE) is a potent irreversible inhibitor of steroid sulfatase (STS). In order to further expand SAR, the compound was substituted at the 2- and/or 4-positions and its 17-carbonyl group was also removed. The following general order of p

Full text of DOI:10.1016/j.bmc.2012.03.007

Bioorganic & Medicinal Chemistry 20 (2012) 2506–2519
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of analogues of estrone-3-O-sulfamate as potent
steroid sulfatase inhibitors
L.W. Lawrence Woo a, Bertrand Leblond a, Atul Purohit b, Barry V.L. Potter a,
⇑
a Medicinal Chemistry, Department of Pharmacy & Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, UK
b Diabetes, Endocrinology & Metabolism, Imperial College London, Section of Investigative Medicine, 6th Floor, Commonwealth Building (6N2B), Hammersmith Hospital,
Du Cane Road, London W12 0NN, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Estrone sulfamate (EMATE) is a potent irreversible inhibitor of steroid sulfatase (STS). In order to further
expand SAR, the compound was substituted at the 2- and/or 4-positions and its 17-carbonyl group was
also removed. The following general order of potency against STS in two in vitro systems is observed for
the derivatives: The 4-NO2 > 2-halogens, 2-cyano > EMATE (unsubstituted) > 17-deoxyEMATE > 2-
NO2 > 4-bromo > 2-(2-propenyl), 2-n-propyl > 4-(2-propenyl), 4-n-propyl > 2,4-(2-propenyl) = 2,4-di-n-
propyl. There is a clear advantage in potency to place an electron-withdrawing substituent on the A-ring
with halogens preferred at the 2-position, but nitro at the 4-position. Substitution with 2-propenyl or n-
propyl at the 2- and/or 4-position of EMATE, and also removal of the 17-carbonyl group are detrimental
to potency. Three cyclic sulfamates designed are not STS inhibitors. This further confirms that a free or
N-unsubstituted sulfamate group (H2NSO2O–) is a prerequisite for potent and irreversible inhibition of
STS as shown by inhibitors like EMATE and Irosustat. The most potent derivative synthesized is 4-
nitroEMATE (2), whose IC50s in placental microsomes and MCF-7 cells are respectively 0.8 nM and
0.01 nM.
Received 9 January 2012
Revised 28 February 2012
Accepted 1 March 2012
Available online 8 March 2012
Keywords:
Steroid sulfatase
Inhibitors
EMATE
Endocrine therapy
Breast cancer
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Despite the large number of compounds that have been explored
in this category, none of these compounds was deemed to be po-
It is reasoned that steroid sulfatase (STS) has a significant role in
fueling the growth and development of hormone-dependent dis-
eases, since the enzyme hydrolyses biologically inactive steroid
sulfates back to their active unconjugated forms. In hormone-
dependent breast cancer for example, STS is responsible for the
conversion of estrone sulfate (E1S) to estrone (E1) and also dehy-
droepiandrosterone sulfate (DHEA-S) to DHEA. The formation of
DHEA by the STS pathway constitutes the production of 90% of
androstenediol which is an estrogen (ca. 100-fold weaker than
estradiol), despite structurally being classified as an androgen.1–4
On this basis, the inhibition of STS in this type of cancer might lead
to estrogen deprivation and hence render therapeutic intervention
in the malignancy.
Once STS had been identified as a target for delivering a poten-
tial new form of endocrine therapy, considerable attention was
placed on designing inhibitors of this enzyme over the past two
decades. One approach was to replace the sulfate group ðOSO3ꢀÞ
of E1S with other sulfate surrogates or mimics that are not meta-
bolically labile or hydrolysable by the enzyme in the course of
competing with E1S for binding to the enzyme active site.5–9
tent enough or adequately attractive pharmaceutically for further
development as an inhibitor of STS.
The breakthrough in the design of potent STS inhibitors came
when the sulfate group of E1S was replaced by a sulfamate moiety
(–OSO2NH2).10 Estrone 3-O-sulfamate (EMATE) (Fig. 1) was found
to be a highly potent inhibitor with an IC50 of 18 nM in a human pla-
cental microsomes preparation.11 More exceptionally, EMATE
inhibits STS in a time- and concentration-dependent manner, indi-
cating that the mechanism of action is of an irreversible nature.
EMATE is orally active in vivo. In one study,12 administration at
10 mg/kg inhibited rat liver STS activity almost completely (99%)
when given by the oral or subcutaneous route and the inhibition
persisted (>95%) for up to 7 days after a single dosing. However,
EMATE was unexpectedly shown to be more estrogenic than ethiny-
lestradiol when administered orally in rats,13 although it was re-
vealed subsequently that estrogen sulfamates per se do not bind
to the estrogen receptor.14 A follow-up study demonstrated that
STS has a crucial role in regulating the estrogenicity associated with
EMATE as its estrogenicity is abolished when STS is inhibited by co-
administration of the non-estrogenic STS inhibitor Irosustat (STX64,
Fig. 1).15 Nonetheless, this unwanted estrogenic property rendered
the inhibitor undesirable for use as an anti-endocrine agent for hor-
mone-dependent cancers. However, a congener of EMATE, estradiol
⇑
Corresponding author. Tel./fax: +44 1225 386114.
E-mail address: b.v.l.potter@bath.ac.uk (B.V.L. Potter).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.007

L. W. Lawrence Woo et al. / Bioorg. Med. Chem. 20 (2012) 2506–2519
2507
O
geometrical isomers obtained was particularly cumbersome. 2-
(2-Propenyl)estrone (4a, Scheme 2) was only isolated in poor yield
upon repeated and slow (over two weeks) fractional recrystalliza-
tions of the crude material in diethyl ether. Despite careful frac-
tional recrystallization of the crude 4-(2-propenyl)estrone (5a,
Scheme 2), this isomer could not be obtained in a pure state and
the best sample in our hands contained approximately 25% of 4a
as shown by 1H NMR. When a mixture of 2-(2-propenyl)- and
4-(2-propenyl)estrones in DMF was heated with 3-bromoprop-1-
ene in the presence of NaH, a mixture of the 3-O-(2-propenyl)
derivatives of 4a and 5a (6 and 7, respectively, Scheme 2) was
obtained. When this mixture was subjected to Claisen rearrange-
ment conditions 2,4-di-(2-propenyl)estrone (8a, Scheme 2) was
obtained in good yield. Compounds 4a, 5a and 8a were sulfamoy-
lated in the same manner as 1a to give sulfamates 4, 5 and 8,
respectively.
Initially, 2-n-propyl- (9), 4-n-propyl- (10) and 2,4-di-n-propyl-
(11) EMATEs were obtained by sulfamoylating the corresponding
parent phenols that were obtained upon hydrogenation of the (2-
propenyl)estrones. However, it was found subsequently that the
sulfamate group is not labile to the hydrogenation conditions em-
ployed and hence 2-(2-propenyl)-(4), 4-(2-propenyl)-(5) and 2,4-
n-di-(2-propenyl)-(8) EMATEs were hydrogenated directly to give,
respectively, 2-n-propyl-(9) and 4-n-propyl- (10) and 2,4-di-n-pro-
pyl-(11) EMATEs (Scheme 3). It is worth noting that while repeated
attempts to recrystallize 5 had failed to afford a pure sample other
than a batch that was co-recrystallized with 4, pure 10 without
contaminating by isomer 9 was obtained when the crude material
was recrystallized from ethyl acetate/hexane.
H2NO2SO
H2NO2SO
O
O
Irosustat
EMATE
Figure 1. Structures of EMATE (estrone 3-O-sulfamate) and the non-steroidal STS
inhibitor, Irosustat (STX64, BN83495).
3-O-sulfamate, entered human clinical trials in the late 90s as an or-
ally active pro-drug for estradiol and good activity has also been re-
ported in animal models of endometriosis, as an STS inhibitor.16 It is
currently in Phase IIa clinical trial for endometriosis.
As EMATE remains an interesting compound because of its
diverse pharmacological properties, we further expand the SAR
studies in this work. To this end, we substituted at the 2- and/or
4-position of A-ring with a nitro group, halogens, alkyl groups
and a cyano group. The D-ring was modified by removal of the
C17 carbonyl group. In addition, three compounds were prepared
which can be loosely described as ‘cyclic sulfamate’ derivatives of
EMATE as the N-atom of the sulfamate group of EMATE is bonded
to either the 2- or 4-position of the steroid scaffold. The synthe-
sized compounds were tested for STS inhibitory activity in a pla-
cental microsomes preparation and also in MCF-7cells.
2. Chemistry
2-Nitroestrone (1a, Scheme 1) and 4-nitroestrone (2a, Scheme
1) were prepared by nitrating estrone according to the method of
Tomson and Horwitz.17 Compound 2a came out of the reaction
mixture overnight and was deposited as a yellow precipitate
whereas 1a was isolated from the filtrate and purified by several
cycles of recrystallization. An earlier method was employed for
the sulfamoylation of these nitrated estrones (Scheme 1). This in-
volved treating a solution of 1a or 2a in anhydrous N,N-dimethyl-
formamide (DMF) with sodium hydride (NaH) followed by addition
of a freshly concentrated solution of sulfamoyl chloride in toluene,
which was prepared according to the method of Woo et al.18
The synthesis of 2- and 4-alkylestrone was carried out as de-
scribed by Patton.19 A Claisen rearrangement of the intermediate
3-O-(2-propenyl)estrone (3, Scheme 2) to give 2- and 4-(2-prope-
nyl)estrone was straightforward although the separation of the
The route to 2-fluoroestrone 12b (Scheme 4) was described by
Page et al.20 and involved an electrophilic fluorination of estrone
by Umemoto’s reagent, N-fluoropyridinium triflate, followed by a
direct acetylation using acetic anhydride in pyridine in order to
facilitate purification of the fluorinated estrone derivative 12a. In
our hands, a low yield (12%, after recrystallization) of 12a was ob-
tained from estrone, primarily as a result of competing formation
of other by-products (presumably 2- and 4-chloroestrones). Upon
deacetylation of 12a using potassium carbonate in methanol, 2-
fluoroestrone (12b) was obtained which was purified by prepara-
tive TLC (45% yield). Sulfamoylation of 12b with sulfamoyl chloride
in the presence of 2,6-di-tert-butyl-4-methylpyridine (DBMP) as
base gave 2-fluoroEMATE (12) in 57 % yield.
Several procedures for preparing the 2-chloro, 2-bromo, 2-iodo
and 2-cyano derivatives of estrone are available but few of these
O
O
O
O2N
O2N
HO
b
1
2
1a
H2NO2SO
O
O
b
a
H2NO2SO
HO
2a
HO
NO2
NO2
O
O2N
HO
not isolated
NO2
Scheme 1. Nitration of estrone to give predominately 2-nitroestrone (1a) and 4-nitroestrone (2a) which upon sulfamoylation gave their respective sulfamates (1) and (2).
Reagents and conditions: (a) concd HNO3/glacial acetic acid, 70–75 °C to rt; (b) NaH/DMF, H2NSO2Cl, 0 °C.

