Structure-activity relationships of C-17-substituted estratriene-3-O- sulfamates as anticancer agents

Article abstract of DOI:10.1021/jm200483x

The synthesis and antiproliferative activities of analogues of 2-substituted estradiol-3,17-O,O-bis-sulfamates (E2bisMATEs) are discussed. Modifications of the C-17 substituent confirm that an H-bond acceptor is essential for high activity; its optimal linkage to C-17 and the local environment in which it resides are defined. In the non-sulfamoylated series 17β-acyl substitution delivers 48b, the most potent compound identified to date. In the sulfamate series a number of permutations of linker and H-bond acceptor deliver excellent activity, with 55, 61, 65, 49a, and 49b proving especially promising. The in vivo potential of these compounds was explored in the NCI hollow fiber assay and also in a mouse Matrigel model of antiangiogenesis in which 49 and 55 show significant inhibitory activity.

Full text of DOI:10.1021/jm200483x

ARTICLE
pubs.acs.org/jmc
StructureꢀActivity Relationships of C-17-Substituted
Estratriene-3-O-sulfamates as Anticancer Agents
Fabrice Jourdan,† Mathew P. Leese,† Wolfgang Dohle,† Eric Ferrandis,‡ Simon P. Newman,§
Surinder Chander,§ Atul Purohit,§ and Barry V. L. Potter*,†
†Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath, BA2 7AY, U.K.
‡Institut de Recherche Henri Beaufour, Ipsen, 91966 Les Ulis Cedex, France
§Diabetes, Endocrinology and Metabolism, Imperial College London, Hammersmith Hospital, London, W12 0NN, U.K.
S Supporting Information
b
ABSTRACT: The synthesis and antiproliferative activities of analogues of
2-substituted estradiol-3,17-O,O-bis-sulfamates (E2bisMATEs) are discussed.
Modifications of the C-17 substituent confirm that an H-bond acceptor is
essentialforhigh activity;its optimallinkageto C-17 and the local environment
in which it resides are defined. In the non-sulfamoylated series 17β-acyl
substitution delivers 48b, the most potent compound identified to date. In the
sulfamate series a number of permutations of linker and H-bond acceptor
deliver excellent activity, with 55, 61, 65, 49a, and 49b proving especially
promising. The in vivo potential of these compounds was explored in the NCI
hollow fiber assay and also in a mouse Matrigel model of antiangiogenesis in
which 49 and 55 show significant inhibitory activity.
’ INTRODUCTION
combining antiangiogenic agents with other anticancer agents
encourage studies into compounds that combine an antiangio-
genic effect with a second antitumor activity.5,9 Furthermore,
there is considerable interest in the development of agents that
cause a disruption of tumor vasculature through alternative, non-
VEGF based mechanisms.
The hypothesis that inhibition of angiogenesis, by blocking
blood vessel formation and thus the blood supply required to
support the growth of solid tumors, might constitute a new
strategy for the treatment of cancer was made four decades ago.1
Recently, a number of drugs disrupting tumor vasculature by
targeting vascular endothelial growth factor (VEGF), like the
monoclonal antibody bevacizumab2 and the small molecule
vascular endothelial growth factor receptor (VEGFR) inhibitors
sorafenib3 and sunitinib,4 have gained approval for the treatment
of cancer, including various combinations with other anticancer
agents.5 Clinical studies have shown that the success of anti-
VEGF therapy is highly dependent on cancer phenotype and
clinical history. Through eradicating excess systemic VEGF,
bevacizumab treatment initially effects a maturation of the tumor
vasculature; this allows for an enhanced delivery of cytotoxic
agent to the tumor and, consequently, improved efficacy relative
to single agent cytotoxic therapy.6 However, tumors are able to
circumvent blockage of VEGF signaling by, for example, switch-
ing to fibroblast growth factor (FGF) to sustain their blood vessel
formation:7 anti-VEGF therapy also results in considerable side
effects, and periodic dosing regimes are required to avoid dose
limiting toxicity. As a result, rapid tumor regrowth is often
observed in the off-dose periods in these regimes after initial
tumor regression. In preclinical models there are indications that
anti-VEGF therapy may generate more aggressive disease; in-
hibition of angiogenesis with sunitinib, for example, has been
correlated with increased tumor metastasis and decreased overall
survival in mice.8 Nevertheless, the promising results obtained by
In previous studies, we detailed the discovery of a number
of sulfamoylated 2-substituted estratriene derivatives that, like
the endogenous estrogen metabolite 2-methoxyestradiol 1
(2-MeOE2), inhibit cancer cell proliferation and angiogenesis. 2-Sub-
stituted-estradiol-3,17-O,O-bis-sulfamates 3 (2-MeOE2bisMATE)
and 4 (2-EtE2bisMATE),10 and the 2-substituted-estradiol-
3-O-sulfamates 5 (2-MeOE2MATE) and 6 (2-EtE2MATE)11
(Figure 1) are differentiated from 1 by their enhanced biological
activities and superior druglike properties. The excellent oral
bioavailability of 3 (>85% in rodents),12 for example, appears to
derive from the ability of a sulfamate group both to block
inactivating metabolism and deactivating conjugation, while also
allowing the molecule to interact reversibly with carbonic
anhydrase.12,13 This latter reversible interaction, which has been
characterized by protein crystallography, may minimize first pass
liver metabolism through sequestration of the sulfamate drugs in
red blood cells.14 Moreover, as with other aryl sulfamates, 3ꢀ6
are irreversible inhibitors of steroid sulfatase (STS), itself a target
for the treatment of hormone dependent cancer.15 Although a
full mechanistic picture for the activity of these compounds
Received: April 21, 2011
Published: May 23, 2011
r
2011 American Chemical Society
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Figure 1. Structures of 2-methoxyestradiol 1, 2-ethylestradiol 2, 2-methoxyestradiol-3,17-O,O-bis-sulfamate 3, 2-ethylestradiol-3,17-O,O-bis-sulfamate
4, 2-methoxyestradiol-3-O-sulfamate 5, 2-ethylestradiol-3-O-sulfamate 6, 2-methoxyestrone-3-O-sulfamate 7, 2-ethylestrone-3-O-sulfamate 8,
2-methoxy-3-hydroxy-17β-cyanomethyl-1,3,5(10)-estratriene 9, 2-ethyl-3-hydroxy-17β-cyanomethylestra-1,3,5(10)-triene 10, 2-methoxy-3-O-
sulfamoyl-17β-cyanomethylestra-1,3,5(10)-triene 11, 2-ethyl-3-O-sulfamoyl-17β-cyanomethylestra-1,3,5(10)-triene 12.
