Synthesis, antitubulin, and antiproliferative SAR of analogues of 2-methoxyestradiol-3,17- O, O -bis-sulfamate

Article abstract of DOI:10.1021/jm9018806

The synthesis and antiproliferative activity of analogues of estradiol 3,17-O,O-bis-sulfamates (E2bisMATEs) are discussed. Modifications of the C-17 substituent reveal that an H-bond acceptor is essential for high antiproliferative activity. The local env

Full text of DOI:10.1021/jm9018806

2942 J. Med. Chem. 2010, 53, 2942–2951
DOI: 10.1021/jm9018806
Synthesis, Antitubulin, and Antiproliferative SAR of Analogues
of 2-Methoxyestradiol-3,17-O,O-bis-sulfamate
Fabrice Jourdan,‡ Mathew P. Leese,‡ Wolfgang Dohle,‡ Ernest Hamel,§ Eric Ferrandis, Simon P. Newman,^ Atul Purohit,^
Michael J. Reed,†,^ and Barry V. L. Potter*,‡
‡Department of Pharmacy and Pharmacology & Sterix Ltd., University of Bath, Claverton Down, Bath BA2 7AY, U.K., §Screening Technologies
Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick, National
Institutes of Health, Frederick, Maryland 21702, Institut de Recherche Henri Beaufour, 91966 Les Ulis Cedex, France, and ^Endocrinology and
Metabolic Medicine, Imperial College, St. Mary’s Hospital, London W2 1NY, U.K. †Deceased.
Received December 21, 2009
The synthesis and antiproliferative activity of analogues of estradiol 3,17-O,O-bis-sulfamates
(E2bisMATEs) are discussed. Modifications of the C-17 substituent reveal that an H-bond acceptor
is essential for high antiproliferative activity. The local environment in which this H-bond acceptor lies
can be varied to an extent. The C-17-oxygen linker can be deleted or substituted with an electronically
neutral methylene group, and replacement of the terminal NH2 with a methyl group is also acceptable.
Mesylates 10 and 14 prove equipotent to the E2bisMATEs 2 and 3, while sulfones 20 and 35 display
enhanced in vitro antiproliferative activity. In addition, the SAR of 2-substituted estradiol-3-O-
sulfamate derivatives as inhibitors of tubulin polymerization has been established for the first time.
These agents inhibit the binding of radiolabeled colchicine to tubulin.
Introduction
In previous reports, we and others described the discovery of
sulfamoylated derivatives of 2-methoxyestradiol 1 (Figure 1) as
promising anticancer agents with dual activity against cancer
cell proliferation and angiogenesis.1-7 The appeal of agents that
act against these two targets has been underlined by the positive
outcomes of clinical trials combining cytotoxic agents with
the anti-VEGF antibody bevacizumab, an inhibitor of angio-
genesis.8-12 2-Methoxyestradiol-3,17-O,O-bis-sulfamate 2 and
2-ethylestradiol-3,17-O,O-bis-sulfamate 3 (E2bisMATEsa) dif-
fer from 1 because of their enhanced biological activity and
superior drug-like properties. The excellent oral bioavailability
of 2 (>85% in rodents) appears to derive from the ability of the
sulfamate group to block inactivating metabolism and deacti-
vating conjugation and to interact reversibly with carbonic
anhydrase.2,7,13-15 This latter reversible interaction, which has
Figure 1. Structures of 2-methoxyestradiol 1, 2-methoxyestradiol-
3,17-O,O-bis-sulfamate 2, 2-ethylestradiol-3,17-O,O-bis-sulfamate
3, 2-methoxyestrone-3-O-sulfamate 4, 2-methoxy-3-O-sulfamoyl-17β-
cyanomethyl estra-1,3,5(10)-triene 5, and 2-ethyl-3-O-sulfamoyl-17β-
cyanomethyl estra-1,3,5(10)-triene 6.
been characterized by protein crystallography, may minimize
first pass liver metabolism through sequestration of the sulfa-
mates in red blood cells. Moreover, as with other aryl sulfa-
mates, 2 and 3 are irreversible inhibitors of steroid sulfatase
(STS), itself a target for the treatment of hormone dependent
cancer.7,16 Although a full mechanistic picture for the activity of
these compounds continues to emerge, all evidence collected
to date suggests that their ability to disrupt the tubulin-
microtubule equilibrium in cells is critical for their antitu-
mor activity.2 It is also important to note, first, that these
compounds are not substrates for the P-glycoprotein pump and
are thus active against taxane-resistant tumors,17,18 and second,
that their activity is independent of the estrogen receptor even
though they are estrogen derivatives.
A-ring modified estrogen-3-O-sulfamates were initially
synthesized with the goal of obtaining potent nonestrogenic,
STS inhibitors.19 It was subsequently discovered that 2-meth-
oxyestrone-3-O-sulfamate 4 also exhibited an antiprolifera-
tive effect against a range of estrogen-independent human
cancer cells in vitro.3 SAR studies on the A-ring estrogen-3-
O-sulfamates demonstrated that optimal antiproliferative
activity is obtained with a 2-methoxy, 2-ethyl, or 2-methyl
sulfanyl group.1 Further SAR studies focused on D-ring
modifications, principally at the C-17 position of the estratriene
*To whom correspondence should be addressed. Phone: þ44 (0)1225
386639. Fax: þ44 (0)1225 386114. E-mail: B.V.L.Potter@bath.ac.uk.
a Abbreviations: E2bisMATEs, estradiol-3,17-O,O-bis-sulfamates;
VEGF, vascular endothelial growth factor; STS, steroid sulfatase;
m-CPBA, 3-chloroperoxybenzoic acid; DMA, N,N-dimethylacetamide;
TEA, triethylamine; GTP, guanosine triphosphate.
r
pubs.acs.org/jmc
Published on Web 03/12/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2943
Figure 2. Proposed modifications to E2bisMATEs and 17β-cyanomethyl estradiol-3-O-sulfamates at C-17.
Scheme 1a
skeleton. A number of modifications were explored, including
variation of the oxidation level of C-17 and substitution at this
position. While 17β-hydroxy, 17-keto, and 17-oximino deriva-
tives show similar antiproliferative activity, the 17-deoxy and
17R-benzyl analogues were considerably less active.20 Sulfa-
moylation of the 17β-hydroxy group gave the E2bisMATEs 2
and 3 that exhibit slightly enhanced in vitro activity and greatly
improved in vivo antitumor effects.2 In addition, a SAR study
of C-17 cyanated analogues revealed that 2-methoxy-3-
O-sulfamoyl-17β-cyanomethyl estra-1,3,5(10)-triene 5 and 2-
ethyl-3-O-sulfamoyl-17β-cyanomethyl estra-1,3,5(10)-triene 6
offered further enhanced antiproliferative and antiangiogenic
activities relative to 2 and 3.21 We have also shown that a high
level of antiproliferative activity can be retained when a
heterocycle possessing a well exposed H-bond acceptor is
installed at C-17.22 It thus appears that optimal activity results
a Reagents and conditions: (i) CH3SO2Cl, pyridine; (ii) H2, Pd/C,
THF, MeOH; (iii) H2NSO2Cl, DMA.
from the combined presence of a C-2 XMe group (X = O, CH2
or S), 3-O-sulfamate, and a H-bond acceptor around the C-17
position.