2508
L. W. Lawrence Woo et al. / Bioorg. Med. Chem. 20 (2012) 2506–2519
O
O
4
X
O
O
HO
O
c
a
O
b
6
8a
HO
O
4a
O
HO
a
O
b
+
+
3
O
c
O
7**
5a
O
HO
O
c
8
X
5*
X
Scheme 2. Synthesis of 2-(2-propenyl)estrone 3-O-sulfamate (4), 4-(2-propenyl)estrone 3-O-sulfamate (5) and 2,4-di-(2-propenyl)estrone 3-O-sulfamate (8) via various
intermediates (3), (4a), (5a), (6), (7) and (8a). Reagents and conditions: (a) NaH/DMF, 3-bromoprop-1-ene, 80 °C, 2 h; (b) N,N-diethylaniline, N2, reflux, 6 h; (c) NaH/DMF,
H2NSO2Cl, 0 °C. X = OSO2NH2 ⁄contains ca. 25% of 4a as shown by 1H NMR; ⁄⁄impure, contains 6.
O
of bromine in acetic acid in the presence of a small amount of pow-
dered iron. In contrast, when aqueous acetic acid was replaced
with glacial acetic acid, the authors reported that the bromination
reaction yielded predominately 2-bromoestrone. However, in our
hands, 19a was obtained as the main product when a solution of
estrone in glacial acetic acid (Scheme 4) was brominated under
the conditions used. The sulfamoylation of 19a to give sulfamate
19 was carried out in the same manner as 1a.
The carbonyl group of estrone was reduced to a methylene
group under Wolff–Kishner conditions for reduction of steroid ke-
tones.22 The product 3-hydroxy-1,3,5(10)-estratriene (20a, Scheme
5) was sulfamoylated in the same manner as 1a to give
1,3,5(10)estratriene 3-O-sulfamate (20).
O
a
a
9
4
X
X
X
X
O
O
10*
5
O
O
The chemistry developed by Andersen et al.23,24 for the synthe-
sis of 1,2,3-benzoxathiazole-2,2-dioxides was applied to the syn-
thesis of ‘cyclic sulfamates’ (21), (22) and (23). The starting
2-amino (21a) and 4-aminoestrone (23a) were easily obtained by
hydrogenating 2-nitro- (1a) and 4-nitroestrone (2a) respectively
in the presence of palladium-charcoal. Whilst 23a appears to be
stable, decomposition of 21a upon standing and exposure to air
at room temperature was observed, as reported by Kraychy,25
and hence 21a was used without further purification. The selective
N-tosylation of 21a and 23a with tosyl chloride to give 2- (21b)
and 4-tosylamidoestrone (23b), respectively, was carried out in
the presence of pyridine using conditions reported by Kurita26
for the selective N-tosylation of o-aminophenol. Ring closure of
21b and 23b to give the respective cyclic compounds 21c and
23c was carried out with sulfuryl chloride in dichloromethane. Re-
moval of the protecting tosyl group of 21c and 23c with an aque-
ous solution of potassium fluoride gave 21 and 23 in moderate to
good yields. Deprotonation of 21 in DMF with NaH followed by
refluxing with methyl iodide gave the N-methylated derivative 22.
a
8
11
X
X
Scheme 3. Hydrogenation of (2-propenyl)estrone sulfamates (4), (5) and (8) to give
respectively 2-n-propylestrone 3-O-sulfamate (9), 4-n-propylestrone 3-O-sulfamate
(10) and 2,4-di-n-propylestrone 3-O-sulfamate (11). Reagents: (a) Pd-C(10%)/
absolute EtOH, H2, 50 psi. X = OSO2NH2 ⁄impure.
are regioselective, that is functionalizing predominantly at position
2. Compounds 15b–18b (Scheme 4) were prepared as described by
Page et al.20 The direct arene thalliation with thallic trifluoroace-
tate (or triacetate) in trifluoroacetic acid and subsequent displace-
ment
of
the
estrogen–thallium(III)
bis(trifluoroacetate)
intermediate with copper(I) halides (halides = Cl, Br, I) and cop-
per(I) cyanide gave a convenient regioselective method for the syn-
thesis of 2-substituted estrones. Sulfamoylation of compounds
15b–18b was carried out in a similar manner to 1a to give the sul-
famates 15–18.
3. Biological results and discussion
Slaunwhite and Neely21 reported that 4-bromoestrone (19a)
was obtained in high yield when a solution of estrone in aqueous
acetic acid (contained 15–20% of water) was treated with 5% v/v
The in vitro inhibitory activities of compounds against STS in a
placental microsomes preparation are shown in Table 1. We re-
ported the biological activity of 1, 2, 4, 5, 8–11 earlier27 but herein

L. W. Lawrence Woo et al. / Bioorg. Med. Chem. 20 (2012) 2506–2519
2509
O
O
O
O
F
F
d
F
c
a,b
12b
12a
12
X
HO
AcO
HO
O
e
O
O
R
15a
16a
17a
18a
Cl
Br
I
R
(F3COCO)2Tl
AcO
g
f
i
13
CN
14
AcO
AcO
c
O
O
O
R
R
15b
16b
17b
18b
15
Cl
Br
I
Cl
Br
I
R
R
X
16
17
18
h
R
R
19a
19
OH
X
h
HO
CN
CN
Br
Scheme 4. Synthesis of 2- and 4-substituted derivatives of EMATE. Reagents and conditions: (a) N-fluoropyridinium triflate/1,2-trichloroethane, reflux, N2, 24 h; (b) pyridine/
acetic anhydride, reflux, 2 h; (c) K2CO3/MeOH, reflux, 3 h; (d) DBMP/DMF, H2NSO2Cl, 0 °C; (e) (CH3CO)2O/pyridine, reflux, 2 h; (f) Tl(OCOCF3)3/CF3COOH, 0–5 °C, N2, 24 h; (g)
CuX/1,4-dioxane (X = Cl, Br, I, CN); (h) NaH/DMF, H2NSO2Cl, 0 °C; (i) Br2/glacial acetic acid/powdered Fe, rt. X = OSO2NH2.
O
chemically and/or less effective in inactivating the enzyme via sul-
famoylation.27 Due to steric hindrance presented by the neigh-
bouring hydrogen atom(s) at C6, the nitro group at the 4-position
of 2 might not be amenable to having potential interactions(s) with
its sulfamate group at the 3-position, rendering it relatively more
stable and effective in sulfamoylating the enzyme.27 Another pos-
sible explanation for the higher STS inhibitory activity observed for
2 is that its 4-nitro group might interact with the enzyme active
site favorably. A recent study carried out by Taylor and co-work-
R
a
OH 20a
b
X
20
R
HO
Scheme 5. Wolff–Kishner reduction of estrone and synthesis of sulfamate 20.
Reagents and conditions: (a) NH2NH2ꢁH2O, KOH/diethylene glycol, reflux; (b) NaH/
DMF, H2NSO2Cl, 0 °C. X = OSO2NH2.
ers42 has shown that 2-nitroestrone (1a, IC50 = 17
fold less potent in a purified STS assay as an STS inhibitor than its
4-nitro isomer (2a, IC50 2.4 M). The authors attributed this finding
lM) was seven-
report full experimental data. Importantly, in order to assess the
ability of compounds to cross the cell membrane and inhibit STS
under conditions that closely resemble the tissue/physiological sit-
uation, we newly evaluate compounds (apart from 21–23) in intact
MCF-7 cells. The results are shown in Table 1.
l
to factors such as hydrogen bonding ability and/or other interac-
tions of the nitro group placed at the 4-position of the steroid ring.
Favorable interactions between a substituent and neighboring
amino acid residue(s) in the active site have been observed before.
For instance, 2-difluoromethylestrone 3-O-sulfamate was found to
have an IC50 of 100 pM against STS in a placental microsomes prep-
aration.28 It has been reasoned that the electron-withdrawing ef-
fect of the 2-fluoromethyl group as well as the potential of the
fluorine atoms in forming hydrogen bond with residues lining
the catalytic active site of STS are contributive factors to the high
potency observed.
Derivatives 12 and 15–17, which are substituted with a halogen
at the 2-position, are clearly more potent STS inhibitors than
EMATE when tested in placental microsomes. The activities of these
halogenated compounds are of the same order of magnitude,
although the bromo congener is the most potent inhibitor of the
series with an IC50 of 1.65 nM. When tested in MCF-7 cells, only
15 and 16 are significantly more potent than EMATE. The activities
of 12 and 17 are considered to be similar to that of EMATE. Given
the pKa value of their respective parent phenol is similar at around
8.5 (Table 2), other factors such as the lipophilicity of compounds
might influence the different in vitro activity observed. In general,
a more lipophilic inhibitor has a higher affinity for the enzyme
active site and hence exhibits better inhibitory activity than one
that is less lipophilic. As fluoro derivative 12 is the least lipophilic,
whereas iodo derivative 17 is the most lipophilic, one might expect
the potency of these 2-halogenated derivatives of EMATE against
STS to be in an ascending order from 12 to 17. However, the fact that
17 (IC50(P.M.) = 6.1 nM and IC50(MCF-7) = 0.81 nM, Table 1) is a weaker
STS inhibitor than its bromo congener 16 (IC50(P.M.) = 1.65 nM and
We have demonstrated repeatedly in previous work that the
STS inhibitory activity of an aryl sulfamate in general can be in-
creased by either placing an electron-withdrawing group adjacent
to the sulfamate group,30–38 or by having the sulfamate group at-
tached to an aryl ring system such as coumarin,39–41 that renders
the parent phenol a better leaving group by virtue of lowering its
pKa value. With the exception of 1, the same effect is observed
for 2, 12, 15–18 when tested in a placental microsome preparation
since these sulfamates were found to be more potent than EMATE
as STS inhibitors. A similar trend is observed for STS inhibition in
MCF-7 cells with the exception of 17 and 18 which are comparable
and slightly weaker inhibitors than EMATE, respectively. As shown
in Table 2, the pKa values of various substituted phenols, 2-nitroe-
strone (1a) and 4-nitroestrone (2a) are between 1 and 3 log units
lower than that of an unsubstituted phenol such as estrone. We
postulate in general that this lowering of pKa value of the parent
phenol improves its leaving group ability which in return facili-
tates the inactivation of the enzyme by its corresponding sulfamate
through sulfamoylation of an essential amino acid residue in the
active site.
Unexpectedly, 2-nitroEMATE (1) is less potent than EMATE as
an STS inhibitor while its congener 4-nitroEMATE (2) is signifi-
cantly more potent than EMATE despite their parent phenols shar-
ing a similar calculated pKa value of around 7 (Table 1). We reason
that this result might be due to potential interaction(s) between
the nitro group at the 2-position and the sulfamate group at the
3-position in 1, rendering the sulfamate group either less stable

2510
L. W. Lawrence Woo et al. / Bioorg. Med. Chem. 20 (2012) 2506–2519
Table 1
Inhibition of STS in a placental microsomes (P.M.) preparation and in MCF-7 cells by derivatives of EMATE 1, 2, 4, 5, 8–12, 15–18, 19 and 20, and ‘cyclic sulfamates’ 21–23
O
R1
20
H2NO2SO
H2NO2SO
O
R2
O
O
H
N
N
O
23
O
O2S
O2S
O2S
O
21
22
NH
Compdd
R1
R2
P.M. IC50 (nM)
MCF-7 IC50 (nM)/% inhibitiona
a
a
EMATE
1
2
H
NO2
H
2-Propenyl
H
2-Propenyl
n-Propyl
H
H
NO2
H
2-Propenyl
2-Propenyl
18b
0.83d
8.3
0.01
37
153
<10%@10
236
<10%@10
<10%@10
0.62
0.44
0.11
0.81
1.2
42
97%@0.01
nd
70c
0.8c
4
2500c
9000c
>10,000c
2900c
>10,000c
>10,000c
5.6
5e
8
9
lM
H
10
11
12
15
16
17
18
19
20
21
22
23
H
n-Propyl
n-Propyl
l
l
M
M
n-Propyl
F
H
H
H
H
H
Br
Cl
Br
I
CN
H
—
—
—
—
3.4
1.65
6.1
5.2
1200
199
>10,000
>10,000
>10,000
lM
nd
nd
a
b
c
Unless stated otherwise, errors are <5% of the reported value (from triplicate experiments).
Ref. 11, cf. 0.4 nM27, 9.1 nM28
Ref. 27.
.
d
e
IC50 0.065 nM.29
Contains 25% of 2-(2-propenyl)EMATE (4); — not applicable; nd: not determined.
to our work here, it is not clear whether halogen-oxygen bonding
plays a role in the binding of halogenated EMATEs to the STS active
site.
While brominating EMATE at the 2-position results in a highly
potent STS inhibitor 16, substitution of EMATE at the 4-position
with a bromine atom is detrimental to activity. As shown in Table
2, the IC50 values of the isomer 4-bromoEMATE (19) against STS in
placental microsomes (1200 nM) and MCF-7 cells (42 nM) are a
Table 2
pKa values of various phenols, 2-nitroestrone (1a) and 4-nitroestrone (2a) as
determined by ACD/Labs Software v 11.02
Compound
pKa
Phenol
9.86 0.10
8.71 0.10
8.50 0.10
8.43 0.10
8.52 0.10
7.17 0.10
7.14 0.14
7.43 0.40
7.10 0.40
2-Fluorophenol
2-Chlorophenol
2-Bromophenol
2-Iodophenol
2-Cyanophenol
2-Nitrophenol
1a
few orders of magnitude lower than those of EMATE (IC50(P.M.)
=
18 nM and IC50(MCF-7) = 0.83 nM) and 16 (IC50(P.M.) = 1.65 nM and
IC50(MCF-7) = 0.11 nM). Given the electron-withdrawing effect of
the bromine atom in 16 and 19 is expected to be similar, it is pos-
sible that any beneficial interaction provided by a bromine atom is
better recognized by the enzyme active site when it is substituted
at the 2-position of EMATE.
2a
In placental microsomes, the IC50 value observed for 20 (17-
deoxyEMATE, IC50 = 199 nM, Table 2) is 11-fold higher than that
of EMATE (IC50 = 18 nM, Table 2). Hence, removal of the 17-
carbonyl group of EMATE significantly reduces STS inhibitory
activity, demonstrating an important role for hydrogen bonding
between the D-ring of steroidal inhibitor and the active site.
However, not all disruptions made to the D-ring of EMATE are
detrimental. Previous work has shown that 3-sulfamoyloxy-
16,17-seco-estra-1,3,5(10)-triene-16,17-imide, a steroid-like deriv-
ative of EMATE, is equipotent to EMATE as an STS inhibitor.11
As shown in Table 1, all derivatives of EMATE substituted at the
2- and/or 4-positions with an aliphatic group are weak STS inhib-
itors. However, substitution of 2-propenyl or n-propyl group at the
2-position is better tolerated by the enzyme than that at the
IC50(MCF-7) = 0.11 nM, Table 1) suggests that steric hindrance ren-
dered by the bulkier iodine atom of 17 interferes with the binding
of its sulfamate group to the catalytic site, preventing the inhibitor
from inactivating the enzyme effectively. Hence, for derivatives of
EMATE substituted at the 2-position with a halogen, it appears that
having a bromine atom is most optimized for STS inhibition and
binding of the inhibitor to STS active site. It is worth noting that a
recent study carried out by Lu et al.43 provides evidence for weak
halogen-oxygen bonding between halogenated ligands and protein
kinases, with the strength of the interactions calculated decreases
in the order H ꢂ I > Br > Cl. Although it is much weaker than hydro-
gen bonding, the authors argue that halogen-bonding may play an
important role in inhibitor recognition and binding. With reference