continues to emerge, it was recently shown that their ability to
disrupt the tubulinꢀmicrotubule equilibrium in cells is critical
for their antitumor activity.16 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 tumors17 and,
second, that their activity is independent of the estrogen receptor
even though they are estrogen derivatives.18
The 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-methoxyes-
trone-3-O-sulfamate 7 (2-MeOEMATE) and 2-ethylestrone-3-
O-sulfamate 8 (2-EtEMATE) (Figure 1) also exhibited an
antiproliferative effect against a range of estrogen-independent
human cancer cells in vitro.20 SAR studies on the A-ring
estrogen-3-O-sulfamates demonstrated that optimal antiproli-
ferative activity is obtained with a 2-methoxy, 2-ethyl, or
2-methylsulfanyl group.11,21 Further SAR studies focused on
D-ring modifications, principally at the C-17 position of the
estratriene 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
derivatives show similar antiproliferative activity, the 17-deoxy
and 17R-benzyl analogues are considerably less active.22 Sulfa-
moylation of the 17β-hydroxy group gave the bis-sulfamates 3
and 4 that exhibit slightly enhanced in vitro activity and greatly
improved in vivo antitumor effects.10 It was recently established
that deletion or substitution of the C-17-oxygen linker of the
17-O-sulfamate group with an electronically neutral methylene
group and replacement of the sulfamate’s terminal NH2 with
methyl are well tolerated.16 In addition, an SAR study of C-17
cyanated analogues revealed that 2-methoxy-3-O-sulfamoyl-17β-
cyanomethylestra-1,3,5(10)-triene 11 and the corresponding
2-ethyl compound 12 offer further enhanced antiproliferative
and antiangiogenic activities relative to 3 and 4.23 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-17.24 All data accrued to date indicate that optimal
in vitro activity results from three key pharmacophore elements,
namely, the C-2 XMe group (X = O, CH2 or S), the 3-O-
sulfamate, and an H-bond acceptor around the C-17 position.
Translation of in vitro activities into the in vivo environment is
somewhat more complex. For example, when evaluated for
activity against the growth of MDA-MB-231 xenografts in female
nude athymic mice, 11 proved to be less active than the
corresponding bis-sulfamate 3, despite being 4-fold more potent
in vitro. This difference in in vitro and in vivo activities observed
for 3 and 11 is dependent on their respective oral bioavailability,
Figure 2. Proposed modifications at C-17 of the 2-substituted estra-
triene nucleus.
Scheme 1. Synthesis of 17-Hydroxy- and Alkoxymethyl
Derivativesa
a Reagents and conditions: (i) Mg, TiCl4, DCM, THF, 0 °C; (ii) 9-BBN,
THF, 0 °C, then 37% H2O2, 10% NaOH, 0 °C; (iii) H2, Pd/C, THF,
MeOH, room temp; (iv) H2NSO2Cl, DMA, room temp; (v) NaH, MeI,
THF, reflux; (vi) TBDPSCl, imidazole, DMF, room temp; (vii) TBAF,
THF, room temp.
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Scheme 2. Synthesis of C-17 Ethyl Derivativesa
Scheme 3. Synthesis of Various C-17 Olefin Derivativesa
a Reagents and conditions: (i) (EtO)2POCH2CO2Et, NaH, THF,
reflux; (ii) TBAF, THF, 0 °C; (iii) H2NSO2Cl, DMA, room temp;
(iv) DIBAL-H, THF, ꢀ78 to 0 °C; (v) H2NSO2Cl, 2,6-di-tert-butyl-4-
methylpyridine, toluene, DCM, room temp.
a Reagents and conditions: (i) (EtO)2POCH2CO2Et, NaH, THF,
reflux; (ii) H2, Pd/C, THF/MeOH, room temp; (iii) H2NSO2Cl,
DMA, room temp; (iv) BnBr, K2CO3, DMF, room temp; (v) LiAlH4,
THF, 0 °C; (vi) MeI, NaH, THF, room temp; (vii) TBDPSCl,
imidazole, DMF, room temp; (viii) TBAF, THF, 0 °C.
disruption.10,11,16,22ꢀ25 Aiming to further refine the series, we set
out to tether various oxygen-containing H-bond acceptor func-
tions (e.g., OH, OSO2NH2, OMe, and COCH3) to C-17 of the
estratriene nucleus through short linkers. 2-Ethyl-17β-hydroxy-
methyl-3-O-benzylestra-1,3,5(10)-triene 14 serves as a common
intermediate for the synthesis of the 17β-sulfamoyloxy 16, 17
β-methoxymethyl 18, and the 17 β-hydroxymethyl derivatives 20
(Scheme 1). Methylenation of 2-ethyl-3-O-benzylestrone 1326
followed by a stereoselective hydroboration using 9-BBN yielded
14. Hydrogenolysis of 14 delivered phenol 15 which was then
treated with sulfamoyl chloride to give the bis-sulfamate deriva-
tive 16.27 Alkylation of 14 with methyl iodide and subsequent
hydrogenolysis afforded the 17β-methoxymethylphenol 17
which was then converted to sulfamate 18. Access to compound
20 first required a sequence of silyl protection of the aliphatic
alcohol and debenzylation. This was found to be best achieved
with tert-butyldiphenylsilyl chloride as silylating agent, as other
silyl protecting groups like the TBS and TIPS group proved less
stable to the following hydrogenolysis. Phenol 19 was then
successively sulfamoylated and desilylated to give the desired
sulfamate 20.
itself a function of their uptake, transport, metabolism, conjuga-
tion, and clearance profiles.
In light of these results, we set out to examine whether further
C-17 modifications could deliver improvements in both in vitro
and, more importantly, in vivo activity and thus embarked on
more extensively defining the SAR of this series by synthesizing
C-17 modified 2-methoxyestratriene-3-O-sulfamates and 2-ethy-
lestratriene-3-O-sulfamates projecting an oxygen-containing
H-bond acceptor (“Z”, see Figure 2) around C-17. The linker
“Y” between the estratriene ring system and the H-bond acceptor
group was varied in length in order to map onto the region of
space occupied by the SO2 group of the bisMATES 3 and 4 and
that of the cyano group of 11 and 12. We describe here our
synthetic approaches to these compounds together with their in
vitro biological activities as antiproliferative agents and in vivo
data for two selected compounds.