Scheme 2a
Having shown that there is a reasonable degree in flexibility
in the nature of the C-17 substituent and that the in vivo
profile of 2 and 3 remained the most promising of the
structural class, we were drawn to modify the 17-O-sulfamate
group in the hope of discovering additional antitumor com-
pounds with improved activity. We thus set out to syntheti-
cally replace the constituent atoms of the sulfamate group
with isosteric groups as outlined in Figure 2. In addition,
amino and nitro derivatives were elaborated to compare their
activities with those of the cyanomethyl compounds 5 and 6.
We describe here our synthetic approaches to these com-
pounds together with their in vitro biological activity. We
also report herein the structure-activity relationships of this
compound class as inhibitors of tubulin polymerization and
colchicine binding.
a Reagents and conditions: (i) NaH, CH3SCH2PPh3Cl, THF; (ii) H2,
Pd/C, THF, MeOH; (iii) m-CPBA, DCM; (iv) TBAF, THF; (v) H2N-
SO2Cl, DMA.
Results and Discussion
Chemistry. Our previous SAR studies on C-17 modified
estradiol-3-O-sulfamates showed that E2bisMATEs 2 and 3
possess an excellent in vivo profile and are the most promis-
ing of their class of compounds.2 To assess the contribution
of the sulfamate NH2 group to activity, the 17-β-O-metha-
nesulfonyl analogues of 10 and 14 were prepared (Scheme 1).
2-Methoxy-3-O-benzyl estradiol 7 was reacted with metha-
nesulfonyl chloride to afford the 2-methoxy-3-O-benzyl-17-
O-methanesulfanylestra-1,3,5(10)-triene 8 in excellent yield.
Hydrogenolysis of 8 furnished the corresponding phenol 9,
which was then treated with sulfamoyl chloride to afford the
corresponding sulfamate derivative 10. 2-Ethyl-3-O-benzyl
estradiol 11 was transformed to give compounds 12-14
under analogous conditions.
Having substituted the sulfamate NH2 group for a methyl,
we proceeded to additionally substitute the 17β-oxygen atom
with a methylene group and thus prepared sulfone 20. Wittig
reaction of (methylthiomethyl)triphenylphosphonium chlo-
ride with 2-ethyl-3-O-tert-butyldimethylsilylestrone 15 gave
methyl vinyl sulfide 16 as a 3:2 mixture of E- and Z-isomers
(Scheme 2). Hydrogenation delivered 17 which, upon
m-CPBA oxidation and silyl deprotection, gave sulfone 19.

2944 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Jourdan et al.
Scheme 3a
aReagents and conditions: (i) hydroxylamine hydrochloride, pyridine, reflux; (ii) NaBH4, MoO3, THF, MeOH, 0 ꢀC; (iii) H2, Pd/C, THF, MeOH;
(iv) sulfamide, 1,4-dioxane, reflux; (v) H2NSO2Cl, DMA; (vi) CH3SO2Cl, pyridine, 0 ꢀC to rt.
Scheme 4a
17β-N-Methylsulfonyl analogues of bis-sulfamates 2 and 3
were synthesized from intermediates 21 and 25 (Scheme 3) by
reaction with methanesulfonyl choride. Deprotection and
sulfamoylation yielded phenols (28 and 30) and sulfamate
derivatives (29 and 31) in succession.
We also wished to evaluate the impact of deletion of the
17β-oxygen linker to the sulfamate on antiproliferative
activity. We thus prepared 17β-methylsulfonyl-3-O-sulfa-
moylestra-1,3,5(10)-triene 34, following the route outlined
in Scheme 4. Treatment of 2-ethyl-3-O-benzylestrone 16 with
Lawesson’s reagent in refluxing toluene/xylene, followed by
reduction with sodium borohydride in THF, afforded thiol
32. S-Methylation with methyl iodide in the presence of
sodium methoxide and subsequent oxidation with m-CPBA
in DCM produced sulfone 33. Conversion to the correspond-
ing phenol 34 and sulfamate 35 was as described for the other
a Reagents and conditions: (i) Lawesson’s reagent, THF, 150 ꢀC,
analogues in the series.
microwave; (ii) NaBH4, THF, 0 ꢀC, 1 h; (iii) MeONa, MeI, THF, MeOH,
As noted above, screening of various C-17 modified 2-sub-
rt, 16 h; (iv) m-CPBA, DCM, 0 ꢀC, 2 h; (v) H2, Pd/C, THF, MeOH;
stituted estrogen-3-O-sulfamates for antiproliferative activ-
(vi) H2NSO2Cl, DMA.
ity indicated that hydrogen bonding interactions around
Subsequent sulfamoylation under standard conditions
afforded sulfamate 20.
C-17 are key to a high level of activity. We therefore
investigated whether this positive interaction could be ac-
cessed by a N,N-dimethylamino group linked to C-17 by an
alkyl linker. Nitrile 3622 was thus converted to 17β-(2-(N,N-
dimethylamino)ethyl)-2-ethyl-3-O-sulfamoylestra-1,3,5(10)-
triene 36, as shown in Scheme 5. Reduction of 37 with lithium
aluminum hydride in THF afforded 37, which was doubly
methylated in a refluxing mixture of formic acid and aqueous
formaldehyde. Hydrogenolysis of the resultant 17β-(2-(N,N-
dimethylamino)ethyl)-2-ethyl-3-O-benzylestra-1,3,5(10)-triene
37 gave the phenol 38, which was converted to the sulfamate 39
as described above.
To access 17β-(N,N-dimethylaminomethyl)-2-ethyl-3-
O-sulfamoylestra-1,3,5(10)-triene 45, a Henry-type reac-
tion under conditions modified from Tamura et al.25 was
used to give 41. This compound was then reduced with
lithium aluminum hydride to give 17β-aminomethyl-2-
ethyl-3-O-benzylestra-1,3,5(10)-triene 42 in 40% overall
yield. Reductive alkylation of 42 was carried out under
the conditions described for the synthesis of 38. Deprotec-
tion of 43 and sulfamoylation delivered successively phenol
44 and sulfamate 45. Hydrogenation of 41 over Pd/C was
also carried out to afford 17β-nitromethyl derivative 46,
We had earlier established that the C-17 oxygen is not
essential for activity21 and were thus drawn to explore what
effect its replacement with a nitrogen would have while
otherwise retaining all other elements of the C-17 sulfamate.