L. W. Lawrence Woo et al. / Bioorg. Med. Chem. 20 (2012) 2506–2519
2511
4-position as shown by the higher inhibitory activity observed for
4 (IC50(P.M.) = 2500 nM and IC50(MCF-7) = 37 nM) and 9 (IC50(P.M.)
2900 nM and IC50(MCF-7) = 236 nM) than 5 (IC50(P.M.) = 9000 nM
and IC50(MCF-7) = 153 nM) and 10 (IC50(P.M. MCF-7)s:
cyclic sulfamate moiety of 19, 20 and 21 did not bind or align in
a similar manner to the sulfamate group of EMATE in the catalytic
site of STS for potential attack by a nucleophilic amino acid. Alter-
natively, the lack of an unsubstituted sulfamate group in 19, 20 and
21 might be the culprit, as it has been shown earlier that a free sul-
famate group (H2NSO2OAr) is prerequisite for potent irreversible
inhibition of STS.10,41,46
=
and
>10,000 nM). One possible reason for this difference in activity be-
tween 2- and 4-alkylated derivatives is the steric hindrance ex-
erted by the hydrogen atom(s) at the neighboring C6 atom on
the substituent at the 4-position, restricting the rotation of alkyl
groups and hence making them less tolerated by the enzyme.
When considering substitution made at either the 2- or 4-positon
alone, apparently there is no significant difference in the inhibitory
activities observed between the more rigid 2-propenyl group and
the more flexible n-propyl group (4 vs 9, and 5 vs 10), suggesting
that both groups are not well tolerated by the enzyme active site.
Clearly, the steric hindrance posed by these groups is expected to
be even more prominent when they are substituted at both the
2- and 4-positions of EMATE (8 and 11). The shielding of the sulfa-
mate group from interacting effectively with the catalytic site of
the enzyme by these aliphatic substituents might explain the over-
all weak STS inhibitory activity observed for compounds 4, 5, 8, and
9–11. It is interesting to note that 2-methylEMATE, a smaller chain
relative of 9, was found to be even a weaker STS inhibitor with an
IC50 of over 10,000 nM in a placental microsomes preparation.28
Despite a methyl group often being viewed to be isosteric and iso-
lipophilic with the chlorine atom,44 which means steric hindrance
might not be the main detrimental factor, the hyperconjugative
electron-donating effect of the methyl group of 2-methylEMATE
on the A-ring, as reflected by the calculated pKa of 10.31 for 2-
methylphenol, might render the sulfamate group less effective in
inactivating STS via sulfamoylation. The best 2-alkyl derivative of
EMATE reported to date as an inhibitor of STS is 2-ethylEMATE.
Its IC50 in a placental microsomes preparation was found to be
820 nM.45
We postulated in previous work several mechanisms through
which an aryl sulfamate (e.g., EMATE) might inhibit STS.7,40 Two
of which are (i) nucleophilic attack on the sulfur atom of the sulfa-
moyl group by the hydrated form of formylglycine residue (gem-
diol) in the STS active site; and (ii) specific or non-specific sulfa-
moylation by an aryl sulfamate of an essential nucleophilic amino
acid residue in the STS active site. Andersen et al.23,24 reported that
some 1,2,3-benzoxathiazole 2,2-dioxides (e.g., 22 and 23, Fig. 2)
are reactive toward nucleophiles. When the candidate was 3-to-
syl-1,2,3-benzoxathiazole 2,2-dioxides (22), hydroxide ion and
amines attacked the endocyclic sulfur atom of cyclic sulfamate,
resulting in ring opening. Given this observation, we prepared
the cyclic sulfamate derivatives of EMATE, 19, 20 and 21
(Scheme 6). The rationale is that if a nucleophilic residue in the en-
zyme active site attacks the sulfur atom of cyclic sulfamate, these
cyclic sulfamates might exhibit STS inhibitory activities. In contrast
to acyclic sulfamates like EMATE, where we expect the parent phe-
nol to be released upon sulfamoylation of the enzyme by the sulfa-
mate group, there should be no release of estrone expected for
cyclic sulfamates 19, 20 and 21 upon ring opening as their steroid
skeleton should remain attached to the sulfamoyl group via its N-
atom. Unfortunately, as shown in Table 2, none of these cyclic sul-
famates was found to be active against STS. It is possible that the
4. Conclusions
A series of 2- and/or 4-substituted derivatives of EMATE was pre-
pared and its 17-carbonyl group removed. The following general or-
der of potency against STS is observed: The 4-NO2 > 2-halogens, 2-
cyano > unsubstituted
(EMATE) > 17-deoxyEMATE > 2-NO2 > 4-
bromo > 2-(2-propenyl), 2-n-propyl > 4-(2-propenyl), 4-n-pro-
pyl > 2,4-(2-propenyl) = 2,4-di-n-propyl. The general SAR derived
from the studies indicates that EMATE derivatives that have elec-
tron-withdrawing substituents (4-nitro, 2-halogens and 2-cyano)
on the A-ring, show a higher STS inhibition than EMATE in vitro,
with the exception of having a nitro group at the 2-position which
results in a significant lowering in potency. This reinforces the rela-
tionship between the pKa value/leaving group ability of a parent
phenol and the potential of an aryl sulfamate in the inactivation of
STS, presumably through sulfamoylation of an essential amino acid
residue. The introduction of a bromine at the 4-position of or re-
moval of the 17-carbonyl group of EMATE is detrimental to activity.
The derivatives resulted are significantly weaker STS inhibitors than
EMATE. When the 2- and/or 4-positions of the A-ring are substituted
with the bulkier 2-propenyl or 2-n-propyl groups, the potent STS
inhibition shown by EMATE is significantly reduced or abolished.
For an aryl sulfamate to be effective as an STS inhibitor, inter alia,
its sulfamate group must be presented to the catalytic site of the en-
zyme unhindered and the required interactions with various key
amino acid residues in the active site uninterrupted. Three cyclic
sulfamates prepared are not STS inhibitors. This further confirms
that a free or N-unsubstituted sulfamate group (H2NSO2O–) is a pre-
requisite for potent and irreversible inhibition of STS as shown by
inhibitors like EMATE and Irosustat.
5. Experimental
5.1. General
All chemicals were purchased from either Aldrich Chemical Co.
(Gillingham, UK) or Lancaster Synthesis (Morecombe, Lancashire,
UK). All organic solvents of A.R. grade were supplied by Fisher Sci-
entific (Loughborough, UK). Anhydrous N,N-dimethylformamide
(DMF), dichloromethane and pyridine were purchased from Al-
drich. Sulfamoyl chloride was prepared by an adaptation of the
method of Appel and Berger47 and was stored as a solution under
N2 in toluene as described by Woo et al.48
Thin layer chromatography (TLC) was performed on pre-coated
plates (Merck TLC aluminium sheets Silica Gel 60 F254, Art. No.
5554). Product(s) and starting material were detected by viewing
under UV light and/or treating with a methanolic solution of phos-
phomolybdic acid followed by heating. Flash column chromatogra-
phy was performed using gradient elution (solvents indicated in
text) on wet-packed silica gel (Sorbsil C60). IR spectra were deter-
mined by a Perkin–Elmer 782 infrared spectrophotometer, and
Ts
peak positions are expressed in cmꢀ1 1H, 13C and 19F NMR spectra
.
N
O
N
O
O2S
O2S
were recorded with either a Jeol Delta 270 MHz or a Varian Mer-
cury VX 400 MHz spectrometer. Chemical shifts are reported in
parts per million (ppm, d) relative to tetramethylsilane (TMS) as
an internal standard. Coupling constants J are given in Hz. Mass
spectra were recorded at the Mass spectrometry Service Center,
22
23
Figure 2. Structures of 3-methyl-1,2,3-benzoxathiazole 2,2-dioxide (22) and 3-
tosyl-1,2,3-benzoxathiazole 2,2-dioxide (23).

2512
L. W. Lawrence Woo et al. / Bioorg. Med. Chem. 20 (2012) 2506–2519
O
O
O
O
O
Ts
N
TsHN
HO
O2N
HO
H2N
HO
c
a
b
O2S
O2S
21a
O
1a
21b
21c
21
d
O
H
N
N
e
O2S
HO
22
O
O
O
O
O
O
c
b
a
23c
23b
23a
2a
HO
O
O2S
HO
NTs
NH
NHTs
NH2
NO2
d
O
23
O
O2S
Scheme 6. Synthesis of ‘cyclic’ derivatives of EMATE. Reagents and conditions: (a) Pd/C(10%)/CH2Cl2, H2 (balloon), 20 h; (b) anhydrous pyridine, 0 °C, TsCl; (c) Et3N/CH2Cl2,
SO2Cl2/CH2Cl2 (0.5 M), ꢀ78 °C; (d) MeCN/aqueous KF, reflux, 3 h; (e) NaH/DMF, MeI, 90 °C, 3 h.
University of Bath. FAB mass spectra were measured using m-
nitrobenzyl alcohol as the matrix. Elemental analyses were per-
formed by the Microanalysis Service, University of Bath. Melting
points were determined using a Reichert-Jung Thermo Galen Kofler
block and are uncorrected. HPLC was undertaken using a Waters
717 machine with Autosampler and PDA detector. The column
used, conditions of elution and purity of sample are as indicated
for each compound analysed.
5.3. Syntheses
5.3.1. 2-Nitroestrone (1a) and 4-nitroestrone (2a)
These were prepared according to the method as described by
Tomson and Horwitz.17
5.3.2. 2-Nitroestrone 3-O-sulfamate (1)
To a solution of 1a (2.50 g, 7.94 mmol) in anhydrous DMF
(100 mL) at ice/water temperature and under an atmosphere of
nitrogen was added sodium hydride (60% dispersion in mineral
oil, 350 mg, 8.73 mmol). After the evolution of hydrogen had
ceased, sulfamoyl chloride in toluene (ca. 0.68 M, 18 mL) was
introduced and the resulting mixture was stirred at room temper-
ature for 3 h. The reaction mixture was then concentrated in vacuo
and the resulting residue in ethyl acetate (200 mL) was washed
with hydrochloric acid (0.1 M, 150 mL) and then brine
(4 ꢃ 100 mL). After drying (MgSO4), filtration and evaporation of
the organic layer (water bath temperature 6 40 °C), the yellow res-
idue (3.1 g) obtained was fractionated by flash chromatography
(ethyl acetate/hexane, 1:2–1:1, gradient). The second fraction col-
lected (yellow syrup, 730 mg, 1.85 mmol, 23%) was recrystallized
from ethyl acetate/hexane (1:2) to give 1 as creamy needles
(459 mg). Mp 158–162 °C (dec); Rf (ethyl acetate/hexane, 1:1)
0.28, cf. Rf 0.58 (1a); 1H NMR (270 MHz, CDCl3) d 0.93 (3H, s,
CH3), 1.2–3.1 (15H, m), 5.25 (2H, br s, exchanged with D2O,
OSO2NH2), 7.29 (1H, s, C4H) and 7.98 (1H, s, C1H); LRMS (FAB+)
m/z (%) 548 [30, (M+H+NBA)+], 395 [100, (M+H)+], 378 [37,
(MꢀNH2)+], 315 [30, (M+HꢀH2NSO2)+], 298 [25, (MꢀH2NSO2O)+];
LRMS (FABꢀ) m/z (%) 546 [10, (MꢀH+NBA)ꢀ], 393 [100, (MꢀH)ꢀ],
314 [30, (MꢀH2NSO2)ꢀ], 96 [50, (H2NSO2O)ꢀ]. Found: C, 54.6; H,
5.58; N, 7.10. C18H22N2O6S requires C, 54.81; H, 5.62; N, 7.10.
5.2. Biology
Assays were performed essentially as described previously.49
The ability of the compounds synthesized to inhibit STS activity
was examined using placental microsomal preparations. Placental
microsomes (100,000 g fraction) were prepared from a sulfatase-
positive human placenta from a normal-term pregnancy.50 To
determine the IC50s for the inhibition of STS, activity was measured
in the presence of the inhibitor (0.1–10
l
M) using [3H]E1S
lM with unlabelled substrate. After
(4 ꢃ 105 dpm) adjusted to 20
incubation of the substrate-inhibitor with placental microsomes
(125 lg of protein/mL) for 30 min, the product formed was isolated
from the mixture by extraction with toluene (4 mL), using
[4-14C]E1 to monitor procedural losses.
Intact monolayers of MCF-7 breast cancer cells were incubated
for 20 h at 37 °C with [3H]E1S (5 pmol, 7 ꢃ 105 dpm, 60 Ci/mmol,
NEN Du Pont, Boston, MA) in serum-free minimal essential med-
ium (2.5 mL) with or without inhibitors (0.1–10 lM). After incuba-
tion, medium (1 mL) was removed and productestrone separated
from E1S by solvent partition using toluene (4 mL). [14C]Estrone
(7 ꢃ 103 dpm, 52 mCi/mmol. Amersham International, UK) was
used to correct for procedural losses. An aliquot of the organic
phase was added to scintillation fluid and the 3H and 14C content
measured by scintillation spectrometry. The mass of E1S hydro-
lyzed was calculated from the 3H counts detected (corrected for
the volume of medium and organic solvent used and for recovery
of 14C counts) and the specific activity of the substrate.
5.3.3. 4-Nitroestrone 3-O-sulfamate (2)
This was prepared from 2a (1.0 g, 3.17 mmol) in a similar manner
to the preparation of 1. The crude product was fractionated by flash
chromatography (chloroform/ethyl acetate, 4:1–2:1, gradient) and