A similar synthetic approach was applied to prepare a series of
ether, sulfamate, and hydroxy derivatives incorporating a two-
carbon linker to C-17 (Scheme 2). HornerꢀWadsworthꢀEmmons
olefination of 2-ethyl-3-O-benzylestrone 13 with triethyl phospho-
noacetate afforded a 5:1 mixture of olefins 21 and 22 that were
’ RESULTS AND DISCUSSION
Chemistry. Previous SAR studies on 2-substituted estratriene-
3-O-sulfamates indicated that an H-bond acceptor around C-17
is required for optimal antiproliferative activity and microtubule
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Scheme 4. Synthesis of C-17 Tethered Alkoxy and Hydroxy
Derivativesa
Scheme 6. Synthesis of C-17 2-Hydroxypropyl and 2-Oxo-
propyl Derivativesa
a Reagents and conditions: (i) LiAlH4, THF, 0 °C; (ii) CH3I, NaH,
THF, room temp; (iii) Na, t-BuOH, reflux; (iv) H2NSO2Cl, DMA,
room temp; (v) DIBAL-H, THF, ꢀ78 to 0 °C.
Scheme 5. Synthesis of 17β-Acyl Derivativesa
a Reagents and conditions: (i) DIBAL-H, THF, 0 °C to room temp; (ii)
MeMgBr, THF, 0 °C; (iii) DessꢀMartin periodinane, DCM, 0 °C to
room temp; (iv) H2, Pd/C, THF, MeOH, room temp; (v) H2NSO2Cl,
DMA, room temp.
beingobtained asthe soleproduct. Presumably, the vinyl sulfamate
formedinthereaction isunstable to eliminationunderthe reaction
conditions.
2-Ethyl-17-(E)-(2-methoxyethylidene)-3-O-sulfamoylestra-
1,3,5(10)-triene 41 was accessed from ester 21 (Scheme 4).
Reduction of 21 with LiAlH4 delivered the vinyl alcohol that was
methylated and debenzylated to afford phenol 41. Sulfamoyla-
tion of 41 under standard conditions gave 42. The sulfamoylated
ester 36 was reduced with DIBAL-H to afford the 17-(E)-
hydroxyethylidene derivative 43.
a Reagents and conditions: (i) EtPPh3I, NaH, DMSO, 100 °C; (ii)
BH3ꢀTHF, THF, 0 °C, then NaOH 30%, H2O2, 37%; (iii)
DessꢀMartin periodinane, DCM; (iv) H2, Pd/C, THF, MeOH; (v)
H2NSO2Cl, DMA, room temp.
As mentioned above, the 17β-cyanomethylestra-1,3,5(10)-
triene-3-O-sulfamates 8 and 9 are particularly potent antiproli-
ferative agents in vitro. We therefore investigated isosteric
replacement of the cyano group with a ketone and thus targeted
C-17 acylestra-1,3,5(10)-triene-3-O-sulfamates 49a and 49b. A
Wittig reaction between 13 and ethyltriphenylphosphonium
iodide in DMSO furnished olefin 45a as a mixture of E and Z
isomers (Scheme 5). Hydroboration with borane THF afforded
the C-17β-isomer of the alcohol 46a along with a trace amount of
the corresponding 17R-isomer which could be removed by
chromatography; attempts to achieve complete selectivity with
9-BBN proved unsuccessful in this case. DessꢀMartin oxidation
afforded compound 47a, which was converted to the phenol 48a
and sulfamate 49a under standard conditions. The same set of
reactions was also carried out with 2-methoxy-3-O-benzylestrone
44 to afford the 2-methoxy analogues 48b and 49b for biological
evaluation.
separable by column chromatography. Concomitant debenzylation
and olefin reduction over Pd/C afforded ester 23. Sulfamoylation of
23 afforded sulfamate 24. Benzyl reprotection of 23 followed by
reduction with LiAlH4 afforded 2-ethyl-17β-(2-hydroxyethyl)-3-O-
benzylestra-1,3,5(10)-triene 25 that was converted to 27, 29, and31
in a manner analogous to that described for 16, 18, and 20 above.
In order to prepare the C-17-C-20 olefinic analogues of 27 and
thus explore the effects of the reduced rotational freedom of such
compounds on biological activity, 2-ethyl-3-O-(tert-butyldimethyl-
silyl)estrone 32 was treated with triethyl phosphonoacetate to give
33asa mixtureofits E and Z isomersinexcellent yield (Scheme3).
Desilylation of 33 afforded phenols 34 and 35 that were separated
by flash column chromatography. Each phenol was reacted with
sulfamoyl chloride to afford sulfamates 36 and 37, respectively.
Upon reduction with DIBAL-H, 34 and 35 afforded the corre-
sponding alcohols 38 and 39. Attempts to sulfamoylate both
hydroxyl groups with sulfamoyl chloride in DMA26 or 2,6-di-tert-
butyl-4-methylpyridine in DCM proved unsuccessful in this case
with 17-vinyl-2-ethyl-3-O-sulfamoylestra-1,3,5(10),16-tetraene 40
Projection of the carbonyl group further from C-17 requires a
two-carbon linker, and the 17β-(2-oxopropyl) derivatives 55 and
61 were thus elaborated (Scheme 6). The 17β-cyanomethyl
compound 50 was prepared in analogy to the method described
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Scheme 7. Improved Approach to 17-(2-Oxopropyl)
estratriene Derivativesa
Scheme 8. Synthesis of 17-(Fluoroethyl)estratriene
Derivativesa
a Reagents and conditions: (i) DAST, THF, 0 °C; (ii) H2, Pd/C, THF,
MeOH, room temp; (iii) H2NSO2Cl, DMA, room temp.
receptor negative) prostate cancer cells and MDA-MB-231
(estrogen receptor negative) and MCF-7 (estrogen receptor
positive) breast cancer cells in vitro. The results of these assays
and comparative data for benchmark compounds 1ꢀ9 are
presented in Table 1.
a Reagents and conditions: (i) (EtO)2POCH2CO2Et, NaH, THF,
reflux; (ii) H2, Pd/C, THF, MeOH, room temp; (iii) Tebbe reagent,
pyridine, THF, toluene, ꢀ78 to 0 °C; (iv) 2 M HCl, room temp; (v)
TBAF, THF, room temp; (vi) H2NSO2Cl, DMA, room temp; (vii)
The bis-sulfamates 3 and 4 (Figure 1) are promising multi-
targeted antitumor agents that exhibit excellent activity against
cancer cell proliferation and angiogenesis in tandem with their
high oral bioavailability.10,12,30 An SAR study on this class of
2-substituted estratriene sulfamates led to the discovery of 11 and
12 and indicated that a sterically unhindered hydrogen bond
acceptor attached to C-17 is essential for activity.23 This finding
was confirmed in a further SAR study wherein a systematic
substitution or deletion of the constituent atoms the C-17
O-sulfamate group, deletion or substitution of the C-17-oxygen
linker with an electronically neutral methylene group, and/or
replacement of the terminal NH2 with a methyl group is well
tolerated.16
As can be seen from data presented in Table 1, activities of the
compounds across all three cell lines are generally comparable in
magnitude and trend; thus, for clarity, data for antiproliferative
activity against DU-145 cells are discussed herein when possible.