Amination of C-17 was thus required to access the C-17
sulfamides. This was effected by synthesizing the corre-
sponding oxime, followed by diasteroselective reduction
with sodium borohydride and molybdenum trioxide
(Scheme 3), as described by Gonschior et al.,23 to afford
17β-amine 21. Deprotection of the benzyl ether by hydro-
genolysis gave phenol 22. Attempts to directly install the two
sulfamate groups with sulfamoyl chloride in DMA24 or 2,6-
di-tert-butyl-4-methylpyridine in DCM proved unsuccess-
ful, and we thus adopted a two-step approach toward the
sulfamate/sulfamide derivatives. First, 22 was N-sulfamoyl-
ated with sulfamide in refluxing 1,4-dioxane to afford
2-methoxy-17β-aminosulfonamide-3-hydroxyestra-1,3,5(10)-
triene 23 in 61% yield. Next, compound 23 was sulfamoy-
lated with sulfamoyl chloride in DMA to give 24 in 23%
yield. The same approach was used in the ethyl series, but the
target compound could not be isolated in satisfactory purity.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2945
Scheme 5a
Table 1. Antiproliferative Activities of 2-Ethyl- and 2-Methoxyestra-
diol Derivatives against DU-145 Human Prostate Cancer Cells and
MDA-MB-231 Human Breast Cancer Cells in Vitroa
GI50 (μM)
MDA
DU-145 MB-231
compd R1
R2
X
Y
Z
1
MeO
MeO SO2NH2
Et SO2NH2
H
OH
na
na
1.22
0.94
0.28
0.21
0.071
0.141
0.12
2.37
0.23
nd
2
O
O
SO2
SO2
CN
CN
na
NH2 0.34
NH2 0.21
3
4
MeO SO2NH2 CH2
Et SO2NH2 CH2
MeO SO2NH2 dO
na
na
0.062
0.054
0.46
4.2
5
a Reagents and conditions: (i) LiAlH4, THF, rt; (ii) 37% aq formal-
dehyde/HCOOH/reflux; (iii) H2, Pd/C, THF, MeOH; (iv) H2NSO2Cl,
DMA.
6
na
9
MeO
MeO SO2NH2
H
O
O
O
O
SO2
SO2
SO2
SO2
SO2
na
CH3
CH3
CH3
CH3
CH3
na
10
13
14
20
22
23
24
26
27
28
29
30
31
34
35
39
40
44
45
46
47
48
0.52
5.58
0.57
0.19
2.64
Scheme 6a
Et
Et
H
SO2NH2
0.20
0.23
3.56
nd
Et
MeO
MeO
SO2NH2 CH2
H
H
NH2
NH
SO2
SO2
na
NH2 58.6
NH2 6.13
MeO SO2NH2 NH
5.6
na
Et
Et
H
H
H
NH2
NH
NH
na
NH2 >100
60.8
SO2
SO2
SO2
SO2
SO2
SO2
SO2
nd
nd
MeO
CH3
CH3
CH3
CH3
CH3
CH3
>100
9.9
MeO SO2NH2 NH
5.2
nd
Et
Et
H
NH
SO2NH2 NH
na
SO2NH2 na
>100
0.84
>100
0.11
7.96
3.54
10.1
3.05
12.2
0.14
0.49
3.52
>100
0.23
4.74
2.96
9.5
Et
Et
H
Et
Et
H (CH2)2 NMe2 na
SO2NH2 (CH2)2 NMe2 na
Et
Et
H
CH2
SO2NH2 CH2
CH2
SO2NH2 CH2
H CH2
NMe2 na
NMe2 na
4.03
10.4
0.17
0.12
Et
Et
H
NO2
NO2
CN
na
na
na
MeO
a Data for compounds 1-6 are taken from the literature.2,21,26
Abbreviations: na, not applicable; nd, not determined.
moiety.2 In an attempt to find a bioisosteric replacement of
the 17-sulfamate, we also showed that 17β-carbamoyloxy-
2-ethyl estradiol-3-O-sulfamate essentially retained the anti-
proliferative activity of 2 and 3. In contrast, introduction of a
17β-O-acetate led to greatly reduced inhibition of cancer cell
proliferation.26
aReagents and conditions: (i) CH3NO2, cat. (CH3)2N(CH2)2NH2,
reflux; (ii) LiAlH4, THF, rt; (iii) 37% aq formaldehyde, HCOOH, reflux;
(iv) H2, Pd/C, THF, MeOH; (v) H2NSO2Cl, DMA.
which was subsequently converted into sulfamate 47
(Scheme 6).
Biology. To assess their potential as anticancer agents, we
evaluated the series of novel 2-ethyl- and 2-methoxyestra-
diol derivatives for their ability to inhibit the prolifera-
tion of DU-145 (androgen receptor negative) prostate
cancer cells and MDA-MB-231 (estrogen receptor nega-
tive) breast cancer cells in vitro. The results of these assays
and comparative data for compounds 1-6 are presented in
Table 1.
We reported earlier on the promising multitargeted anti-
tumor agents 2 and 3 (Figure 1), both of which exhibit
excellent activities against cancer cell proliferation and an-
giogenesis and possess excellent oral bioavailability.2,6,15
These studies showed that N,N-bismethylation of the
17β-sulfamate causes a marked decreased in antiproliferative
activity, the detrimental effect being attributed to steric
factors, resulting from the size of the N,N-dimethylsulfamate
In an attempt to better understand the role played by each
constituent (O, SO2, NH2) of the 17β-O-sulfamate group in
the antitumor activity of compounds 2 and 3, and to possibly
achieve enhanced activity, we designed a number of analo-
gues. First, we replaced the NH2 group with a methyl group.
Hence, phenols (9, 13) and sulfamates (10, 14) were prepared
and screened against DU-145 and MDA-MB-231 cancer
cells. As can be seen from the data presented in Table 1,
phenols 9 and 13 proved 3-4 fold less active than 2-methox-
yestradiol 1. With the notable exception of compound 48,21
which is the only C-17 substituted estratriene-3-ol derivative
that possesses significantly enhanced antiproliferative activ-
ity relative to 1, these results are in agreement with those
of previous studies wherein simple modification of position
C-17 alone does not yield greatly improved activities relative
to the parent estradiol.1,2,20,21,26,27 Sulfamates 10 and 14,

2946 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Jourdan et al.
Table 2. GI50 (μM) and MGM (μM) Values Obtained from the NCI Screening Panela
lung
HOP-62
colon
HCT-116
CNS
SF-539
melanoma
UACC-62
ovarian
OVCAR-3
renal
SN12-C
compd
MGM
1
0.7
0.051
<0.01
6.9
0.47
0.045
nd
0.32
0.036
<0.01
4.6
0.36
<0.01
<0.01
14.4
0.21
<0.01
<0.01
2.9
0.95
0.126
0.028
19.9
1.3
0.087
0.018
5.9
2
3
8
5.9
10
14
20
28
29
31
44
45
47
1.17
0.78
<0.01
<0.01
60.3
5.6
0.34
0.65
<0.01
10.7
0.54
3.1
1.58
0.028
0.030
89.1
7.1
<0.01
<0.01
>100
9.6
<0.01
<0.01
>100
3.2
<0.01
<0.01
>100
3.5
<0.01
<0.01
>100
23.4
>100
7.1
4.17
4.37
11.5
9.1
0.47
5.89
0.71
6.92
2.69
20.9
10.2
0.071
21.4
13.5
16.2
12.6
18.6
11.7
39.8
12.6
43.7
18.2
0.031
0.041
0.021
0.063
0.025
0.066
a Results are micromolar GI50 values. Data for 1, 2, and 3 are taken from the literature.2,30 The MGM represents the mean concentration that caused
50% growth inhibition in all 60 cell lines. Abbreviation: nd, not determined.
on the other hand, displayed excellent antiproliferative
activities, being slightly less potent than 2 and 3 in DU-145
cells but equipotent in MDA-MB-231 cells. Thus, the C-17
terminal NH2 can be replaced by a small alkyl group and
the amino group’s potential H-bond donating properties
are not required for a compound to maintain good anti-
proliferative activity. Compounds 10 and 14, together
with a selection of other phenols and sulfamates, were also
tested for antiproliferative activity in the NCI 60-cell line
assay.28,29 Data obtained for six individual cell lines are
presented in Table 2. Compound 14 caused >50% growth
inhibition at concentrations below 10 nM in the majority of
the cell lines, with MGM value of 28 nM, which correlates
well with our DU-145 and MDA-MB-231 assay results.