L. W. Lawrence Woo et al. / Bioorg. Med. Chem. 20 (2012) 2506–2519
2513
the second fraction collected (pale yellow syrup, 300 mg, 761
l
mol,
0.53 (4a); IR m
max/cmꢀ1 (KBr) 3420, 3340, 3240, 3080, 2940,
24%) was recrystallized from chloroform/hexane (1:1) to give 2 as
pale yellow crystals (167 mg). Mp 176–178 °C; Rf (chloroform/ethyl
acetate, 4:1) 0.20, cf. Rf 0.49 (2a); 1H NMR (270 MHz, CDCl3) d 0.93
(3H, s, CH3), 1.2–3.0 (15H, m), 5.1 (2H, br s, exchanged with D2O,
OSO2NH2), 7.43 (1H, d, J ꢄ 9 Hz, ArH) and 7.49 (1H, d, J = 9 Hz,
ArH); LRMS (FAB+) m/z (%) 395 [100, (M+H)+]; LRMS (FABꢀ) m/z
(%) 393 [100, (MꢀH)ꢀ], 314 [72, (MꢀH2NSO2)ꢀ], 96 [38,
(H2NSO2O)ꢀ]. Found: C, 54.6; H, 5.57; N, 7.02. C18H22N2O6S requires
2870, 1720, 1500, 1380, 1180; 1H NMR (270 MHz, CDCl3) d 0.91
(3H, s, CH3), 1.0–2.7 (13H, m), 2.91 (2H, m, C6-H2), 3.45 (2H, d,
J = 6.4 Hz, C@CH–CH2), 4.99 (2H, br s, exchanged with D2O,
OSO2NH2), 5.08–5.20 (2H, m, H2C@CH–), 5.97 (1H, m, H2C@CH–),
7.14 (1H, s, ArH) and 7.18 (1H, s, ArH); LRMS (FAB+) m/z (%)
389.1 [88, M+], 147.1(60), 72.9(100); LRMS (FABꢀ) m/z (%) 542.3
[20, (M+NBA)ꢀ], 388.1 [100, (MꢀH)ꢀ]; HRMS (FAB+) Found:
389.1674. C21H27NO4S requires 389.1661. Found: C, 64.9; H, 7.02;
N, 3.49. C21H27NO4S requires C, 64.75; H, 6.99; N, 3.60.
C, 54.81; H, 5.62; N, 7.10. HPLC: Spherisorb ODS2 (5 lm,
25 ꢃ 4.6 cm), methanol/water (75:25), 1 mL/min, kmax 281 nm,
tR = 3.82 min.
5.3.7. 4-(2-Propenyl)estrone 3-O-sulfamate (5) (contained ca.
25% of 4)
5.3.4. 3-O-(2-Propenyl)estrone (3)
This was prepared from 5a (contained ca. 25% of 4a by 1H NMR,
648 mg, 2.09 mmol) in a similar manner to the sulfamoylation of
1a. The crude product obtained was fractionated by flash chroma-
tography (ethyl acetate/hexane, 1:4–1:1 gradient) and the second
fraction collected corresponded to an inseparable mixture of ca.
75% of 5 and 25% of 4 as fluffy creamy residue (523 mg). Mp 75–
80 °C; Rf (ethyl acetate/hexane, 2:1) 0.64, cf. Rf 0.73 (4a and 5a);
To a solution of estrone (10.0 g, 36.98 mmol) in DMF (70 mL) at
ice/water temperature was treated cautiously with sodium hydride
in portions (60% in mineral oil, 1.63 g, 40.68 mmol). The resulting
mixture was heated with 3-bromoprop-1-ene (3.9 mL,
44.38 mmol) at 80 °C for 2 h and then stirred under an atmosphere
of nitrogen at room temperature overnight. The reaction mixture
was concentrated in vacuo and the resulting slurry diluted with
ethyl acetate (300 mL). The organic layer was washed with brine
(4 ꢃ 100 mL), dried (MgSO4) and evaporated to give a yellow resi-
due. Upon recrystallization of this crude product from hot absolute
IR
m
max/cmꢀ1 (KBr) 3480, 3280, 3080, 2940, 2870, 1730, 1480,
1380, 1185; 1H NMR (400 MHz, CD2Cl2) d 0.89 (3H, s, CH3), 1.2–
3.0 (15H, m), 3.48 (2H, m, C@CH–CH2), 5.06 (4H, m, 2H exchanged
with D2O, H2C@CH– and OSO2NH2), 5.93 (1H, m, H2C@CH–), 7.11
(ꢄ0.25H, s, ArH of 2-(2-propenyl) isomer 4), 7.20 (ꢄ0.25H, ArH of
2-(2-propenyl) isomer 4), 7.24 (ꢄ0.75H, d, J = 8.9 Hz, ArH of 4-(2-
propenyl) isomer 5), 7.28 (ꢄ0.75H, d, J = 8.9 Hz, ArH of 4-(2-prope-
nyl) isomer 5); LRMS (FAB+) m/z (%) 389.1 [88, M+], 310.2 [31,
(MꢀHNSO2)+], 133.1(45), 72.9(64); LRMS (FABꢀ) m/z (%) 542.3
[20, (M+NBA)ꢀ], 388.2 [100, (MꢀH)ꢀ]; HRMS (FAB+) Found:
389.1671. C21H27NO4S requires 389.1661. Found: C, 64.8; H, 7.08;
N, 3.48. C21H27NO4S requires C, 64.75; H, 6.99; N, 3.60.
alcohol,
3 was obtained as pale yellow crystals (9.48 g,
30.54 mmol, 83%). Mp 106–108 °C [Lit.19 (aq ethanol) 108–
109 °C]; 1H NMR (400 MHz, CDCl3) d 0.90 (3H, s, CH3), 1.3–2.6
(13H, m), 2.88 (2H, m), 4.51 (2H, m, CH2O), 5.28 (1H, m,
HAHBC@CH), 5.40 (1H, m, HAHBC@CH), 6.04 (1H, m, H2C@CH),
6.66 (1H, d, J = 2.7 Hz, C4H), 6.73 (1H, dd, J = 2.8 and 8.5 Hz, C2H)
and 7.19 (1H, d, J = 8.5 Hz, C1H). 13C NMR (100.4 MHz, CDCl3) d
13.84 (q), 21.56 (t), 25.79 (s), 25.88 (t), 26.54 (t), 29.65 (t), 31.57
(t), 35.84 (t), 38.31 (d), 43.95 (d), 47.99 (s), 50.37 (d), 68.75 (t),
112.29 (d), 114.78 (d), 117.45 (t), 126.28 (d), 132.17 (s), 133.50
(d), 137.71 (s) and 156.58 (s).
5.3.8. 2,4-Di-(2-propenyl)estrone (8a)
To a solution of a mixture of 4a and 5a (3.14 g, 10.11 mmol) in
DMF (20 mL) at ice/water temperature was added cautiously so-
dium hydride (60% in mineral oil, 485 mg, 12.14 mmol). The result-
ing mixture was heated with 3-bromoprop-1-ene (1.05 mL,
12.14 mmol) at 80 °C for 2 h and then stirred under an atmosphere
of nitrogen at room temperature overnight. The reaction mixture
was diluted with ethyl acetate (300 mL) and the organic layer sep-
arated, washed with brine (300 mL, 4 ꢃ 100 mL), dried (MgSO4)
and evaporated to give a brown syrup (3.63 g). The Claisen rear-
rangement of this mixture of 2-(2-propenyl)estrone-3-O-(2-prope-
nyl) ether (6) and 4-(2-propenyl)estrone-3-O-(2-propenyl) ether
(7) was carried out according to the method as described by Pat-
ton.19 The crude 8a obtained was fractionated by flash chromatog-
raphy (silica 200 g; ethyl acetate/hexane, 1:3–1:2 gradient). The
third fraction collected gave a light brown residue which upon
recrystallization from hot cyclohexane yielded 8a as creamy bar-
shaped crystals (2.64 g, 7.53 mmol, 75%). Mp 119–120 °C [Lit.19
(ethanol) 121.5–122 °C]; Rf (ethyl acetate/hexane, 1:3) 0.43, cf. Rf
0.51 (4a and 4b); 1H NMR (270 MHz, CD2Cl2) d 0.90 (3H, s, CH3),
1.0–3.0 (15H, m), 3.40 (4H, m, 2 ꢃ C@CH–CH2), 4.9–5.3 (5H, m,
1H exchanged with D2O, 2 ꢃ H2C@CH– and OH), 6.0 (2H, m,
H2C@CH–) and 6.98 (1H, s, C1H).
5.3.5. 2-(2-Propenyl)estrone (4a) and 4-(2-propenyl)estrone (5a)
These were prepared according to the method as described by
Patton19 via the Claisen rearrangement of 3. A solution of 3
(7.68 g, 24.74 mmol) in diethylaniline (50 mL) was heated under
an atmosphere of nitrogen at reflux temperature for 6 h. The cooled
reaction mixture was diluted with ethyl acetate (300 mL), washed
with hydrochloric acid (1 M, 4 ꢃ 100 mL) and then brine
(4 ꢃ 100 mL), dried (MgSO4) and evaporated to give a crude prod-
uct (8.04 g). A 5.0 g portion of which was fractionated by flash
chromatography (silica 400 g, ethyl acetate/hexane, 1:2). The sec-
ond fraction collected gave a creamy residue (4.24 g) which was
recrystallized from ether/hexane (3:2, 50 mL) to give 4a as pale
yellow crystals (721 mg, 2.32 mmol, 15%). Mp 175–182 °C [Lit.19
186–187 °C]; 1H NMR (270 MHz, CDCl3) d 0.91 (3H, s, CH3), 1.2–
2.6 (13H, m), 2.82 (2H, m, C6-H2), 3.38 (2H, d, J = 6.4 Hz, C@CH–
CH2), 4.91 (1H, br s, exchanged with D2O, OH), 5.16 (2H, m,
H2C@CH–), 5.99 (1H, m, H2C@CH–), 6.57 (1H, s, ArH) and 7.02
(1H, s, ArH).
The mother liquor of 4a was evaporated to give a creamy resi-
due (3.45 g). A 2.45 g portion of which was recrystallized from
ether/hexane (2:1, 42 mL) slowly in the refrigerator. After a week,
a crop of yellow crystals (461 mg) was obtained which contained
approximately 75% of 5a and 25% of 4a according to 1H NMR.
5.3.9. 2,4-Di-(2-propenyl)estrone 3-O-sulfamate (8)
This was prepared from 8a (1.0 g, 2.85 mmol) in a similar man-
ner to the sulfamoylaton of 1a. The crude product obtained was
fractionated by flash chromatography (ethyl acetate/hexane, 1:2)
to give 8 as fluffy pale yellow residue (722 mg, 1.68 mmol, 59%).
Mp 75–80 °C; Rf (ethyl acetate/hexane, 2:1) 0.64, cf. Rf 0.72 (8a);
1H NMR (270 MHz, CDCl3) d 0.91 (3H, s, CH3), 1.1–3.0 (15H, m),
3.55 (4H, m, 2 ꢃ C@CH–CH2), 4.9–5.3 (6H, m, 2H exchanged with
D2O, 2 ꢃ H2C@CH– and OSO2NH2), 5.97 (2H, m, 2 ꢃ H2C@CH–)
5.3.6. 2-(2-Propenyl)estrone 3-O-sulfamate (4)
This was prepared from 4a (600 mg, 1.93 mmol) in a similar
manner to the sulfamoylation of 1a. The crude product obtained
was fractionated by flash chromatography (ethyl acetate/hexane,
1:4–1:1 gradient) to give 4 as fluffy creamy residue (540 mg,
72%). Mp 153–155 °C; Rf (ethyl acetate/hexane, 1:1) 0.42, cf. Rf