In agreement with our foregoing studies, the 3-hydroxy deriva-
tives generally exhibit only modest activity when compared with
the corresponding 3-O-sulfamoyl derivatives. However, a num-
ber of functional groups at C-17 are found to confer greater
antiproliferative activity than the 17β-hydroxyl of the estradiol
derivatives (e.g., 2-MeOE2 1). For example, insertion of a two-
carbon linker between the hydroxyl group and C-17 (in 26, 38,
and 39) delivers a 4- to 15-fold enhancement in antiproliferative
activity relative to the parent estradiol derivative 2. This observa-
tion reflects that made for 2-substituted estradiol derivatives
1 and 2 and their 17β-cyanomethyl analogues 9 and 10,23
respectively, wherein a two-carbon linker also separates C-17
and the hydrogen bond acceptor. In contrast 16, which features a
one-carbon linker between the H-bond acceptor and C-17, is
only equipotent to its parent estradiol derivative 2, in DU-145
cells. Interestingly, the C-17-(2-fluoroethyl) derivative 68 dis-
plays similar activity to the C-17-(2-hydroxyethyl) derivative 26
(albeit the compounds were assessed in different cell lines),
indicating that the fluorine is a reasonable bioisostere of hydroxyl
in this case. Furthermore, the relative activities of 68 and 71 show
NaBH4, i-PrOH, THF, room temp; (viii) H2NOH HCl, NaOAc,
3
MeOH/H2O, room temp.
earlier for the synthesis of 56.23 A DIBAL-H reduction of 50 then
delivered aldehyde 51 in good yield. 51 was reacted with
methylmagnesium bromide to afford the secondary alcohol 52
that was then oxidized to ketone 53 with DessꢀMartin period-
inane. Hydrogenolysis of 53 and subsequent sulfamoylation
delivered phenol 54 and sulfamate 55 derivatives. 2-Ethyl-3-O-
benzyl-17β-cyanomethylestra-1,3,5(10)-triene 56 was trans-
formed to compounds 57ꢀ61 under analogous conditions.
A shorter and higher yielding route to 55 was later developed
(Scheme 7). 2-Methoxy-3-O-triisopropylsilyloxyestrone 62 was
converted to the ester 63 by sequential HornerꢀWadsworthꢀ
Emmons olefination and hydrogenation as described above for
the synthesis of 23. A Tebbe methylenation28 followed by acidic
hydrolysis of the intermediate enol ether then delivered ketone
64 in excellent yield. Deprotection of 64 and subsequent
sulfamoylation furnished sulfamate 55 in 61% overall yield
starting from 2-methoxyestrone. Reduction of 55 with NaBH4
afforded 17β-(2-hydroxypropyl) derivative 65, while reaction of
55 with hydroxylamine delivered oxime 66.
We also wished to assess the antiproliferative activity of C-17
fluorinated analogues of sulfamates 31, 55, and 61, since fluorine
has long proven to be a successful bioisostere of the hydroxyl in
various contexts.29 We therefore treated compound 25 with
DAST to afford 67 that, after successive debenzylation and
sulfamoylation, gave access to phenol 68 and sulfamate 69,
respectively (Scheme 8). Similarly, aldehyde 57 could be trans-
formed into the difluoro compounds 70ꢀ72.
Biology. To assess their potential as anticancer agents, the
series of novel 2-ethyl- and 2-methoxyestratriene derivatives was
evaluated for antiproliferative activity against DU-145 (androgen
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Table 1. Antiproliferative Activities of 2-Ethyl and 2-Methoxyestratriene Derivatives against DU-145, MDA-MB-231, and MCF-7
Human Cancer Cells in Vitroa
GI50 (μM)
MDA
compd
R1
R2
R3
DU-145
MB-231
MCF-7
1
MeO
Et
H
H
OH
OH
1.22
10.3
0.34
0.21
0.49
2.5
0.94
8.0
2.35
10.5
0.25
0.07
0.30
>100
0.07
0.06
2
3
MeO
Et
SO2NH2
SO2NH2
H
OSO2NH2
0.28
0.21
0.12
4
OSO2NH2
9
MeO
Et
CH2CN
10
11
12
15
16
17
18
20
23
24
26
27
28
29
31
34
35
36
38
39
41
43
48a
49a
48b
49b
54
55
60
61
65
66
68
69
71
72
H
CH2CN
MeO
Et
SO2NH2
SO2NH2
H
CH2CN
0.062
0.054
10.1
0.29
25.3
0.33
0.10
0.071
0.141
6.3
CH2CN
Et
CH2OH
Et
SO2NH2
H
CH2OSO2NH2
CH2OCH3
0.43
9.2
Et
Et
SO2NH2
SO2NH2
H
CH2OCH3
0.53
0.11
Et
CH2OH
Et
CH2CO2Et
26.4
4.19
2.52
5.64
24.8
Et
SO2NH2
H
CH2CO2Et
Et
CH2CH2OH
CH2CH2OSO2NH2
CH2CH2OCH3
CH2CH2OCH3
CH2CH2OH
(E)-CHCO2Et
(Z)-CHCO2Et
(E)-CHCO2Et
(E)-CHCH2OH
(Z)-CHCH2OH
(E)-CHCH2OCH3
(E)-CHCH2OCH3
COCH3
Et
SO2NH2
H
Et
Et
SO2NH2
SO2NH2
H
3.4
Et
<0.5
30.6
31.1
1.85
0.77
0.67
>10
10
Et
Et
H
Et
SO2NH2
H
Et
Et
H
Et
H
Et
SO2NH2
H
Et
2.0
Et
SO2NH2
H
COCH3
0.062
0.30
0.046
2.4
0.019
0.39
0.026
MeO
MeO
MeO
MeO
Et
COCH3
SO2NH2
H
COCH3
0.048
CH2COCH3
CH2COCH3
CH2COCH3
CH2COCH3
CH2CH(OH)CH3
CH2CdN(OH)CH3
CH2CH2F
SO2NH2
H
0.089
18.9
0.16
0.14
2.09
3.4
0.068
<0.005
5.79
Et
SO2NH2
SO2NH2
SO2NH2
H
0.18
0.14
2.35
0.32
MeO
MeO
Et
Et
SO2NH2
H
CH2CH2F
0.78
60
Et
CH2CHF2
Et
SO2NH2
CH2CHF2
1.81
a Data for compounds 1ꢀ12 are taken from the literature.14,23,27 GI50 values are the mean values obtained from experiments performed in triplicate;
SEM, (7%.
the monofluoro derivative to be superior to the difluoro
analogue.