Compound 10 was much less active, with a MGM value of
1.58 μM.
The next change we made in the 17-O-sulfamate moiety
was replacement of the oxygen linker between C-17 and the
SO2 group with a methylene group (sulfamate 20) or a NH
group (sulfamates 24, 29, 31). Compound 20 was equipotent
with derivatives 2 and 3 in both the DU-145 and MDA-MB-
231 cells. It was also highly active in the NCI cell lines,
yielding a MGM value of 30 nM. Conversely, the 17-β-
aminosulfonamide 24 (X = NH) was 20- to 30-fold less
active than E2bisMATEs 2 and 3. When both the oxygen
linker and terminal NH2 were replaced, as in 17-β-amino-
methanesulfonyl derivatives 29 and 31 (X = NH, Z = CH3),
the two resulting compounds had different activities. Com-
pound 29 was dramatically less potent than 2 and 3 in the
DU-145 and MDA-MB-231 cells and in the NCI cell lines.
Compound 30 was more active, with a submicromolar GI50
in the DU-145 cells. In summary, while an H-bond acceptor
group (X = O) and an electronically neutral group (X =
CH2) were tolerated, an H-bond donating group (X=NH)
led to a marked decrease in antiproliferative activity.
Interestingly, sulfamate 35, in which the methylsulfonyl
group is directly linked to C-17, was slightly more active
than 2 and 3 in DU-145 cells and equipotent with them in
MDA-MB-231 cells. Thus, the oxygen linker to the sulfa-
mate at C-17 is not required for antiproliferative activity.
These results also illustrate the importance of the SO2
hydrogen-bond accepting group because replacement
of both the oxygen linker and final NH2 group (com-
pound 20) or deletion of the oxygen linker (compound
35), can still lead to compounds displaying remarkable
antiproliferative activities.
In light of the above results, we sought to evaluate the
impact of the H-bond accepting group Y (see Figure 2) on the
antiproliferative activity of the resulting sulfamates by re-
placing the SO2 group with a N,N-dimethylamino group
(sulfamates 39 and 44) and a nitro group (sulfamate 46). As
can be seen from Table 1, compounds 39 and 44 proved 10- to
15-fold less active than the E2bisMATEs 2 and 3 and
between 20- and 60-fold less active than the cyanomethyl
derivatives 4 and 5, indicating that the N,N-dimethylamino
group is not a suitable replacement for the SO2 and the cyano
groups, regardless of the length of the alkyl linker (CH2 or
(CH2)2). This might be due to the size of the NMe2 group,
with steric hindrance around the nitrogen atom reducing its
accessibility and in turn its ability to form the requisite
H-bond. This postulate is supported by the excellent anti-
proliferative activities in DU-145 and MDA-MB-231 cells
shown by the 17β-nitromethyl derivative 46, which had a
MGM value of 71 nM in the NCI panel.
The antiproliferative effect of 2-methoxyestradiol 1 has
been shown to stem from its ability to disrupt tubulin
polymerization by interacting at the colchicine site of
tubulin.31 Likewise, mechanistic studies carried out on 2-
methoxyestrone-3-O-sulfamate 4 and its 2-ethyl analogue
have shown that they induce mitotic arrest and apoptosis,
properties that are most likely attributable to their antimi-
crotubule activity.4 To explore their likely mechanism of
action and construct an on target SAR for this class of
compounds, the E2bisMATEs 2 and 3 and a number of the
most active 2-substituted estradiol-3-O-sulfamate deriva-
tives were evaluated for their inhibitory effects on tubulin
polymerization and on the binding of [3H]colchicine to
tubulin (Table 3). With the exception of compounds 50, 53,
and 54, which had little effect on tubulin assembly (and
indeed are only very modest inhibitors of cell proliferation),
all the tested derivatives were strong inhibitors of tubulin
polymerization, with compounds 3, 5, 14, 47, 49, and 52
essentially equipotent to combretastatin A-4 in this assay
and 2- to 5-fold more active than 2-methoxyestradiol 1. A
clear correlation between the ability to inhibit tubulin
assembly and cell proliferation is evident. Indeed, all of the
compounds with submicromolar GI50’s against DU-145 and
MDA-MB-231 proliferation were good inhibitors of tubulin
assembly (IC50 values between 1.3 and 3.6 μM), while the
compounds with weak antiproliferative activity (50, 53, and
54) had negligible effects on tubulin assembly. Importantly,
sulfamates 2 and 51 are markedly more active than their

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2947
Table 3. Inhibition of Tubulin Polymerization and Colchicine Binding by 2-Ethyl and 2-Methoxyestradiol Derivatives and Combretastatin A-4 (CA-4)
inhibition of tubulin
assembly c IC50 (μM) ( SD
colchicine binding,d
compd
R1
na
R2
R3
GI50(μM) DU-145
% inhibition ( SD
CA-4
1
na
H
na
1.2 ( 0.2
7.0 ( 0.8
3.0 ( 0.4
1.3 ( 0.04
3.3 ( 0.2
>40
99 ( 1
17 ( 4
29 ( 6
57 ( 0.8
22 ( 5
nd
MeO
MeO
Et
OH
OH
OH
dO
dO
1.22
0.19a
<0.01a
0.32a
>10b
18.3a
0.34
51
52
4
SO2NH2
SO2NH2
SO2NH2
SO2NMe2
H
MeO
MeO
MeO
MeO
Et
53
54
2
OSO2NH2
OSO2NH2
OSO2NH2
OSO2NH2
OSO2NH2
OSO2CH3
CH2SO2CH3
SO2CH3
>40
nd
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
2.2 ( 0.3
1.3 ( 0.01
1.3 ( 0.2
20 ( 1
1.6 ( 0.2
2.1 ( 0.2
3.6 ( 0.2
1.3 ( 0.08
1.7 ( 0.3
28 ( 3
45 ( 4
46 ( 4
nd
3
0.21
0.38
49
50
14
20
35
5
Me
nPr
Et
3.4
0.57
49 ( 0.2
37 ( 5
28 ( 5
78 ( 0.9
74 ( 2
Et
Et
0.19
0.11
MeO
Et
CH2CN
CH2NO2
0.062
0.14
47
a Data taken from the literature.1,2 b GI50 in MCF-7 cells.1 c Tubulin was at 10 μM, and compounds were evaluated at various concentrations.
Inhibition of the extent of assembly at 20 min was determined. d Inhibition of [3H] colchicine binding: tubulin was at 1.0 μM, colchicine was at 5.0 μM,
and tested compounds were at 5.0 μM. na: not applicable, nd: not determined.
binding. Among the most active inhibitors of assembly, the
greatest inhibition of colchicine binding was observed with
compound 5 (78%) and the least with compound 3 (45%).