2514
L. W. Lawrence Woo et al. / Bioorg. Med. Chem. 20 (2012) 2506–2519
and 7.15 (1H, s, C1H); LRMS (EI, 70 eV) m/z (%) 429.1 [7, M+], 350.2
[48, (MꢀHN@SO2)+] 149.0(40), 43.0[100, (C3H7)+]; HRMS (EI)
Found: 429.1999. C24H31NO4S requires 429.1974. Found: C, 66.8;
H, 7.28; N, 3.22. C24H31NO4S requires C, 67.10; H, 7.28; N, 3.26.
s, CH3) 2.46–2.57 (1H, m), 2.80–2.95 (2H, m, C6H), 6.83 (1H, d,
J = 8.3 Hz, C4H) and 7.07 (1H, d, J = 13.2 Hz, C1H); LRMS (FAB+)
m/z (%) 331.1 (59, M++1), 288.1 (100) and 270.1 (22); HRMS
(FAB+) Found: 331.1705. C20H23FO3 (M++1) requires 331.1709.
5.3.10. 2-n-Propylestrone 3-O-sulfamate (9)
5.3.14. 2-Fluoroestrone (12b)
To a solution of 4 (250 mg, 642
l
mol) in absolute ethanol
This was prepared according to the method as described by
(50 mL) was added a suspension of Pd–C (10%, 250 mg) in absolute
ethanol (2 mL) and the resulting suspension was stirred at room
temperature overnight under an atmosphere of hydrogen at
50 psi. After removal of the supported catalyst by filtration and
evaporation of the filtrate, the crude product obtained was frac-
tionated by flash chromatography (ethyl acetate/hexane, 1:4–2:1
Page et al.20 through heating
a
solution of 12a (185 mg,
560
lmol) in methanol (5 mL) with K2CO3 (387 mg, 5 equiv) un-
der reflux for 3 h. The crude residue obtained was recrystallized
from petroleum ether (bp 60–80 °C)/absolute ethanol (2:8) to
give 12b (121 mg, 75% purity as estimated by 1H NMR) as color-
less crystals (mp 220–227 °C). Further purification by semi-pre-
parative HPLC (Waters Radialpak C18 100 ꢃ 25 mm, MeOH/H2O
(60:40), 20 mL/min, kmax 254 and 270 nm) gave 12b as a white
solid (72 mg, 45%). Mp 220–223 °C [Lit.20 (petroleum ether/abso-
lute ethanol) 220–222 °C]; 1H NMR (400 MHz, CDCl3) d 0.78 (3H,
s, CH3), 1.37–1.79 (6H, m), 1.91–2.40 (6H, m), 2.46–2.56 (1H, m),
2.80–2.90 (2H, m, C6H), 5.02 (1H, d, ex. with D2O, J = 3.9 Hz, OH),
6.72 (1H, d, J = 9.4 Hz, C4H) and 6.98 (1H, d, J = 12.5 Hz, C1H);
19F NMR (376 MHz, CDCl3) d ꢀ144.50 (1F, t, J = 9.2 Hz); LRMS
(FAB+) m/z (%) 289.0 [100, (M++1)], 149.0 (33) and 120.0 (35);
LRMS (FABꢀ) m/z (%) 287.1.0 [100, (Mꢀꢀ1)], 200.0 (25), 140.0
(25) and 123.0 (43); HRMS (FAB+) Found 288.1520. C18H21FO2 re-
quires 288.1526. HPLC: Waters Radialpak C18 8 ꢃ 100 mm,
MeOH/H2O (60:40), 2 mL/min, kmax 270 nm, tR = 11.69 min, pur-
ity: 96.9.
gradient) to give 9 as fluffy light yellow residue (220 mg, 562
88%). Mp 75–80 °C; Rf (ethyl acetate/hexane, 1:1) 0.53, cf. Rf 0.44
(4); IR
max/cmꢀ1 (KBr) 3380, 3280, 3100, 2960, 2880, 1730,
lmol,
m
1500, 1380, 1200; 1H NMR (270 MHz, CDCl3) d 0.91 (3H, s,
C18H3), 0.96 (3H, t, J = 7.3 Hz, CH3), 1.2–3.0 (19H, m), 4.98 (2H,
br s, exchanged with D2O, OSO2NH2), 7.12 (1H, s, Ar) and 7.18
(1H, s, Ar); LRMS (FAB+) m/z (%) 390.1 [88, (MꢀH)+], 147.1(55),
73.0(100); LRMS (FABꢀ) m/z (%) 544.2 [17, (M+NBA)ꢀ], 390.1
[100, (MꢀH)ꢀ]. Found: C, 64.3; H, 7.61; N, 3.42. C21H29NO4S re-
quires C, 64.42; H, 7.47; N, 3.58.
5.3.11. 4-n-Propylestrone 3-O-sulfamate (10)
This was prepared from 5 (250 mg, 481 lmol, contained ca. 25%
of 4) in a similar manner to the preparation of 9. The crude product
obtained was fractionated by flash chromatography (ethyl acetate/
hexane, 1:4–1:1 gradient) and the second fraction isolated gave a
mixture of 10 and 9 (ca. 3:1) as creamy residue (234 mg). A portion
of this residue (145 mg) was recrystallized from ethyl acetate/hex-
5.3.15. 2-Fluoroestrone-3-O-sulfamate (12)
To a stirred solution of 12b (65 mg, 230
DMF (7 mL) under an atmosphere of nitrogen was added at room
temperature 2,6-di-tert-butyl-4-methylpyridine (139 mg,
lmol) in anhydrous
ane (1:3) to give pure 10 as soft white crystals (61 mg, 157 lmol,
yield: 70% based on the amount of 5 present in the crude). Mp
194–198 °C; Rf (ethyl acetate/hexane, 1:1) 0.42, cf. Rf 0.40 (5); IR
0.68 mmol) then a solution of sulfamoyl chloride (0.68 M in tol-
uene, 1.61 mL, 1.13 mmol) via a syringe. After stirring the reac-
tion mixture at room temperature for 18 h, ethyl acetate
(60 mL) was added and the organic layer separated was washed
with brine (3 ꢃ 50 mL), dried (MgSO4), filtered and evaporated.
The crude product obtained was purified by flash chromatogra-
phy (chloroform/acetone, 8:1) to afford 12 as a white solid,
(47 mg, 57%). Mp 189–193 °C; Rf (chloroform/acetone, 8:1) 0.32;
1H NMR (400 MHz, CDCl3) d 0.91 (3H, s, CH3), 1.36–1.70 (6H,
m), 1.92–2.36 (6H, m), 2.46–2.57 (1H, m), 2.80–2.95 (2H, m,
C6H), 5.03 (2H, br s, ex. with D2O, –OSO2NH2), 7.11 (1H, d,
J = 9.7 Hz, C4H) and 7.13 (1H, d, J = 15.5 Hz, C1H); 19F NMR
(CDCl3, 376 MHz) d ꢀ113.24 (1F, dd, J = 15.5 Hz and J = 9.7 Hz);
LRMS (FAB+) m/z (%) 367.0 (100, M+), 350.0 (30), 288.1 (49),
258.0 (32), 243.1 (38), 178 (34), 133.0 (30) and 97.0 (35); LRMS
(FABꢀ) m/z (%) 366.0 [100, (MꢀH)ꢀ] and 77.9 (21); HRMS (FAB+)
Found: 367.1267. C18H22NO4S requires 367.1254. HPLC: Waters
m
max/cmꢀ1 (KBr) 3400, 3320, 3240, 3000–2860, 1720, 1470, 1390,
1180; 1H NMR (400 MHz, CD2Cl2) d 0.89 (3H, s, C18-H3), 1.00
(3H, t, J = 7.3 Hz, CH3), 1.2–3.0 (19H, m), 5.11 (2H, br s, exchanged
with D2O, OSO2NH2) and 7.21 (2H, s, Ar); LRMS (FAB+) m/z (%)
391.1 [100, M+]; LRMS (FABꢀ) m/z (%) 544.2 [20, (M+NBA)ꢀ],
390.1 [100, (MꢀH)ꢀ]. Found: C, 64.1; H, 7.45; N, 3.51. C21H29NO4S
requires C, 64.42; H, 7.47; N, 3.58.
5.3.12. 2,4-Di-n-propylestrone 3-O-sulfamate (11)
This was prepared from 8 (200 mg, 466 lmol) in a similar man-
ner to the preparation of 9. The crude product obtained was frac-
tionated by flash chromatography (ethyl acetate/hexane, 1:4–1:2
gradient) and the second fraction collected gave 11 as fluffy crea-
my residue (190 mg, 438
tate/hexane, 1:1) 0.64, cf. Rf 0.59 (8); IR
l
mol, 94%). Mp 72–82 °C; Rf (ethyl ace-
m
max/cmꢀ1 (KBr) 3380,
3300, 3100, 2970, 2940, 2880, 1730, 1470, 1380, 1180; 1H NMR
(270 MHz, CDCl3) d 0.91 (3H, s, C18-H3), 0.96 (3H, t, J = 7.3 Hz,
CH3), 1.00 (3H, t, J = 7.3 Hz, CH3), 1.3–3.0 (23H, m), 5.02 (2H, br s,
exchanged with D2O, OSO2NH2) and 7.08 (1H, s, C1H); LRMS
(FAB+) m/z (%) 433.1 [100, M+], 353.2 [43, (MꢀH2NSO2)+],
255.2(50), 173.1(80); LRMS (FABꢀ) m/z (%) 432.2 [100, (MꢀH)ꢀ],
353.1 [8, (MꢀH2NSO2)ꢀ]; HRMS (FAB+) Found: 433.2311.
‘Symmetry’ C18 (packing: 3.5
MeCN/H2O (90/10) isocratic; flow rate, 1 mL/min; tR = 2.19 min;
purity: 95%.
l
m, 4.6 ꢃ 150 mm); mobile phase
5.3.16. Estrone acetate (13)
This was prepared from estrone according to the method as de-
scribed by Page et al.20
C
24H35NO4S requires 433.2287. Found: C, 66.6; H, 8.37; N, 2.97.
24H35NO4S requires C, 66.48; H, 8.14; N, 3.23.
C
5.3.17. 2-Chloroestrone acetate (15a)
This was prepared according to the method as described by
Page et al.20 To a solution of 13 (500 mg, 1.60 mmol) in trifluoro-
acetic acid (TFA, 20 mL) was added thallic trifluoroacetate
(1.74 g, 3.21 mmol) was and the resulting mixture was stirred at
0 °C under nitrogen for 24 h. Upon removal of TFA under reduced
pressure at <40 °C, the crystalline estrone–thallium(III) complex
(14) obtained was washed twice with 1,2-dichloroethane. 1,4-
Dioxane (10 mL) and copper chloride (475 mg, 4.80 mmol) were
5.3.13. 2-Fluoroestrone acetate (12a)
This was prepared according to the method as described by
Page et al.20 through heating
a mixture of estrone (1.50 g,
5.55 mmol) and N-fluoropyridinium triflate (2.74 g, 11.0 mmol) in
1,1,2-trichloroethane (24 mL) at reflux temperature under nitrogen
for 24 h. Mp 83–88 °C [Lit.20 88–90 °C]; 1H NMR (CDCl3, 270 MHz) d
0.91 (3H, s, C18H3), 1.38–1.75 (6H, m), 1.90–2.45 (6H, m), 2.32 (3H,