In the case where the hydrogen bond acceptor is a ketone, a
one-carbon linker proved optimal (cf. 48a and 48b with 60
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Table 2. GI50 and MGM Obtained for Representative Cell Lines in the NCI-60 Screening Panela
GI50 (μM)
lung
colon
CNS
melanoma
UACC-62
ovarian
renal
compd
HOP-62
HCT-116
SF-539
OVCAR-3
SN12-C
MGM (μM)
1
0.7
0.47
0.32
0.36
0.21
0.95
1.3
3
0.051
<0.01
0.061
0.045
<0.01
0.59
0.045
nd
0.036
<0.01
0.056
<0.01
<0.01
0.22
<0.01
<0.01
0.022
nd
<0.01
<0.01
0.019
<0.01
<0.01
0.22
0.126
0.028
0.309
0.072
<0.01
0.63
0.087
0.018
0.085
0.042
0.018
0.52
4
16
18
20
24
29
31
43
49a
55
65
69
72
0.038
0.022
<0.01
0.35
<0.01
0.45
4.90
2.70
5.01
2.34
4.07
4.68
4.57
0.25
0.27
0.35
2.89
11.48
0.23
2.45
1.73
0.42
0.27
0.22
0.47
0.58
0.49
<0.01
<0.01
0.048
3.72
<0.01
<0.01
0.025
3.39
<0.01
<0.01
0.023
5.25
<0.01
<0.01
<0.01
3.89
<0.01
<0.01
<0.01
22.9
<0.01
<0.01
0.052
7.59
0.015
0.021
0.028
6.31
3.89
2.82
1.95
4.90
3.47
5.37
3.09
a Results are GI50 values in micromolar. Data for compounds 1ꢀ4 are taken from the literature.14,21,30 The MGM represents the mean concentration that
caused 50% growth inhibition in all 60 cell lines.
and 54, respectively). The 2-methoxy-17β-acylestratriene deri-
vative 48b exhibits the best antiproliferative activity of any
3-hydroxy 2-substituted estratriene to our knowledge with a
GI50 of 300 nM against DU-145 cells, 4-fold better than the
activity of 2-MeOE2 1 and over 30-fold better than 2-MeOE1,
which has GI50 > 10 μM against this cell line. Likewise, a 5-fold
improvement in activity is seen for the corresponding 2-ethyl
compound 48a relative to 2-ethylestradiol 2. Ketones 54 and 60
in contrast proved to be 2-fold less active the corresponding
estradiol derivatives 1 and 2. The significant antiproliferative
activities of compounds such as 26, 48a, and 39, assuming similar
cellular uptake properties, are likely to stem from the ability of
their C-17 tethered H-bond acceptor groups (CN, OH, CdO)
to project into a very specific area of space around C-17 favorable
for optimum interaction in the colchicine binding site of tubulin
where these compounds are believed to bind. These results
suggest that such an interaction is accessible when a one- or two-
carbon linker is inserted between C-17 and the H-bond acceptor.
When considering the activities of the 3-O-sulfamate deriva-
tives, it is useful to first consider the effect of variation of the
linker “Y” while the same hydrogen bond acceptor “Z” is kept
constant. In the case where Z is a hydroxyl group, both one- and
two-carbon linkers “Y” deliver excellent antiproliferative activity
(see 20, 31, and 65). This result reflects the essential equipotency
of the cyano and hydroxyl groups as H-bond acceptors31 and also
their ability to access the same interactions around C-17 with
these linkers. Methylation of the hydroxyl group proves delete-
rious to activity, and although this is not as pronounced when the
linker “Y” is a methyl group (cf. 20 and 18), a greater than 6-fold
reduction in activity is apparent between 29 and 31 when “Y” is
an ethyl group. The negative impact of methylation of the
hydroxy group on antitumor activity indicates, as observed in
earlier studies,16,23 that steric bulk around the H-bond acceptor is
often detrimental to the necessary interaction in the binding site.
Likewise when a one-carbon linker is introduced between the
sulfamate and C-17, little difference in antiproliferative activity
results (cf. 4 210 nM and 16 290 nM) while the corresponding
analogue 27 featuring a two-carbon linker displays only modest
micromolar activity. In the ketone series (Z equals CdO) both
one- and two-carbon linkers are well tolerated though. As with
the 3-hydroxy derivatives discussed above, the one-carbon linker
appears optimal, for example, with 49b exhibiting a GI50 of 62
nM. In general, a longer linker brings unfavorable steric factors
into play, as demonstrated by the activities obtained for com-
pounds such as sulfamate 27 and ester 36.
The present study also allows for a comparison between
hydrogen bond acceptors Z with constant linker Y. One series
of compound that is instructive to compare is the two-carbon-
linked series of ketone 55, alcohol 65, oxime 66, ester 24, and
methyl ether 29. The ketone 55 and alcohol 65 with their
unsubstituted H-bond acceptors display excellent activity, with
55 proving slightly more active in vitro (GI50 = 89 nM). In
contrast, the larger oxime, ester, and methyl ether display activity
in the low micromolar range, once more illustrating the steric
restrictions to interaction around C-17. Similarly, with a one-
carbon linker, ketone 49a and alcohol 20 are of equivalent
activity (62 and 100 nM, respectively) while the methyl ether
18 (GI50 = 330 nM) is less active. Once more, substitution of the
hydrogen bond acceptor proved detrimental to activity, albeit
here, with a shorter linker, this substitution is better tolerated
than in the longer two-carbon-linked series.