Conclusions
This study was aimed at identifying the minimum require-
ments to maintain or enhance the high level of antiprolifera-
tive activity of E2bisMATEs 2 and 3 by modification of the
C-17β-O-sulfamate. A summary of the in vitro SAR of the
2-substituted estradiol-3-O-sulfamate compounds as antipro-
liferative agents and tubulin polymerization inhibitors is
presented in Figure 3.
Figure 3. In vitro SAR of 2-substituted estradiol-3-O-sulfamate
derivatives as antiproliferative agents and tubulin polymerization
inhibitors.
We first showed that the hydrogen bonding potential of the
terminal NH2 group of the sulfamate is not essential and that
high antiproliferative activity is retained when it is replaced by
a CH3, as illustrated by compounds 10, 14, and 20. We also
established that, while the 17β-aminosulfamate 24 and 17β-
aminomesylates 29 and 31 show much reduced antiprolifera-
tive activities, substitution of the oxygen linker remains
possible. This was shown by the substantial antiproliferative
activity obtained with sulfone 20. We also demonstrated that
the oxygen linker of the 17β-sulfamate can be deleted, as
demonstrated by the antiproliferative potency of compound
35. The high antiproliferative activity shown by 2-ethyl-17β-
nitromethyl-3-O-sulfamoylestra-1,3,5(10)-triene 46 contrasts
with the 20-25-fold lower antiproliferative activity observed
with the N,N-dimethylaminomethyl derivative 45 and the N,
N-dimethylaminoethyl derivative 40. This result is in concert
with the excellent antiproliferative activity obtained with
compounds 10, 14, 20, and 35, all of which possess a SO2
group able to function as a H-bond acceptor moiety, which
thus far is required at position C-17. Finally, evaluation of a
number of 2-substituted estradiol-3-O-sulfamate derivatives
that possess high antiproliferative activity demonstrated that
this activity probably derives from their ability to inhibit
tubulin assembly. Over the course of a number of studies,
corresponding phenols 1 and 54, a result that also correlates
with our previous observation that sulfamoylation at the
phenolic position is a key factor for obtaining a good level of
antiproliferative activity.1-3,20-22,26,27 Similarly, in the bis-
sulfamate series, optimal activity was obtained for the C-2
methyl, ethyl, and methoxy derivatives (compounds 49, 2
and 3), while the bulkiest group in the series (n-propyl,
compound 49) was detrimental to activity, with a sharp,
>10-fold decrease in the GI50 obtained as well as sharply
reduced activity as an inhibitor of tubulin assembly. N,N-
Dimethylation of sulfamate 52 (IC50 = 3.3 μM) to yield
compound 53 led to a sharp decrease in tubulin assembly
activity, in line with the deleterious effect of this substituent
on antiproliferative activity.1
Study of the effects of this series of compounds on inhibi-
tion of colchicine binding was performed with the agents that
inhibited tubulin assembly with IC50’s in the low micromolar
range, with a comparison to the known weak effect of 1 and
the known potent effect of combretastatin A-4 (CA-4). All
the sulfamates examined were more potent than 1 but
substantially less active than CA-4. When present in equi-
molar concentration with [3H]colchicine (5.0 μM each), there
was a reasonable correlation between potency as an inhibitor
of tubulin assembly and potency as an inhibitor of colchicine

2948 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Jourdan et al.
we have found that the sulfamate group yields strong inter-
actions with enzymes such as sulfatase and carbonic anhy-
drase and, in the present case, proteins such as tubulin. The
results presented here serve to define which components of the
sulfamate group are key to its interaction with tubulin and
should thus facilitate the design of further microtubule dis-
ruptors and optimization of sulfamate bearing lead com-
pounds in the future.
analyses were performed by the Microanalysis Service, Univer-
sity of Bath. Melting points were determined using a Stuart
SMP3 melting point apparatus and are uncorrected. HPLC
analyses were performed on a Waters Millenium 32 instrument
equipped with a Waters 996 PDA detector. A Waters Sunfire
C18 reversed-phase column (8 mm ꢀ 100 mm) was eluted with a
methanol/water gradient at 2 mL/min. The purity of final
compounds was greater than 95% unless specified otherwise.
2-Methoxy-3-O-benzyl-17β-O-methanesulfonylestra-1,3,5(10)-
triene 8. Methanesulfonyl chloride (0.09 mL, 1.2 mmol) was
added to an ice-cooled solution of 2-methoxy-3-O-benzyl-estra-
diol 7 (390 mg, 1 mmol) in dry pyridine (5 mL) under nitrogen.
The solution was stirred at 0 ꢀC for 2 h, and the reaction mixture
was quenched with water (20 mL). The organics were extracted
with ethyl acetate (2 ꢀ 60 mL), and the organic layer was washed
successively with water and brine and then dried over MgSO4.
After evaporation of the solvents under vacuum, the resultant
crude solid was purified by flash chromatography (hexane/
ethyl acetate 5:1) to give 8 as a white powder (0.44 g, 93%),
mp 163-164 ꢀC. 1H NMR (270 MHz, CDCl3): δ 0.86 (3H, s),
1.19-1.52 (6H, m), 1.70-1.91 (3H, m), 2.05 (1H, m), 2.17-2.32
(3H, m), 2.72 (2H, m), 3.00 (3H, s), 3.85 (3H, s), 4.56 (1H, dd,
J 8.6 and 8.1 Hz), 5.09 (2H, s), 6.60 (1H, s), 6.82 (1H, s),
7.28-7.46 (5H, m). HRMS (ESþ) m/z found 471.2188; C27H35-
O5S (Mþ þ H) requires 471.2200.
Experimental Section
Biology. In Vitro Studies: Cell Lines. DU-145 (brain metas-
tasis carcinoma of the prostate) and MDA-MB-231 (metastatic
pleural effusion of breast adenocarcinoma) established human
cell lines were obtained from the ATCC Global Bioresource
Center. Cells were maintained in a 5% CO2 humidified atmo-
sphere at 37 ꢀC in RPMI-1640 medium, supplemented with 10%
fetal bovine serum, penicillin, and streptomycin.
Antiproliferative Assays. DU-145 and MDA-MB-231 cells
were seeded into 96-well microtiter plates (5000 cells/well) and
treated with 10-9-10-4 M compounds or with vehicle control.
At 96 h post-treatment, live cell counts were determined by the
WST-1 cell proliferation assay (Roche, Penzberg, Germany) as
per the manufacturer’s instructions. Viability results were ex-
pressed as a percentage of mean control values resulting in the
calculation of the 50% growth inhibition (GI50). All experi-
ments were performed in triplicate.