L. W. Lawrence Woo et al. / Bioorg. Med. Chem. 20 (2012) 2506–2519
2515
then added to this washed complex and the resulting mixture was
5.3.21. 2-Bromoestrone (16b)
heated under reflux for 3 h. Upon cooling and evaporation of sol-
vent, the residue obtained was treated with water (30 mL) and
the organic components were extracted into dichloromethane
(3 ꢃ 50 mL). The combined organic extracts were washed with
water, dried (MgSO4), filtered and evaporated. The crude product
obtained was fractionated by flash chromatography (ethyl ace-
tate/hexane, 1:4) and the pale yellow solid obtained (526 mg)
was further purified by recrystallization from methanol to give
15a as white crystals (436 mg, 78%). Mp 195–197 °C; Rf (chloro-
This was prepared from 16a (1.5 g, 3.83 mmol) in the same
manner as the preparation of its chloro analogue 15b. The pale yel-
low crude product (1.3 g) obtained was purified by recrystalliza-
tion from ethyl acetate/petroleum ether 60–80 °C (3:2) to give
16b as pale yellow crystals (1.23 g, 3.52 mmol, 92%). Mp 190–
193 °C [Lit.20 194–195 °C]; Rfs (chloroform/acetone, 8:1 and 4:1)
0.51 and 0.64 respectively; IR
m
max/cmꢀ1 (KBr) 1710 (C@O); 1H
NMR (400 MHz, CDCl3) d 0.87 (3H, s, C18H3), 1.4–2.52 (13H, m),
2.84 (2H, m, C6H), 5.32 (1H, br s, ex. with D2O, OH), 6.73 (1H, s,
C4H) and 7.52 (1H, s, C1H); 13C NMR (100.4 MHz, CDCl3) d 13.81
(q, C18), 21.56 (t), 25.89 (t), 26.26 (t), 29.9 (t), 31.54 (t), 31.91 (t),
38.04 (d), 43.6 (d), 47.93 (s, C13), 50.38 (d), 114.94 (d, C4),
135.04 (d, C-1), 128.84 (s), 130.92 (s), 139.02 (s), 152.7 (s, C3)
and 220 (s, C@O); LRMS (FAB+) m/z (%) 502.1 [20, (M+NBA)+],
349.1 [100, (M)+] and 271.0 (20); LRMS (FABꢀ) m/z (%) 502.2 [40,
(M+NBA)ꢀ] and 348.1 [100, (MꢀH)ꢀ]; HRMS (FAB+) Found:
348.0753. C18H21BrO2 requires 348.0725.
form/acetone, 8:1) 0.78; IR
m
max/cmꢀ1 (KBr) 1770, 1730 (C@O);
1H NMR (400 MHz, CDCl3) d 0.90 (3H, s, C18H3), 1.38–2.29 (12H,
m), 2.33 (3H, s, CH3CO), 2.5 (1H, m), 2.87 (2H, m, C6H), 6.85 (1H,
s, C4H) and 7.34 (1H, s, C1H).
5.3.18. 2-Chloroestrone (15b)
This was prepared according to the method as described by
Page et al.20 A mixture of 15a (400 mg, 1.15 mmol), K2CO3
(800 mg, 5.80 mmol) in MeOH (20 mL) was heated under reflux
for 3 h. Upon cooling and evaporation of the solvent, water
(20 mL) was added to the residue obtained and the crude product
was extracted into dichloromethane (3 ꢃ 20 mL). The combined
organic extracts were washed with water, dried (MgSO4), filtered
and evaporated to give a yellow crude product which was purified
by recrystallization from methanol to give 15b as white crystals
(320 mg, 90%). Mp 221–223 °C [Lit.20 223–225 °C]; Rf (chloro-
5.3.22. 2-Bromoestrone 3-O-sulfamate (16)
This was prepared from 16b (500 mg, 1.43 mmol) in a similar
manner to the sulfamoylation of 1a. The crude product (620 mg)
obtained was fractionated by flash chromatography (chloroform/
acetone, 8:1). The beige residue obtained (510 mg) was further
purified by recrystallization from acetone/hexane (1:2) to give 16
as beige crystals (405 mg, 66%). Mp > 155 °C (dec); Rfs (chloro-
form/acetone, 8:1) 0.70; IR
m
max/cmꢀ1 (KBr) 3400 (OH), 1730
form/acetone, 8:1 and 4:1) 0.43 and 0.61 respectively; IR mmax/
(C@O); 1H NMR (270 MHz, CDCl3) d 0.91 (3H, s, CH3), 1.4–2.54
(13H, m), 2.84 (2H, m, C6H), 5.27 (1H, s, ex, with D2O, OH), 6.71
(1H, s, C4H) and 7.31 (1H, s, C1H).
cmꢀ1 (KBr) 3500, 3300 (NH2), 1730 (C@O), 1390 (SO2); 1H NMR
(400 MHz, acetone-d6) d 0.92 (3H, s, CH3), 1.41–2.49 (13H, m),
2.86 (2H, m, C6H), 7.27 (1H, s, C4H), 7.32 (2H, br s, ex. with D2O,
OSO2NH2) and 7.55 (1H, s, C1H); LRMS (FAB+) m/z (%) 428.1 [100,
(M)+] and 349.2 [40, (M+H-SO2NH2)+]; LRMS (FABꢀ) m/z (%)
580.2 [MꢀH+NBA], 428.1 [100, (M)ꢀ] and 349.1 [30, (M+H-
SO2NH2)ꢀ]; HRMS (FAB+) Found: 428.0430. C18H23BrNO4S requires
5.3.19. 2-Chloroestrone 3-O-sulfamate (15)
This was prepared from 15b (200 mg, 655 lmol) in a similar
manner to the sulfamoylation of 1a. The crude product obtained
was fractionated by flash chromatography (chloroform/acetone,
8:1) to give a beige residue (190 mg) which was further purified
by recrystallization from acetone/hexane (1:2) to give 15 as white
crystals (170 mg, 68%). Mp > 163 °C (dec); Rfs (chloroform/acetone,
428.0531. HPLC: Waters ‘Symmetry’ C18 (packing: 3.5 lm,
4.6 ꢃ 150 mm); mobile phase MeCN/H2O (90/10) isocratic; flow
rate, 1 mL/min; tR = 2.19 min; purity: 98%.
8:1 and 4:1) 0.63 and 0.74 respectively; IR
m
max/cmꢀ1 (KBr) 3500,
5.3.23. 2-Iodoestrone acetate (17a)
3200 (NH2), 1720 (C@O), 1390 (SO2); 1H NMR (400 MHz, CDCl3/
DMSO-d6: 10:1) d 0.93 (3H, s, CH3), 1.4–2.51 (13H, m), 2.86 (2H,
m, C6H), 7.01 (2H, br s, ex. with D2O, OSO2NH2), 7.25 (1H, s,
C4H) and 7.35 (1H, s, C1H). LRMS (FAB+) m/z (%) 537.2 (5), 383.2
(17), 206.2 (100), 97.1 (22); LRMS (FABꢀ) m/z (%) 536.2 (45),
383.2 (100), 250.0 (52), 96.9 (49), 77.9 (34); HRMS (FAB+) Found:
383.0956. C18H22ClNO4S requires 383.0958. HPLC: Waters ‘Sym-
This was prepared from 13 (5.0 g, 16.01 mmol) in the same
manner as for the preparation of 15a except that copper iodide
(18.29 g, 96.03 mmol) was used. The crude product obtained was
purified by flash chromatography with ethyl acetate/hexane (1:4)
and the yellow solid obtained (6.25 g) was further purified by
recrystallization from methanol to give 17a as pale yellow crystals
(5.89 g, 84%). Mp 170–172 °C (crystals turned brown at >145 °C); Rf
metry’ C18 (packing: 3.5
MeCN/H2O (96/4) isocratic; flow rate, 1 mL/min; tR = 2.01 min;
l
m, 4.6 ꢃ 150 mm); mobile phase
(chloroform/acetone, 8:1) 0.8; IR m
max/cmꢀ1 (KBr) 1770, 1730
(C@O); 1H NMR (400 MHz, CDCl3) d 0.92 (3H, s, C18H3), 1.4–2.31
(12H, m), 2.34 (3H, s, CH3CO), 2.5 (1H, m), 2.87 (2H, t, J = 4.27 Hz,
C6H), 6.82 (1H, s, C4H) and 7.69 (1H, s, C1H); LRMS (FAB+) m/z
(%) 592.1 [20, (M+H+NBA)+], 439.1 [80, (M+H)+], 396.1 [100,
(M+H-CH3CO)+] and 270.2 (40); HRMS (FAB+) Found: 439.0767.
purity: 98%.
5.3.20. 2-Bromoestrone acetate (16a)
This was prepared from 13 (2.0 g, 6.40 mmol) in the same man-
ner as the preparation of 15a except that copper bromide (8.58 g,
38.41 mmol) was used. The crude product obtained was fraction-
ated by flash chromatography (ethyl acetate/hexane, 1:4) and the
pale white solid isolated (2.05 g) was further purified by recrystal-
lization from methanol to give 16a as colorless crystals (1.79 g,
4.58 mmol, 72%). Mp 229–231 °C; Rfs (chloroform/acetone, 8:1
and ethyl acetate/hexane, 4:1) 0.89 and 0.62 respectively; IR
C20H24IO3 requires 439.0770.
5.3.24. 2-Iodoestrone (17b)
This was prepared from 17a (4.0 g, 9.13 mmol) in the same
manner as for the preparation of its chloro analogue 15b. The yel-
low crude product obtained was recrystallized from methanol to
give 17b as pale yellow crystals (3.29 g, 91%). Mp 207–209 °C
(crystals turned brown at >169 °C) [Lit.20 167–168 °C]; Rf (chloro-
m
max/cmꢀ1 (KBr) 1770, 1730 (C@O) cmꢀ1 1H NMR (400 MHz,
;
CDCl3) d 0.91 (3H, s, C18H3), 1.25–2.3 (12H, m), 2.34 (3H, s, CH3CO),
2.5 (1H, m), 2.86 (2H, t, J = 4.6 Hz, C6H), 6.85 (1H, s, C4H) and 7.49
(1H, s, C1H); LRMS m/z (FAB+) 545.2 [90, (M+H+NBA)+], 391.2 [100,
(M)+], 348.1 [90, (MꢀCH3CO)+] and 330.1 (10); HRMS (FAB+)
Found: 391.0856. C20H24BrO3 requires 391.0909. Found: C, 61.2;
H, 5.89. C20H23BrO3 requires C, 61.39; H, 5.92.
form/acetone, 8:1) 0.75; IR
m
max/cmꢀ1 (KBr) 3400 (OH), 1730
(C@O) cmꢀ1 1H NMR (270 MHz, CDCl3) d 0.91 (3H, s, CH3), 1.4–
;
2.56 (13H, m), 2.83 (2H, m, C6H), 5.24 (1H, s, ex. with D2O, OH),
6.74 (1H, s, C4H) and 7.52 (1H, s, C1H); 13C NMR (100.4 MHz,
CDCl3) d 13.82 (q, C-18), 21.57 (t), 25.89 (t), 26.26 (t), 29.98 (t),
31.57 (t), 31.81 (t), 39.0 (d), 43.9 (d), 48.3 (s, C13), 50.38 (d),