A selection of compounds was also tested in the NCI 60-cell
line assay (Table 2) which allows the activity across a wide range
of cancer types to be assessed. Data from six cell lines are
presented along with the mean activity across the whole panel
(MGM value). Screening is conducted at concentrations ranging
from 10 nM upward, with maximal activity (where a 50% growth
inhibition was obtained in all cell lines at 10 nM) being indicated
by an MGM value of 10 nM. The data obtained in the assay are
consistent with those obtained in the preliminary antiprolifera-
tive screens discussed above and confirm the potential of
these compounds against a broad range of cancer phenotypes.
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or absence of inhibitor.30 Mice were thus dosed orally with 49a
and 55 at 10 and 20 mg/kg for 4 days, starting 24 h after Matrigel
injection. Visual inspection of the plugs after removal clearly
indicated that a marked increased in vascularization, as indicated
by increasing red intensity of the plug, occurred in plugs of
untreated animals (Figure 3b). For treated animals a noticeable
reduction in the level of vascularization was observed and
quantification of angiogenesis through FITC staining revealed
a very significant inhibitory effect of both 49a and 55 at 10 and 20
mg/kg (Figure 3a). This study confirms the in vivo antiangio-
genic potential of these new derivatives, which reflects observa-
tions made for foregoing members of this series such as 3 and 11.
’ CONCLUSIONS
In this study we set out to further refine the anticancer activity
of 2-substituted estratriene-3-O-sulfamates by optimizing the
C-17 substituent. Variation of the hydrogen bond acceptor “Z”
and the linker group “Y” (Figure 2) that tethers “Z” to C-17
provided further insights into the steric and electronic factors that
deliver optimal antiproliferative activity. We have identified a
number of C-17 substituents that confer enhanced antiprolifera-
tive activity to non-sulfamoylated derivatives relative to the
corresponding 2-substituted estradiols; these new derivatives
include the 2-methoxy-17β-acyl estratriene 48b which is the most
active D-ring modified 2-methoxyestradiol analogue identified to
date. These C-17 substituents could conceivably be combined
with alternative C-2 substitution to yield yet more potent non-
sulfamoylated estratriene derivatives. Results obtained for the
3-O-sulfamates revealed the tolerance to, and benefit of, introdu-
cing a linker group between the hydrogen bond acceptor group
tethered to C-17. The hydroxymethyl 20, acyl 49a and 49b,
2-hydroxypropyl 65, and 2-propoxy 55 and 61 derivatives all
exhibit excellent antiproliferative activity in vitro across a broad
range of cancer cell lines with MGM values below 30 nM across
the NCI 60-cell line panel. The potential of three of these
compounds as in vivo antitumor and antiangiogenic agents has
been established. Promising activity in the NCI hollow fiber assay
for compound 20 illustrates in vivo antitumor potential, while 49a
and 55 showed good in vivo antiangiogenic activity. On the basis
of these studies, a number of compounds that could supersede 3,
which is currently in preclinical development, are available.
Figure 3. Effect of compounds 49a and 55 on bFGF induced angiogen-
esis in Matrigel plugs in mice. Compounds 49a and 55 were adminis-
tered orally at the doses indicated for 4 days starting 24 h after Matrigel
injection. (a) Quantification of angiogenesis within the Matrigel plugs
was achieved after FITC-dextran injection. Compounds 49a and 55
significantly inhibited FGF-induced angiogenesis at both doses (p <
0.05, unpaired t test). (b) Effect of 49a and 55 on Matrigel plug
neovascularization. Normal (upper) and fluorescent (lower) images of
excised Matrigel plugs.
A number of compounds proves to be exceptionally active, with
six compounds exhibiting an MGM below 100 nM. Of particular
note are the C17-hydroxymethyl compound 20, the ketones 49a
and 55, and the 2-hydroxypropyl derivative 65, whose MGM
values are all below 30 nM.
On the strength of its in vitro antiproliferative activity 20 was
selected for in vivo evaluation at the NCI. Preliminary toxicology
showed that the compound is well tolerated by female athymic
nude mice at doses up to 400 mg/kg (single ip dose) when
formulated in 10% DMSO in saline/Tween 80. Evaluation of 20
in the hollow fiber assay32 involved assessment of activity against
the proliferation of various cancer lines in sealed polyvinylidine
fluoride fibers implanted ip or sc. A 50% net cell growth inhibition
is awarded a score of 2, and over 48 fibers (12 cell lines ꢁ 2 sites ꢁ
2 dose levels) a maximum score of 96 is possible. 20 achieved an
overall score of 18 with the growth of 25% cells in the ip fibers and
12.5% of the cells in the sc fibers being inhibited by 50% or more.
Although this result is promising, a score of 20 or more is required
for further NCI development. 20 may potentially suffer inactivat-
ing conjugation or metabolism of the C-17 group in vivo, both of
which are known issues for the 2-substituted estradiol series.12,13b
Interestingly, the net score of 20 in this system is equal to that
achieved by 2-ethylestrone-3-O-sulfamate.11
’ EXPERIMENTAL SECTION
Biology. In Vitro Studies: Cell Lines. DU145 (brain metastasis
carcinoma of the prostate) and MDA-MB-231 (metastatic pleural
effusion of breast adenocarcinoma) established human cell lines were
obtained from ATCC Global Bioresource Center. Cells were maintained
in a 5% CO2 humidified atmosphere at 37 °C in RPMI-1640 medium,
supplemented with 10% fetal bovine serum, penicillin, and streptomycin.
Antiproliferative Assays. DU145 and MDA-MB-231 cells were
seeded into 96-well microtiter plates (5000 cells/well) and treated with
10ꢀ9ꢀ10ꢀ4 M compounds or with vehicle control. At 96 h after
treatment, live cell counts were determined by WST-1 cell proliferation
assay (Roche, Penzberg, Germany), as instructed by the manufacturer.
Viability results were expressed as a percentage of mean control values
resulting in the calculation of the 50% growth inhibition (GI50). All
experiments were performed in triplicate.