Tubulin Assays. Bovine brain tubulin, prepared as described
previously,32 was used in the studies presented here. Assembly
IC50’s were determined as described in detail elsewhere.33
Briefly, 1.0 mg/mL (10 μM) tubulin was preincubated without
GTP with varying compound concentrations for 15 min at
30 ꢀC. The reaction mixtures were placed on ice, and GTP
(final concentration, 0.4 mM) was added. The reaction mixtures
were transferred to cuvettes, held at 0 ꢀC in a recording spectro-
photometer. Baselines were established at 0 ꢀC, and increase in
turbidity was followed for 20 min following a rapid (<30 s) jump
to 30 ꢀC. Compound concentrations required to reduce the
turbidity increase by 50% were determined. The method for
measuring inhibition of the binding of [3H]colchicine to tubulin
was described in detail previously.34 Reaction mixtures con-
tained 0.1 mg/mL (1.0 μM) tubulin, 5.0 μM [3H]colchicine, and
potential inhibitor at 5.0 μM. Compounds were compared to
CA-4, a particularly potent inhibitor of the binding of colchicine
to tubulin.35 Reaction mixtures were incubated 10 min at 37 ꢀC,
a time point at which the binding of colchicine in control
reaction mixtures is generally 40-60% complete.
Chemistry. All chemicals were either purchased from Aldrich
Chemical Co. (Gillingham, UK) or Alfa Aesar (Heysham, UK).
Organic solvents of A.R. grade were supplied by Fisher Scien-
tific (Loughborough, UK) and used as supplied. DMA was
purchased from Aldrich and stored under a positive pressure of
N2 after use. THF was distilled from sodium with benzophenone
indicator. Sulfamoyl chloride was prepared by an adaptation
of the method of Appel and Berger36 and was stored in the
refrigerator under positive pressure of N2 as a solution in
toluene as described by Woo et al.37 An appropriate volume
of this solution was freshly concentrated in vacuo immediately
before use. Reactions were carried out at rt unless stated
otherwise. Flash column chromatography was performed on
silica gel (MatrexC60).
2-Methoxy-3-hydroxy-17β-O-methanesulfonylestra-1,3,5(10)-
triene 9. A solution of 8 (375 mg, 0.8 mmol) in THF (10 mL) and
methanol (30 mL) was treated with 10% Pd/C (40 mg) and then
stirred under an atmosphere of hydrogen for 24 h. The resultant
suspension was then filtered through celite, washed with ethyl
acetate, and evaporated under reduced pressure. The resultant
crude solid was purified by flash chromatography (hexane/ethyl
acetate 2:1) to give a white powder (260 mg, 86%), mp 167-
168 ꢀC. 1H NMR (400 MHz, CDCl3): δ 0.91 (3H, s), 1.33-1.60
(6H, m), 1.76-1.94 (3H, m), 2.05-2.09 (1H, m), 2.18-2.34 (3H,
m), 2.80 (2H, m), 3.06 (3H, s), 3.90 (3H, s), 4.61 (1H, dd, J 9.1
and 7.7 Hz), 5.52 (1H, s), 6.68 (1H, s), 6.81 (1H, s). HRMS
(ESþ) m/z found 381.1743; C20H29O5S (Mþ þ H) requires
381.1730. Anal. (C20H28O5S) C, H, N.
2-Methoxy-3-O-sulfamoyl-17β-O-methanesulfonylestra-1,3,5(10)-
triene 10. Compound 9 (115 mg, 0.3 mmol) was added to an ice
cold solution of sulfamoyl chloride (0.6 mmol) in DMA (1.0
mL). The resulting mixture was stirred for 16 h at rt and then
diluted with ethyl acetate (50 mL) and washed with water (4 ꢀ 20
mL), brine (20 mL), dried, and evaporated. Flash chromato-
graphy (hexane/ethyl acetate 5:1 to 2:1) gave a white powder
(120 mg, 86%), mp 152-153 ꢀC. 1H NMR (400 MHz, acetone-
d6): δ 0.76 (3H, s), 1.18-1.25 (2H, m), 1.31-1.44 (4H, m),
1.62-1.81 (3H, m), 1.84-1.96 (1H, m), 2.08-2.19 (2H, m),
2.22-2.34 (1H, m), 2.65 (2H, m), 2.96 (3H, s), 3.71 (3H, s), 4.44
(1H, dd, J 8.9 and 7.7 Hz), 6.78 (2H, s), 6.88 (1H, s), 6.90 (1H, s).
HRMS (ESþ) m/z found 459.1372; C20H29NO7S2 (Mþ) requires
459.1385. Anal. (C20H29NO7S2) C, H, N.
2-Ethyl-3-hydroxy-17β-O-methanesulfonylestra-1,3,5(10)-tri-
ene 13. A solution of 12 (330 mg, 0.7 mmol) in THF (10 mL) and
methanol (30 mL) was treated with 10% Pd/C (40 mg) and then
stirred under an atmosphere of hydrogen for 24 h, as described
for the synthesis of 9. The resultant crude solid was purified by
flash chromatography (hexane/ethyl acetate 2:1) to give a white
powder (190 mg, 77%), mp 195-196 ꢀC. 1H NMR (270 MHz,
CDCl3): δ 0.86 (3H, s), 1.21 (3H, t, J 7.7 Hz), 1.25-1.60 (6H, m),
1.71-1.91 (3H, m), 2.03 (1H, m), 2.13-2.38 (3H, m), 2.58 (2H,
q, J 7.7 Hz), 2.79 (2H, m), 3.01 (3H, s), 4.53 (1H, s), 4.56 (1H, dd,
J 9.1 and 7.9 Hz), 6.49 (1H, s), 7.03 (1H, s). HRMS (ESþ) m/z
found. 379.1946; C21H31O4S (Mþ þ H) requires 379.1938. Anal.
(C21H30O4S) C, H, N.
1H NMR and 13C NMR spectra were recorded with either a
JMN-GX 270 at 270 and 67.5 MHz, respectively, or a Varian
Mercury VX 400 NMR spectrometer at 400 and 100 MHz,
respectively, and chemical shifts are reported in parts per million
(ppm, δ) relative to tetramethylsilane (TMS) as internal stan-
dard. Mass spectra were recorded at the Mass Spectrometry
Service Center, University of Bath, UK. FAB-MS were carried
out using m-nitrobenzyl alcohol (NBA) as the matrix. Elemental
2-Ethyl-3-O-sulfamoyl-17β-O-methanesulfonylestra-1,3,5(10)-
triene 14. Compound 13 (115 mg, 0.3 mmol) was reacted with
sulfamoyl chloride (0.6 mmol) in DMA (1 mL), as described for
the synthesis of 10. Flash chromatography (hexane/ethyl acetate

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7 2949
5:1 to 2:1) gave a white powder (105 mg, 77%), mp 179-180 ꢀC.
1H NMR (270 MHz, CDCl3): δ 0.85 (3H, s), 1.20 (3H, t, J 7.4
Hz), 1.30-1.55 (6H, m), 1.73-1.87 (3H, m), 2.04 (1H, m),
2.16-2.36 (3H, m), 2.68 (2H, q, J 7.4 Hz), 2.82 (2H, m), 3.01
(3H, s), 4.57 (1H, dd, J 8.7 and 8.1 Hz), 5.08 (2H, s), 6.49 (1H, s),
7.03 (1H, s). HRMS (ESþ) m/z found 458.1672; C21H32NO6S2
(Mþ þ H) requires 458.1666. Anal. (C21H31NO6S2) C, H, N.