2516
L. W. Lawrence Woo et al. / Bioorg. Med. Chem. 20 (2012) 2506–2519
116.54 (d, C4), 138.14 (d, C1), 129.64 (s), 131.92 (s), 138.02 (s),
153.6 (s, C3) and 220 (s, C17, C@O); LRMS (FAB+) m/z (%) 550.1
[20, (M+H+NBA)+], 396.1 [100, (M)+] and 271.2 (40); LRMS (FABꢀ)
m/z (%) 549.2 [20, (M+NBA)ꢀ] and 395.1 [100, (MꢀH)ꢀ]; HRMS
(FAB+) Found: 397.0651. C18H22IO2 requires 397.0665.
the added bromine. After stirring for an additional 5 min, the reac-
tion mixture was poured into ice and water. The precipitate that
formed was collected by filtration, washed with water and recrys-
tallized from 98% ethanol to give 19a as white powder (300 mg,
858 l
mol, 23%). Mp 260–263 °C (Lit.21 264–265 °C); Rf (chloro-
form/acetone, 4:1) 0.76; 1H NMR (270 MHz, CDCl3) d 0.90 (3H, s,
C18H3), 1.30–3.06 (15H, m), 5.55 (1H, s, ex. with D2O, OH), 6.88
(1H, d, J = 8.6 Hz, C2H) and 7.19 (1H, d, J = 8.6 Hz, C1H); LRMS
(FAB+) m/z (%) 349.1 [84, (M+1)+], 348.1 (100); LRMS (FABꢀ) m/z
(%) 502.1 [64, (MꢀH+NBA)ꢀ], 500.1 (55), 349.1 [100, (MꢀH)ꢀ],
347.1 (88). HRMS (FAB+) Found: 348.0716. C18H21BrO2 requires
348.0725.
5.3.25. 2-Iodoestrone 3-O-sulfamate (17)
This was prepared from 17b (500 mg, 1.26 mmol) in a similar
manner to the sulfamoylation of 1a. The crude product obtained
was fractionated by flash chromatography (chloroform/acetone,
8:1) and the beige residue collected (450 mg) was further purified
by recrystallization from acetone/hexane (1:2) to give 17 as beige
crystals (342 mg, 57%). Mp > 145 °C (dec); Rfs (chloroform/acetone,
8:1 and 4:1) 0.61 and 0.73 respectively; IR
m
max/cmꢀ1 (KBr) 3500,
5.3.29. 4-Bromoestrone sulfamate (19)
3200 (NH2), 1720 (C@O), 1390 (SO2); 1H NMR (400 MHz, ace-
tone-d6) d 0.92 (3H, s, CH3), 1.4–2.53 (13H, m), 2.83 (2H, m,
C6H), 7.25 (1H, s, C4H), 7.32 (2H, br s, ex. with D2O, OSO2NH2)
and 7.76 (1H, s, C1H); LRMS (FAB+) m/z (%) 629.1 [40,
(M+H+NBA)+], 476.1 [100, (M+H)+] and 396.1 [40, (M+H-
SO2NH2)+]; LRMS (FABꢀ) m/z (%) 628.1 [35, (M+NBA)ꢀ], 474.1
[100, (MꢀH)ꢀ] and 395.1 [20, (MꢀSO2NH2)ꢀ]; HRMS (FAB+) Found:
476.0387. C18H23INO4S requires 476.0393. Found: C, 45.8; H; 4.74;
N, 2.85. C18H22INO4S requires C, 45.48; H, 4.66; N, 2.95.
This was prepared from 19a (286 mg, 816 lmol) in a similar
manner to the sulfamoylation of 1a. The crude product obtained
was fractionated by flash chromatography (acetone/hexane, 1:4)
The residue collected was further purified by recrystallization from
ethyl acetate/hexane to give 19 as white solid (90 mg, 26%). Mp
200–201 °C; Rf (chloroform/acetone, 4:1) 0.47, cf. Rf 0.76 (19a);
IR
m
max/cmꢀ1 (KBr) 3361, 3228 (NH2), 1719 (C@O), 1390 (SO2);
1H NMR (400 MHz, CDCl3) d 0.91 (3H, s, CH3), 1.36–1.72 (6H, m),
1.92–2.22 (4H, m), 2.25–2.42 (2H, m), 2.47–2.58 (1H, m), 2.71–
2.84 (1H, m), 3.00–3.10 (1H, m), 4.99 (2H, br s, ex. with D2O,
OSO2NH2) and 7.29 (2H, d, J = 7.3 Hz, C1H and C2H); LRMS
(FAB+) m/z (%) 428.1 [100, (M+H)+], 349.1 [27, (MꢀSO2NH2)+]
348.1 (30), 476.1 [100, (M+H)+] and 396.1 [40, (M+H-SO2NH2)+];
LRMS (FABꢀ) m/z (%) 580.1 [45, (MꢀH+NBA)ꢀ], 428.1 [98, (M)ꢀ],
349.1 (10), 347.1 [8, (M+H-SO2NH2)ꢀ], 77.9 (50); HRMS (FAB+)
Found: 428.0460. C18H23BrNO4S requires 428.0531. Found: C,
50.6; H; 5.21; N, 3.18. C18H22BrNO4S requires C, 50.47; H, 5.18;
N, 3.27.
5.3.26. 2-Cyanoestrone (18b)
2-Cyanoestrone acetate (18a) was prepared from 13 (500 mg,
1.60 mmol) in the same manner as for the preparation of 15a ex-
cept that the estrone–thallium (III) complex was heated with cop-
per cyanide in pyridine instead. Without the isolation of product,
crude 18a obtained was directly deacetylated in the same manner
as the preparation of its chloro analogue 15b. Upon fractionation of
crude 18b by flash chromatography (ethyl acetate/hexane, 1:4),
the pale yellow solid obtained (286 mg) was further purified by
recrystallization from methanol to give 18b as white crystals
(260 mg, 80%). Mp 266–268 °C [Lit.20 265–266 °C]; Rf (chloro-
5.3.30. 3-Hydroxy-1,3,5(10)-estratriene (20a)
This was prepared by heating a mixture of estrone (10.0 g,
36.98 mmol), hydrazine monohydrate (15 mL, mmol), potassium
hydroxide (15 g, mmol) in ethanol (5 mL) and diethylene glycol
(120 ml) under the conditions for a Wolff–Kishner reduction of
steroid ketones. The crude product obtained was purified by flash
chromatography (ethyl acetate/hexane, 1:4) to give 20a (7.0 g,
form/acetone, 8:1) 0.74; IR
m
max/cmꢀ1 (KBr) 1770, 1730 (C@O)
cmꢀ1 1H NMR (400 MHz, CDCl3) d 0.92 (3H, s, CH3), 1.39–2.56
,
(13H, m), 2.86 (2H, m, C6CH2), 5.97 (1H, br s, ex. with D2O,
C3OH), 6.71 (1H, s, C4H) and 7.52 (1H, s, C1H).
5.3.27. 2-Cyanoestrone 3-O-sulfamate (18)
65%). Mp 132–135 °C [Lit.22 (aq ethanol) 134–134.5 °C]; IR mmax
/
This was prepared from 18b (200 mg, 677
l
mol) in a similar
cmꢀ1 (KBr) 3371 (OH), 2926 (ArH); 1H NMR (400 MHz, CDCl3) d
0.74 (3H, s), 1.08–1.94 (13H, m), 2.14–2.30 (2H, m), 2.74–2.90
(2H, m, C6H), 4.47 (1H, s, ex. with D2O, OH), 6.56 (1H, d,
J = 2.7 Hz, C4H), 6.62 (1H, dd, J = 2.7 and 8.6 Hz, C2H) and 7.17
(1H, d, J = 8.6 Hz, C1H); LRMS (FAB+) m/z (%) 256.1 (100, M+),
159.1 (17), 145.0 (8) and 133.0 (11); LRMS (FABꢀ) m/z (%)
408.2 [51, (MꢀH+NBA)ꢀ], 255.2 [100, (MꢀH)ꢀ], 195.1 (21) and
manner to the sulfamoylation of 1a. The crude product obtained
was fractionated by flash chromatography (chloroform/acetone,
8:1) and the pale yellow residue collected (217 mg) was further
purified by recrystallization from acetone/hexane (1:2) to give 12
as beige crystals (181 mg, 70%); Mp > 205 °C (dec); Rfs (chloro-
form/acetone, 8:1 and 4:1) 0.60 and 0.72 respectively; IR mmax
/
cmꢀ1 (KBr) 3500, 3200 (NH2), 1720 (C@O), 1390 (SO2); 1H NMR
(400 MHz, CDCl3/DMSO-d6; 5:1) d 0.91 (3H, s, CH3), 1.4–2.49
(13H, m), 2.99 (2H, m, C6H), 7.33 (1H, s, C4H), 7.57 (1H, s, C1H)
and 8.0 (2H, br s, ex. with D2O, OSO2NH2); LRMS (FAB+) m/z (%)
528.2 [24, (M+H+NBA)+], 375,1 [65, (M+H)+], 206.2 (100); LRMS
(FABꢀ) m/z (%) 527.2 [24, (M+NBA)ꢀ], 373,2 [65, (MꢀH)ꢀ], 294.2
[43, (MꢀH2NSO2)ꢀ]; HRMS (FAB+) Found: 375.1375. C19H23N2O4S
requires 375.1379. HPLC: Waters ‘Symmetry’ C18 (packing:
139.0 (9); HRMS (FAB+) Found: 256.1826. C18H24
256.1827.
O requires
5.3.31. 1,3,5(10)-Estratriene 3-O-sulfamate (20)
This was prepared from 20a (1.0 g, 3.90 mmol) in a similar man-
ner to the sulfamoylation of 1a. The crude product obtained was
fractionated by flash chromatography (ethyl acetate/hexane, 1:3)
and the second fraction collected (911 mg, 69%) was recrystallized
from chloroform/hexane (6:7) to give a crop of 20 as white crystals
(280 mg); Mp 124–125 °C; Rf (ethyl acetate/hexane, 1:2) 0.40, cf. Rf
3.5
l
m, 4.6 ꢃ 150 mm); mobile phase MeCN/H2O (70/30) isocratic;
flow rate, 1 mL/min; tR = 1.12 min; purity: 94%.
0.50 (20a); IR m
max/cmꢀ1 (KBr) 3410 and 3100 (NH2), 3000–2800,
5.3.28. 4-Bromoestrone (19a)
1380, 1360; 1H NMR (270 MHz, CDCl3) d 0.74 (3H, s, CH3), 1.0–
3.0 (17H, m), 4.91 (2H, s, ex. with D2O, OSO2NH2), 7.04 (1H, d,
J = 2.3 Hz, C4H), 7.07 (1H, dd, J = 2.3 and 8.4 Hz, C2H) and 7.32
(1H, d, J = 8.6 Hz, C1H); LRMS (CI) m/z (%) 353.1 [100, (M+NH4)+],
257 (11), 256 (19), 255 (11); HRMS (NH4+) Found: 353.1897.
To 300 mL of glacial acetic acid was added estrone (1.0 g,
3.70 mmol) and the suspension was gently heated with stirring un-
til it became a solution. Upon cooling to ice-water temperature,
powdered iron (10–15 mg) was added to the stirred solution, fol-
lowed by a dropwise addition of a 5% v/v solution of bromine in
glacial acetic acid until the reaction mixture no longer decolorized
C
C
18H29N2O3S requires 353.1899. Found: C, 64.3; H, 7.55; N, 4.16.
18H25NO3S requires C, 64.45; H, 7.51; N, 4.18.

L. W. Lawrence Woo et al. / Bioorg. Med. Chem. 20 (2012) 2506–2519
2517
5.3.32. 2-Aminoestrone (21a)
5.3.35. Estra-1,3,5(10)-trien-17-oxo[3,2d]-1,2,3-oxathiazole-2,2-
To a solution of 1a (1.0 g, 3.17 mmol) in dichloromethane
(50 mL) was added a suspension of Pd-C (10%, 200 mg) in dichloro-
methane (3 mL). The resulting suspension was stirred at room
temperature under an atmosphere of hydrogen (balloon) for 20 h
at which time the reduction was complete according to TLC. After
removal of the supported catalyst by filtration and evaporation of
filtrate, the crude product obtained (940 mg) [Rf (chloroform/ace-
tone, 4:1) 0.31, cf. Rf 0.76 (1a)] was used for the next reaction with-
out further purification.
dioxide (21)
To a solution of 21c (148 mg, 295 lmol) in acetonitrile (25 mL)
was added a solution of potassium fluoride (171 mg, 2.95 mmol) in
water (4 mL). The resulting white suspension/emulsion was re-
fluxed for 3 h, cooled and evaporated. The white residue obtained
was dissolved in ethyl acetate (50 mL) and the organic layer
washed with hydrochloric acid (1 M, 50 mL) followed by brine
(4 ꢃ 50 mL). After drying (MgSO4) and filtration, the filtrate on
evaporation gave a light purple residue (141 mg) which was
recrystallized from ethyl acetate/hexane (1:3, 8 mL) to give 21 as
light purple powder (68 mg, 196
m
lmol, 66%). Mp 130–137 °C; IR
5.3.33. 2-Tosylamidoestrone (21b)
max/cmꢀ1 (KBr) 3700–2500, 2920, 2860, 1720, 1490, 1370, 1190;
To a stirred suspension of the freshly prepared crude 21a
(900 mg, 3.15 mmol) in anhydrous dichloromethane (30 mL) at
ice/water temperature was added anhydrous pyridine (0.5 mL) fol-
lowed by tosyl chloride (605 mg, 3.17 mmol). After stirring at room
temperature for 3 h, the reaction mixture was filtered and the fil-
trate thus obtained was diluted with ethyl acetate (50 mL). The or-
ganic layer was washed with hydrochloric acid (1 M, 2 ꢃ 50 mL)
followed by brine (4 ꢃ 50 mL), dried (MgSO4), filtered and evapo-
rated to give a yellow residue (1.23 g). This was recrystallized from
ethyl acetate/hexane (4:3) to give a crop of slightly impure 21b as
fine bright yellow crystals (390 mg). The yellow residue (720 mg)
obtained from the evaporation of the mother liquor was fraction-
ated by flash chromatography (ethyl acetate/hexane, 1:2 to 4:1
1H NMR (400 MHz, CDCl3) d 0.92 (3H, s, C18H3), 1.40–1.70 (6H,
m), 1.90–2.40 (6H, m), 2.52 (1H, dd, J 8.8 and ꢄ19 Hz), 2.90 (2H,
m, C6H2), 6.73 (1H, s, ex. with D2O, NH), 6.86 (1H, s, Ar) and 6.97
(1H, s, Ar); LRMS (FAB+) m/z (%) 500.9 [7, (M+H+NBA)+], 346.9
(100, M+); LRMS (FABꢀ) m/z (%) 693.4[10, (2 MꢀH)ꢀ], 346.2 [100,
(MꢀH)ꢀ]; HRMS (FAB+) Found: 347.1196. C18H21NO4S requires
347.1191. HPLC: Waters ‘Symmetry’ C18 (packing: 3.5 lm,
4.6 ꢃ 100 mm); mobile phase MeCN/H2O (96/4), gradient elution:
50:50 MeCN/H2O to 95:5 MeCN/H2O over 5 min; flow rate, 1 mL/
min; tR = 2.01 min; purity: 98%.
5.3.36. Estra-1,3,5(10)-trien-17-oxo[3,2d]-1,2,3-oxathiazole-3-
methyl-2,2-dioxide (22)
gradient). The third fraction obtained gave
(568 mg) which was recrystallized from ethyl acetate/hexane
(2:3) to give 21b as soft creamy crystals (365 mg, 830 mol, overall
yield ca. 45%). Mp 217–220 °C; Rf (ethyl acetate/hexane, 2:1) 0.45,
cf. Rf 0.58 (1a); IR
max/cmꢀ1 (KBr) 3380, 3240, 3000–2800, 1710,
a white residue
To a solution of 21 (32 mg, 92
room temperature was added sodium hydride (60% dispersion in
mineral oil, 4 mg, 100 mol). After 10 min, the resulting yellow mix-
lmol) in anhydrous DMF (5 mL) at
l
l
ture was heated in the presence of methyl iodide (0.2 mL,
3.21 mmol) at 90 °C for 3 h. Upon cooling and diluting with ethyl
acetate (20 mL), the organic layer that separated was washed with
brine (30 mL, 4 ꢃ 10 mL), dried (MgSO4), filtered and evaporated
to give a light brown residue (37 mg) which was recrystallized from
ethyl acetate/hexane (3:4 , 7 mL) to give 22 as light yellow/brown
m
1340, 1160; 1H NMR (400 MHz, DMSO-d6) d 0.80 (3H, s, C18H3),
1.1–2.5 (16H, m), 2.65 (2H, m, C6H2), 6.42 (1H, s, C4-H), 6.87
(1H, s, C1H), 7.31 (2H, AA0BB0, Tosyl), 7.61 (2H, AA0BB0, Tosyl),
9.01 (1H, s, ex. with D2O, NH or OH) and 9.18 (1H, s, ex. with
D2O, NH or OH); LRMS (FAB+) m/z (%) 439.1 (74, M+), 284.1 [100,
(MꢀTosyl)+]; LRMS (FABꢀ) m/z (%) 592.2 [11, (M+NBA)ꢀ], 438.1
[100, (MꢀH)ꢀ]; HRMS (FAB+) Found: 439.1817. C25H29NO4S re-
quires 439.1817. Found: C, 68.1; H, 6.82; N, 3.13. C25H29NO4S re-
quires C, 68.31; H, 6.65; N, 3.19.
crystals (12 mg, 33 lmol, 36%). Mp turned brown at ca. 230 °C and
melted at >250 °C to form a dark brown syrup; Rf (chloroform/ace-
tone, 1:1) 0.80, cf. Rf 0.51 (21); 1H NMR (400 MHz, CDCl3) d 0.93
(3H, s, C18H3), 1.40–2.60 (ꢄ13H), 2.88 (2H, m, C6H2), 3.26 (3H, s,
NCH3), 6.73 (1H, s, Ar) and 6.84 (1H, s, Ar); LRMS (FAB+) m/z (%)
361.2 (100, M+), 330.2 (8), 298.3 (17), 173.2 (17), 97.1 (15); LRMS
(FABꢀ) m/z (%) no peaks were observed; HRMS (FAB+) Found:
361.1359. C19H23NO4S requires 361.1348. HPLC: Waters 0Symmetry0
5.3.34. Estra-1,3,5(10)-trien-17-oxo[3,2d]-1,2,3-oxathiazole-3-
tosyl-2,2-dioxide (21c)
Sulfuryl chloride (1.40 mL of a 0.5 M solution in dichlorometh-
C18 (packing: 3.5
(96/4) isocratic; flow rate, 1 mL/min; tR = 2.39 min; purity: >99%.
l
m, 4.6 ꢃ 150 mm); mobile phase MeCN/H2O
ane, 700
lmol) was added dropwise over a 5 min period to a stir-
red mixture of 21b (280 mg, 637
l
mol) and triethylamine
(0.27 mL, 1.911 mmol) in dichloromethane (10 mL) at ꢀ78 °C un-
der an atmosphere of nitrogen. After an additional 15 min, the
reaction mixture was allowed to warm to room temperature, con-
centrated and the yellow residue obtained was dissolved in ethyl
acetate (50 mL). The organic layer was washed with hydrochloric
acid (1 M, 50 mL) followed by brine (4 ꢃ 50 mL), dried (MgSO4), fil-
tered and evaporated to give a yellow residue (390 mg) which was
fractionated by flash chromatography (chloroform/acetone, 16:1).
The first fraction isolated upon evaporation gave the product
5.3.37. 4-Aminoestrone (23a)
To a solution of 2a (1.49 g, 4.73 mmol) in tetrahydrofuran (THF,
80 mL) was added a suspension of Pd-C (10%, 300 mg) in THF
(2 mL) and the resulting suspension was stirred at room tempera-
ture under an atmosphere of hydrogen (balloon) for 20 h. After re-
moval of the supported catalyst by filtration and evaporation of
filtrate, the crude product obtained (1.42 g) [Rf chloroform/ace-
tone, 4:1) 0.53, cf. Rf 0.68 (2a)] was used for the next reaction with-
out further purification.
(192 mg, 383
tate/hexane to give 21c as white crystals (90 mg). Mp 245–247 °C
(dec); IR
max/cmꢀ1 (KBr) 3000–2850, 1745, 1400, 1390, 1220; 1H
lmol, 60%) which was recrystallized from ethyl ace-
5.3.38. 4-Tosylamidoestrone (23b)
m
To a stirred suspension of the freshly prepared crude 23a
(500 mg, 1.75 mmol) in anhydrous dichloromethane (15 mL) at
ice/water temperature was added anhydrous pyridine (0.28 mL)
followed by tosyl chloride (334 mg, 1.75 mmol). After stirring at
room temperature for 3 h, the reaction mixture was diluted with
ethyl acetate (100 mL). The organic layer was washed with hydro-
chloric acid (1 M, 50 mL) followed by brine (4 ꢃ 50 mL), dried
NMR (400 MHz, CDCl3) d 0.94 (3H, s, C18H3), 1.40–1.75 (6H, m),
2.0–2.6 (10H, m), 2.87 (2H, m, C6H2), 6.79 (1H, s, C4H), 7.31 (2H,
AA0BB0, Tosyl), 7.57 (1H, s, C1H) and 7.82 (2H, AA0BB0, Tosyl); LRMS
(FAB+) m/z (%) 655.0 [25, (M+H+NBA)+], 501.0 (100, M+), 420.2 (45),
346.0 [70, (MꢀTosyl)+]; LRMS (FABꢀ) m/z (%) 346.2 [100,
(MꢀTosyl)ꢀ]; HRMS (FAB+) Found: 501.1299. C25H27NO6S2 requires
501.1280. Found: C, 59.6; H, 5.46; N, 2.80. C25H27NO6S2 requires C,
59.86; H, 5.43; N, 2.79.
(MgSO4), filtered and evaporated to give
(790 mg) which was fractionated by flash chromatography (ethyl
a yellow residue