A second evaluation of this class of molecule in vivo was
carried out for the ketone derivatives 49a and 55. The in vivo
antiangiogenic activity of compounds 49a and 55 was assessed in
mice using a Matrigel plug assay in which the bFGF stimulated
vessel formation into a Matrigel plug is evaluated in the presence
In Vivo Angiogenesis Assay. The Matrigel plug assay was a
modified version of previously described methods.30,33 Briefly, female
C57BL/6J mice (6ꢀ8 weeks old) were obtained from Charles River UK
Ltd. (Margate, Kent, U.K.). Animals were maintained in positive
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pressure isolators under a 12 h lightꢀdark cycle and allowed access to
food and water ad libitum. The experiments were approved by the
Imperial College Animal Ethical Review Committee and met the
standards required by the UKCCCR guidelines.34 Mice were anesthe-
tized, placed on a heated pad (37 °C), and injected subcutaneously into
the flanks with 0.5 mL of ice-cold Matrigel (Becton Dickinson, Oxford,
Oxon, U.K.) supplemented with 500 ng of basic fibroblast growth factor
(bFGF; R&D Systems, Oxford, Oxon, U.K.). Control mice were
injected with Matrigel without bFGF. After Matrigel injection they were
divided into six groups of five mice: control, bFGF only, bFGF, and 49a
(10 mg/kg, po), bFGF and 49a (20 mg/kg, po), bFGF and 55 (10 mg/
kg, po), and bFGF and 55 (20 mg/kg, po). Control and bFGF only mice
were dosed orally with vehicle (10% THF, 90% PG), while the others
were dosed every day for 4 days with 49a or 55 at the indicated dose.
Before the end of each study vascularization of Matrigel was quantified
by injecting mice with FITC-dextran (125 000 molecular weight,
Sigma), 0.1 mL of a 0.25 mg/mL solution intravenously (iv), which
allowed blood vessels within plugs to be visualized. Animals were
euthanized 20 min after injection, when Matrigel plugs were removed
and photographs showing the extent of vascularization taken using a
dissecting microscope (Nikon SMZ1500). Photographs of blood vessels
within Matrigel plugs were also obtained using a microscope with a
fluorescent light source (Zeiss-Axiovert 200). Quantification of FITC-
dextran in the Matrigel plugs was achieved by incubating plugs in 1 mL pf
Dispase reagent (Becton Dickinson) for 16 h at 37 °C. The resulting
mixture was centrifuged in a microfuge at 13 000 rpm for 30 s. The
fluorescence of the resulting supernatants was measured using a
fluorimeter (Fluostar plus Optima, BCG, Bucks, U.K.), with excitation
at 480 nm and measurement at 520 nm, and quantitated against a
standard curve of FITC dextran (0.4ꢀ25 mg/mL).
(MgSO4), filtered, and concentrated in vacuo. Purification by flash
column chromatography (hexane/EtOAc 50:1) afforded the alkene as
a light yellow oil (1.3 g, 56%). δH 0.88 (3H, s), 1.26 (3H, t, J 7.4),
1.27ꢀ1.70 (7H, m), 1.80ꢀ2.05 (3H, m), 2.22ꢀ2.68 (5H, m), 2.74 (2H,
q, J 7.4), 2.87ꢀ2.95 (2H, m), 4.74 (2H, m), 5.10 (2H, s), 6.70 (1H, s),
7.19 (1H, s), 7.28ꢀ7.50 (5H, m). LC/MS (APCIþ): m/z 387.1 (Mþ þ
H). A solution of this oil (1.24 g, 3.2 mmol) in THF (10 mL) was added
to solution of 9-BBN (1.56 g, 6.4 mmol) in THF (20 mL) at 0 °C, and the
mixture was stirred at 0 °C for 5 h. Sodium hydroxide (10%, 20 mL) was
cautiously added followed by hydrogen peroxide (37%, 10 mL). The
solution was stirred at 0 °C for 2 h and then extracted with EtOAc (2 ꢁ
50 mL). The combined organics were washed with water and brine, then
dried (MgSO4), filtered, and concentrated in vacuo. Purification by flash
column chromatography (hexane/EtOAc 10:1 to 5:1) afforded com-
pound 14 as a white solid (1.05 g, 81%), mp 99ꢀ100 °C. δH 0.68
(3H, s), 1.20 (3H, t, J 7.3 Hz), 1.27ꢀ1.70 (8H, m), 1.62ꢀ1.90 (4H, m),
1.95ꢀ2.04 (1H, m), 2.17ꢀ2.36 (2H, m), 2.66 (2H, q J 7.3), 2.75ꢀ2.90
(2H, m), 3.58 (1H, dd, J 10.4, 7.4), 3.76 (1H, dd, J 10.4, 6.7), 5.03
(2H, s), 6.63 (1H, s), 7.10 (1H, s), 7.26ꢀ7.46 (5H, m). LC/MS
(APCIꢀ): m/z 403.4 (Mꢀ ꢀ H).
2-Ethyl-3-hydroxy-17β-hydroxymethylestra-1,3,5(10)-tri-
ene 15. A solution of compound 14 (202 mg, 0.5 mmol) in THF
(10 mL) and methanol (10 mL) was treated with Pd/C (10%, 30 mg),
degassed, then stirred under an atmosphere of hydrogen at room
temperature for 24 h. The mixture was filtered through Celite, washed
with EtOAc, and concentrated in vacuo. Purification by flash column
chromatography (hexane/EtOAc 8:1 to 5:1) afforded compound 15 as a
white powder (140 mg, 89%), mp 169ꢀ170 °C. δH 0.68 (3H, s),
1.20ꢀ1.61 (11H, m), 1.65ꢀ1.91 (3H, m), 1.94ꢀ1.99 (1H, m),
2.14ꢀ2.32 (2H, m), 2.58 (2H, q, J 7.4), 2.70ꢀ2.88 (2H, m), 3.58
(1H, dd, J 9.9, 7.9), 3.76 (1H, dd, J 9.9, 7.4), 4.65 (1H, s), 6.48 (1H, s),
Chemistry. All chemicals were either purchased from Aldrich
Chemical Co. (Gillingham, U.K.) or Alfa Aesar (Heysham, U.K.).
Organic solvents of A.R. grade were supplied by Fisher Scientific
(Loughborough, U.K.) and used as supplied. Anhydrous DMA and
anhydrous THF were purchased from Aldrich and stored under a
positive pressure of N2 after use. Sulfamoyl chloride was prepared by
an adaptation of the method of Appel and Berger35 and was stored in the
refrigerator under positive pressure of N2 as a solution in toluene as
described by Woo et al.36 An appropriate volume of this solution was
freshly concentrated in vacuo immediately before use. Reactions were
carried out at room temperature unless stated otherwise. Flash column
chromatography was performed on silica gel (Matrex C60).
þ
7.04 (1H, s). HRMS (ESþ): m/z found 315.2324; C21H31O2
(Mþ þ H) requires 315.2319. Anal. (C21H30O2) C, H, N.
2-Ethyl-3-O-sulfamoyl-17β-sulfamoyloxymethylestra-1,3,
5(10)-triene 16. Compound 15 (80 mg, 0.25 mmol) was added to
solution of sulfamoyl chloride (1.0 mmol) in DMA (1.0 mL) at 0 °C.