(E- and Z-)-2-Ethyl-3-O-tert-butyldimethylsilyl-17-methylsul-
fanylmethylene Estrone 16. A rt suspension of hexane-washed
sodium hydride (12 mmol) in THF (20 mL) was treated with
diethyl (methylthiomethyl)phosphonate (2.14 mL, 12 mmol)
and then brought to reflux. On cessation of gas evolution, the
clear colorless solution was treated with a solution of 2-ethyl-
3-O-tert-butyldimethylsilyl estrone (1.68 g, 4 mmol) in THF
(20 mL). After 18 h at reflux, the reaction was cooled, diluted
with ethyl acetate (60 mL), poured onto water (40 mL), and the
aqueous layer separated. The organic layer was then washed
with water (3 ꢀ 35 mL) and brine (50 mL) and then dried and
evaporated. Column chromatography (5% ethyl acetate/
hexane) yielded the desired TBS-protected alkene 16 (1.13 mg,
62%), a colorless oil, as a mixture of isomers, as well as an
amount of the corresponding desilylated product (300 mg,
washed with water (3 ꢀ 10 mL), brine (10 mL), dried, and
evaporated to give a yellow powder. Crystallization from ethyl
acetate/hexane afforded the desired product 20 as white crystals
1
(42 mg, 58%), mp 208-210 ꢀC. H NMR (CDCl3 þ 2 drops
CD3OD) δ 7.17 (1H, s), 7.07 (1H, s), 4.95 (2H, s), 3.10-3.18 (1H,
m), 2.76-2.95 (6H, m, including 2.92 (3H, s)), 2.66 (2H, q, J 7.4),
1.16-2.40 (17H, m including 1.20 (3H, t, J 7.4), 0.64 (3H, s). m/z
[APCI-] 454.29 (Mþ - H, 100%). HRMS [FABþ] 455.1769,
C22H33NO5S requires 455.1800.
2-Ethyl-3-benzyloxyestra-1,3,5(10)-triene-17β-thiol 32. 3-Ben-
zyl-2-ethylestrone (3.1 g, 8.0 mmol), Lawesson’s reagent (1.6 g,
4.0 mmol), and a magnetic stirring bar were placed in an oven-
dried 10 mL microwave vessel. Anhydrous THF (4.0 mL) was
added and the mixture irradiated in the microwave at 150 ꢀC for
30 min. After cooling to rt, additional Lawesson’s reagent (1.6 g,
4.0 mmol) was added and the mixture irradiated once more in
the microwave at 150 ꢀC for 30 min. To gain enough material,
the whole procedure was carried out altogether four times. The
resulting thick slurry was taken up in DCM and filtered through
a silica gel pad. The filtrate was concentrated in vacuo, and the
residue was purified by flash column chromatography (SiO2,
hexane/DCM 9:1 to 4:1 to 1:1) to give 2-ethyl-3-benzyloxyestra-
1,3,5(10)-triene-17-thione as a salmon-red solid (914 mg, 28%),
1
22%). H NMR (CDCl3) δ 0.21 (6H, s), 0.92, 0.81 (3H, 2s),
1
mp 125-129 ꢀC. H NMR (270 MHz, CDCl3) δ 0.93 (3H, s),
1.00 (9H, s), 1.19 (3H, t, J 7.4), 2.26, 2.21 (3H, 2s), 2.56 (2H, q,
J 7.4), 2.75 (2H, m), 5.48-5.55 (1H, 2m, both isomers), 6.54,
6.47 (1H, s, both isomers), 7.05 (1H, s). m/z [FABþ] found
456.2882, C28H44SOSi requires 456.2882.
1.21 (3H, t, J 7.5), 1.34-1.92 (6H, m), 1.96-2.32 (4H, m),
2.39-2.54 (1H, m), 2.59-3.08 (4H, m), 2.68 (2H, q, J 7.5 Hz),
5.05 (2H, s), 6.65 (1H, s), 7.12 (1H, s), 7.27-7.46 (5H, m).
HRMS (ESþ) m/z found 405.2243; C27H33OS (Mþ þ H) re-
quires 405.2247. Starting material was recovered as beige solid
(1.868 g, 60%). The thione (485 mg, 1.2 mmol) was dissolved in
anhydrous THF (6.0 mL) and cooled to 0 ꢀC. Sodium borohy-
dride (138 mg, 3.6 mmol) was added portionwise, and the
reaction mixture was stirred for 1 h at 0 ꢀC. Hydrochloric acid
(1 M, 30 mL) was slowly added at 0 ꢀC, and the mixture was
extracted with ethyl acetate (4 ꢀ 30 mL), washed with water and
brine, dried (MgSO4), filtered, and concentrated to give the title
compound as a light-yellow solid (479 mg, 98%); mp 127-
132 ꢀC. 1H NMR (270 MHz, CDCl3) δ 0.74 (3H, s), 1.10-1.65
(7H, m), 1.21 (3H, t, J 7.5 Hz), 1.70-1.89 (3H, m), 1.93 (1H, dt,
J 12.4, 3.0 Hz), 2.12-2.29 (2H, m), 2.37 (1H, dq, J 13.5, 3.0 Hz),
2.67 (2H, q, J 7.5 Hz), 2.68-2.95 (3H, m), 5.04 (2H, s), 6.63 (1H,
s), 7.11 (1H, s), 7.27-7.47 (5H, m). HRMS (ESþ) m/z found
407.2397; C27H35OS (Mþ þ H) requires 407.2403.
2-Ethyl-3-O-tert-butyldimethylsilyl-17β-methylsulfanylmethyl
Estrone 17. A solution of 16 (1 g, 2.2 mmol) in THF (5 mL)
and ethanol (50 mL) was treated with 10% Pd/C (1 g) at 80 psi
for 18 h. The material was filtered through celite and then
evaporated to give the desired product 17 as a clear colorless
oil (570 mg, 56%). 1H NMR (CDCl3) δ 0.21 (6H, s), 0.65
(3H, s), 0.99 (9H, s), 1.15 (3H, t, J 7.4), 2.10 (3H s), 1.20-2.40
(15H, m), 2.56 (2H, q, J 7.4), 2.65-2.85 (3H, m), 6.47 (1H, s)
and 7.04 (1H, s). HRMS [FABþ], C28H46OSSi requires
458.3039.
2-Ethyl-3-O-tert-butyldimethylsilyl-17β-methanesulfonylmethyl
Estrone 18. A rt solution of 17 (500 mg, 1.09 mmol) in dichloro-
methane (25 mL) was treated with m-CPBA (764 mg, 4 mmol).
The reaction was stirred for 16 h and then washed with aqueous
sodium hydroxide (40 mL, 1 M), water (40 mL), and brine (40
mL), dried, and evaporated. The crude product, a yellow oil, was
purified by column chromatography (4:1 to 3:1 hexane/ethyl
acetate) to give the desired sulfone 18 (170 mg, 35%) as a
2-Ethyl-3-O-benzyl-17β-methanesulfonylestra-1,3,5(10)-triene
33. Compound 32 (406 mg, 1.0 mmol) was dissolved in anhy-
drous THF (15 mL) and methanol (15 mL) and cooled to 0 ꢀC.
Sodium methoxide (51.4 mg, 0.95 mmol) was added portion-
wise, and the reaction mixture was stirred for 15 min. After
warming to rt, iodomethane (131 mg, 0.92 mmol) was added,
and the reaction mixture was stirred for 16 h at rt. After removal
of the solvents under reduced pressure, water (100 mL) and
glacial acetic acid (5 mL) were added, the organics were ex-
tracted with ethyl acetate (3 ꢀ 50 mL), and the combined
organic layers were dried (MgSO4), filtered, and concentrated.