2518
L. W. Lawrence Woo et al. / Bioorg. Med. Chem. 20 (2012) 2506–2519
acetate/hexane, 1:1 to 2:1 gradient). The major fractions isolated
gave a light yellow residue (670 mg) and this was recrystallized
from acetone/diethyl ether (1:25, 52 mL) in the refrigerator to give
References and notes
1. Adams, J. B.; Garcia, M.; Rochefort, H. Cancer Res. 1981, 41, 4720.
2. Dauvois, S.; Labrie, F. Breast Cancer Res. Treat. 1989, 13, 61.
3. Naitoh, K.; Honjo, H.; Yamamoto, T.; Urabe, M.; Ogino, Y.; Yasumura, T.;
Nambara, T. J. Steroid Biochem. 1989, 33, 1049.
4. Chetrite, G. S.; Cortes-Prieto, J.; Philippe, J. C.; Wright, F.; Pasqualini, J. R. J.
Steroid Biochem. Mol. Biol. 2000, 72, 23.
5. Anderson, C. J.; Lucas, L. J. H.; Widlanski, T. S. J. Am. Chem. Soc. 1995, 117, 3889.
6. Nussbaumer, P.; Billich, A. Med. Res. Rev. 2004, 24, 529.
7. Reed, M. J.; Purohit, A.; Woo, L. W. L.; Newman, S. P.; Potter, B. V. L. Endocr. Rev.
2005, 26, 171.
8. Woo, L. W. L.; Purohit, A.; Potter, B. V. L. Mol. Cell. Endocr. 2011, 340, 175.
9. Maltais, R.; Poirier, D. Steroids 2011, 76, 929.
23b as light yellow crystals (185 mg, 421
209 °C; TLC (chloroform/acetone, 4:1) Rf 0.64, cf. Rf 0.49 (23a); IR
max/cmꢀ1 (KBr) 3420, 3300, 3000–2850, 1730, 1600, 1500, 1160;
lmol, 24%), mp 206–
m
1H NMR (400 MHz, DMSO-d6) d 0.83 (3H, s, C18H3), 1.0–2.5 (18H,
m), 6.53 (1H, d, J = 8.6 Hz, C1H), 7.00 (1H, d, J = 8.5 Hz, C2H), 7.30
(2H, AA0BB0, Tosyl), 7.60 (2H, AA0BB0, Tosyl), and 8.87 (2H, br s,
ex. with D2O, NH and OH); LRMS (FAB+) m/z (%) 440.1 [45,
(M+H)+], 284.1 [100, (MꢀTosyl)+]; LRMS (FABꢀ) m/z (%) 592.2
[10, (M+NBA)ꢀ], 438.2 [100, (MꢀH)ꢀ]; HRMS (FAB+) Found:
440.1881. C25H30NO4S requires 440.1896. Found: C, 68.2; H, 6.74;
N, 3.24. C25H29NO4S requires C, 68.31; H, 6.65; N, 3.19.
10. Howarth, N. M.; Purohit, A.; Reed, M. J.; Potter, B. V. L. J. Med. Chem. 1994, 37,
219.
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1995, 63, 106.
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5.3.39. Estra-1,3,5(10)-trien-17-oxo[4,3d]-1,2,3-oxathiazole-3-
tosyl-2,2-dioxide (23c)
Sulfuryl chloride (2.0 mL of a 0.5 M solution in dichlorometh-
ane, 1.00 mmol) was added dropwise with stirring over a 5 min
period to 23b (400 mg, 910 lmol) and triethylamine (0.39 mL,
14. Elger, W.; Barth, A.; Hedden, A.; Redderse, G.; Ritter, P.; Schneider, B.;
Zuchner, J.; Krahl, E.; Muller, K.; Oettel, M.; Schwarz, S. Reprod. Fertil. Dev.
2001, 13, 297.
15. Chander, S. K.; Purohit, A.; Woo, L. W. L.; Potter, B. V. L.; Reed, M. J. Biochem.
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2.73 mmol) in dichloromethane (20 mL) at ice–water temperature
under an atmosphere of nitrogen. After an additional 15 min, the
reaction mixture was allowed to warm to room temperature, con-
centrated and diluted with ethyl acetate (50 mL). The organic layer
was washed with hydrochloric acid (1 M, 100 mL) followed by
brine (4 ꢃ 50 mL), dried (MgSO4), filtered and evaporated to give
a light yellow residue/syrup (550 mg) which was fractionated by
flash chromatography (chloroform/acetone, 16:1). The first fraction
isolated upon evaporation gave 23c as a creamy residue (312 mg).
Mp 105–115 °C; Rf (chloroform/acetone, 8:1) 0.69, cf. Rf 0.48 (23b);
1H NMR (400 MHz, CDCl3) d 0.98 (3H, s, C18H3), 1.4–2.4 (m), 2.45
(3H, s, ArCH3), 2.5–2.6 (ꢄH, m), 3.20 (1H, m), 3.35 (1H, m), 6.86
(1H, d, J = 8.5 Hz, ArH), 7.24 (2H, AA0BB0, Tosyl), 7.34 (1H, d,
J = 8.5 Hz, ArH), 7.51 (2H, AA0BB0, Tosyl); LRMS (FAB+) m/z (%)
879.6 (20), 599.4 (40), 337.2 (15), 91.0 (100); LRMS (FABꢀ) m/z
(%) 346.1 [100, (MꢀTosyl)ꢀ]. This fraction was used for the next
reaction without further purification.
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5.3.40. Estra-1,3,5(10)-trien-17-oxo[4,3d]-1,2,3-oxathiazole-2,2-
dioxide (23)
To a solution of crude 23c (150 mg, 299
(20 mL) was added a solution of potassium fluoride (56 mg,
964 mol) in water (5 mL). The resulting mixture was refluxed for
lmol) in acetonitrile
l
4 h, cooled, concentrated and diluted with ethyl acetate (50 mL).
The organic layer was washed with hydrochloric acid (1 M, 50 mL)
followed by brine (4 ꢃ 50 mL). After drying (MgSO4) and filtration,
the filtrate on evaporation gave a yellow-green syrup (170 mg)
which was fractionated by flash chromatography (ethyl acetate/
hexane, 1:1 to 4:1 gradient) to give a green residue (95 mg). A solu-
tion of this fraction in absolute ethanol was heated with activated
charcoal and upon filtration and evaporation gave 23 as a creamy
powder (85 mg, 245 lmol, 82%); IR m
max/cmꢀ1 (KBr) 3300–3000,
3000–2850, 1710, 1450, 1370, 1190; 1H NMR (400 MHz, CDCl3) d
0.93 (3H, s, C18H3), 1.2–2.8 (ꢄ15H, m), 6.73 (1H, br s, NH), 6.94
(1H, d, J = 8.5 Hz, ArH) and 7.07 (1H, d, J = 8.5 Hz, ArH); LRMS
(FAB+) m/z (%) 879.6 (50), 599.3 (100), 530.3 (20), 337.2 (30),
263.2 (24), 219.1 (20), 97.0 (55); LRMS (FABꢀ) m/z (%) 1086.0 (10),
346.1 [100, (MꢀH)ꢀ], 297.2 (25); HRMS (FABꢀ) Found: 346.1128.
C18H20NO4S requires 346.1113. HPLC: Waters ‘Symmetry’ C18
(packing: 3.5
l
m, 4.6 ꢃ 150 mm); mobile phase MeCN/H2O (96/4)
isocratic; flow rate, 1 mL/min; tR = 1.20 min; purity: >98%.
Acknowledgements
46. Woo, L. W. L.; Purohit, A.; Reed, M. J.; Potter, B. V. L. Bioorg. Med. Chem. Lett.
1997, 7, 3075.
47. Appel, R.; Berger, G. Chem. Ber. 1958, 91, 1339.
We thank A.C. Smith for technical assistance. This work was
supported by Sterix Ltd.

L. W. Lawrence Woo et al. / Bioorg. Med. Chem. 20 (2012) 2506–2519
2519
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Biochem. Mol. Biol. 1996, 57, 79.
50. Duncan, L.; Purohit, A.; Howarth, N. M.; Potter, B. V. L.; Reed, M. J. Cancer Res.
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49. Purohit, A.; Williams, G. J.; Howarth, N. M.; Potter, B. V. L.; Reed, M. J.
Biochemistry 1995, 34, 11508.