The residue was purified by flash column chromatography
(SiO2, hexane/ethyl acetate 95:5 to 90:10) to give 2-ethyl-3-O-
benzyl-17β-methanesulfanylestra-1,3,5(10)-triene as a white so-
lid (142 mg, 35%); mp 186-188 ꢀC. 1H NMR (270 MHz,
CDCl3) δ 0.81 (3H, s), 1.23 (3H, t, J 7.5 Hz), 1.18-1.72 (7H,
m), 1.74-1.93 (2H, m), 2.05 (1H, dt, J 12.1, 3.2 Hz), 2.12-2.29
(2H, m), 2.16 (3H, s), 2.31-2.42 (1H, m), 2.62 (1H, t, J 9.1 Hz),
2.69 (2H, q, J 7.4 Hz), 2.75-2.96 (2H, m), 5.05 (2H, s), 6.64 (1H,
s), 7.12 (1H, s), 7.28-7.49 (5H, m). HRMS (ESþ) m/z found
421.2566; C28H37OS (Mþ þ H) requires 421.2560. The inter-
mediate sulfide (84 mg, 0.2 mmol) was dissolved in anhydrous
DCM and cooled to 0 ꢀC. 3-Chloroperbenzoic acid (70 wt %;
149 mg, 0.6 mmol) was added portionwise. The reaction mixture
was stirred for 2 h at 0 ꢀC and then diluted with DCM (30 mL)
and washed with a 1 M aqueous sodium hydroxide solution (2 ꢀ
15 mL). The organic layer was dried (MgSO4), filtered, and
1
colorless oil. H NMR (CDCl3) δ 0.21 (6H, s), 0.64 (3H, s),
0.99 (9H, s), 1.15 (3H, t, J 7.4), 1.20-2.40 (14H, m), 2.55 (2H, q,
J 7.4), 2.74-2.94 (3H, m), 2.92 (3H, s), 3.10-3.20 (1H, m), 6.47
(1H, s), 7.03 (1H, s). HRMS [FABþ], C28H46O3SSi requires
490.2937.
2-Ethyl-17-methanesulfonylmethyl Estrone 19. A solution of
18 (135 mg, 0.29 mmol) in THF (5 mL) was treated with a
solution of tetra-butyl ammonium fluoride in THF (0.5 mL. 0.5
mmol) and maintained at rt for 16 h. The reaction was then
diluted with ethyl acetate (25 mL), washed with water (20 mL)
and brine (25 mL), and then dried and evaporated. The product
was crystallized from ether/hexane to give the desired sulfone 19
as a white solid (85 mg over 3 crops, 77%). 1H NMR (CDCl3) δ
0.65 (3H, s), 1.21 (3H, t, J 7.4), 1.26-2.38 (14H, m), 2.58 (2H, q,
J 7.4), 2.76-2.84 (2H, m), 2.85-2.92 (1H, m), 2.93 (3H, s), 3.13
(1H, dd, J 13.3 and 2.3), 4.60 (1H, s), 6.48 (1H, s), 7.02 (1H, s).
m/z [ES-] 375.3 (Mþ - H, 100%). HRMS [FABþ] 376.2072,
C22H32SO3 requires 376.2072.
2-Ethyl-3-O-sulfamoyl-17-methanesulfonylmethyl Estrone 20.
Sulfamoyl chloride (150 mg, 1.3 mmol) was cooled to 0 ꢀC,
dissolved in DMA (2 mL) and then after 5 min treated with 19
(60 mg, 0.16 mmol). External cooling was removed after 15 min,
and the reaction was left to stir at rt for 3 h. The reaction was
then diluted in ethyl acetate (15 mL), poured onto brine (15 mL),
and the organic layer was separated. The organic extract was

2950 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 7
Jourdan et al.
concentrated to give the title compound as a white solid (91 mg,
99%); mp 168-174 ꢀC. 1H NMR (270 MHz, CDCl3) δ 1.08 (3H,
s), 1.19 (3H, t, J 7.4 Hz), 1.23-1.62 (6H, m), 1.78-1.99 (2H, m),
2.02-2.41 (4H, m), 2.46 (1H, dd, J 8.9, 2.5 Hz), 2.65 (2H, q, J 7.6
Hz), 2.75-2.91 (2H, m), 2.82 (3H, s), 5.02 (2H, s), 6.61 (1H, s),
7.07 (1H, s), 7.26-7.47 (5H, m). HRMS (ESþ) m/z found
453.2464; C28H37O3S (Mþ þ H) requires 453.2458.
(1H, s). HRMS (ESþ) m/z found 423.1939; C21H31N2O5S
(Mþ þ H) requires 423.1948. Anal. (C21H30N2O5S) C, H, N.
Acknowledgment. This work was supported by Sterix Ltd.,
a member of the IPSEN Group. We thank Alison Smith for
technical support and the NCI DTP program for providing in
vitro screening resources. We thank Dr. G. R. Pettit, Arizona
State University, for providing CA-4.
2-Ethyl-3-hydroxy-17β-methanesulfonylestra-1,3,5(10)-triene
34. A solution of 33 (90 mg, 0.2 mmol) in THF (5 mL) and
methanol (20 mL) was treated with 10% Pd/C (19 mg) as
described for the synthesis of 9. The resultant crude solid was
purified by flash column chromatography (SiO2: hexane/ethyl
acetate 9:1 to 4:1) to give the title compound as a light-yellow
solid (69 mg, 97%); mp 194-197 ꢀC. 1H NMR (270 MHz,
CDCl3) δ 0.93 (3H, s), 1.05 (3H, t, J 7.5 Hz), 1.11-1.43 (6H, m),
1.64-1.84 (1H, m), 1.92-2.23 (4H, m), 2.26-2.33 (1H, m), 2.45
(2H, q, J 7.5 Hz), 2.59-2.72 (2H, m), 2.71 (3H, s), 2.88 (2H, t,
J 9.4 Hz), 6.40 (1H, s), 6.85 (1H, s), 7.77 (1H, s, br). HRMS
(ES-) m/z found 361.1848; C21H29O3S (M- - H) requires
361.1843.
Supporting Information Available: Table of elemental analy-
sis, 13C NMR data, and the experimental details for compounds
12, 21-31, 37-40 and 42-45 are presented. This material is
available free of charge via the Internet at http://pubs.acs.org.
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nitromethane (60 mL) was refluxed in a round-bottom flask
equipped with a Dean-Stark trap and a condenser until 10 mL
of nitromethane was distilled, and then N,N-dimethylethylene-
diamine (0.1 mL, 0.9 mmol) was added and the solution refluxed
for 24 h. After cooling the reaction mixture to rt, the solvent was
evaporated under vacuum and the residual solid was purified by
column chromatography (hexane/ethyl acetate 10:1 to 8:1) and
recrystallized in hexane/ethyl acetate 20:1 to afford a white
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(1H, dd, J 2.5 and 2.2 Hz), 7.10 (1H, s), 7.29-7.46 (5H, m).
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