The reaction mixture was stirred at room temperature for 24 h. Water
(10 mL) was added, and the mixture was extracted with EtOAc (2 ꢁ
30 mL). The combined organics were successively washed with water
and brine, then dried (MgSO4), filtered, and concentrated in vacuo.
Purification by flash column chromatography (hexane/EtOAc 3:2)
afforded compound 16 as a white powder (90 mg, 76%), mp
167ꢀ168 °C. δH (acetone-d6): 0.77 (3H, s), 1.16 (3H, t, J 7.4),
1.28ꢀ1.55 (7H, m), 1.79ꢀ1.97 (3H, m), 2.21ꢀ2.41 (2H, m), 2.68
(2H, q, J 7.4), 2.76ꢀ2.85 (2H, m), 4.08 (1H, dd, J 9.4, 6.4), 4.21 (1H, dd,
J 9.4, 7.4), 6.63 (2H, s, br), 7.07 (1H, s), 7.12 (2H, s, br), 7.23 (1H, s).
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 tetramethyl-
silane (TMS) as internal standard. Deuteriochloroform was used as
solvent unless otherwise stated. Mass spectra were recorded at the Mass
Spectrometry Service Center, University of Bath, U.K. FAB-MS were
carried out using m-nitrobenzyl alcohol (NBA) as the matrix. Elemental
analyses were performed by the Microanalysis Service, University of
Bath. Melting points were determined using a Stuart SMP3 melting
point apparatus and are uncorrected. All compounds were g98% pure
by reverse phase HPLC run with acetonitrile/water and methanol/water
(Sunfire C18 reverse phase column, 4.6 mm ꢁ 150 mm, 3.5 μm pore
size).
2-Ethyl-3-O-benzyl-17β-hydroxymethylestra-1,3,5(10)-tri-
ene 14. Compound 13 (2.35 g, 6 mmol) was added portionwise over 15
min into a mixture of Mg (576 mg, 24 mmol) and TiCl4 (0.63 mL, 6
mmol) in DCM (10 mL) at 0 °C under nitrogen. The reaction mixture
was stirred at 0 °C for 2 h and then at room temperature overnight. After
cooling to 0 °C, the mixture was neutralized with potassium carbonate
(saturated) and extracted with EtOAc (2 ꢁ 60 mL). The combined
organics were successively washed with water and brine, then dried
HRMS (ESþ): m/z found 473.1767; C21H33N2O6S2 (Mþ þ H)
þ
requires 473.1775. Anal. (C21H32N2O6S2) C, H, N.
2-Ethyl-3-hydroxy-17β-methoxymethylestra-1,3,5(10)-tri-
ene 17. A solution of compound 14 (265 mg, 0.65 mmol) in THF
(20 mL) was stirred under nitrogen, and sodium hydride (60% suspen-
sion inoil, 52mg, 1.3 mmol) was added portionwise atroom temperature.
After 15 min iodomethane (0.08 mL, 1.3 mmol) was added and the
mixture was heated to reflux for 2 h, cooled to room temperature. Water
(5 mL) was added, and the mixture was extracted with EtOAc (2 ꢁ
50 mL). The combined organics were washed with water and brine, then
dried (MgSO4), filtered, and concentrated in vacuo. Purification by flash
column chromatography (hexane/EtOAc 20:1) afforded the methyl
ether as a white powder (220 mg, 81%), mp 60ꢀ61 °C. δH 0.66 (3H,
s), 1.21 (3H, t, J 7.3), 1.22ꢀ1.51 (7H, m), 1.71ꢀ2.02 (5H, m), 2.17ꢀ2.33
(2H, m), 2.66 (2H, q J 7.3), 2.75ꢀ2.92 (2H, m), 3.26ꢀ3.31 (1H, m), 3.34
(3H, s), 3.47 (1H, dd, J 9.3, 6.3), 5.03 (2H, s), 6.63 (1H, s), 7.10 (1H, s),
þ
7.28ꢀ7.46 (5H, m). HRMS (ESþ): m/z found 419.2937; C29H39O2
(Mþ þ H) requires 419.2945. The above methyl ether (190 mg, 0.45
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Journal of Medicinal Chemistry
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mmol) was treated with Pd/C (10%, 30 mg) in THF (5 mL) and
methanol (20 mL) following the method described to access compound
15. Purification by flash column chromatography (hexane/EtOAc 10:1)
afforded compound 17 as a white powder (125 mg, 84%), mp
132ꢀ133 °C. δH 0.65 (3H, s), 1.19ꢀ1.55 (10H, m), 1.70ꢀ1.91 (4H,
m), 1.95ꢀ2.03 (1H, m), 2.14ꢀ2.29 (2H, m), 2.56 (2H, q J 7.4),
2.69ꢀ2.88 (2H, m), 3.27 (1H, dd, J 9.2, 7.4), 3.34 (3H, s), 3.47 (1H,
dd, J 9.2, 6.4), 4.61 (1H, s), 6.48 (1H, s), 7.04 (1H, s). HRMS (ESþ): m/
was added, and the mixture was extracted with EtOAc (2 ꢁ 30 mL). The
combined organics were successively washed with water and brine, then
dried (MgSO4), filtered, and concentrated in vacuo. Purification by flash
column chromatography (hexane/EtOAc 5:1 to 1:1) afforded com-
pound 20 as a white powder (85 mg, 68%), mp 176ꢀ177 °C. δH
(acetone-d6) 0.58 (3H, s), 1.09 (3H, t, J 7.4), 1.15ꢀ1.47 (7H, m),
1.57ꢀ1.69 (2H, m), 1.75ꢀ1.82 (2H, m), 1.87ꢀ1.96 (2H, m),
2.12ꢀ2.23 (2H, m), 2.59 (2H, q J 7.4), 2.70ꢀ2.74 (2H, m),
3.44ꢀ3.51 (1H, m), 3.60ꢀ3.69 (1H, m), 5.93 (2H, s), 6.98 (1H, s),
7.07 (1H, s). HRMS (ESþ):m/zfound 394.2052; C21H32NO4Sþ (Mþ þ H)
requires 394.2047. Anal. (C21H31NO4S) C, H, N.
2-Ethyl-3-benzyloxy-17-(2-ethoxy-2-oxoethylidene)estra-
1,3,5(10)-trienes 21 and 22. Sodium hydride (60% dispersion in
mineral oil, 0.8 g, 20 mmol) was washed with hexane (3 ꢁ 1 mL), then
stirred in THF (50 mL) at 0 °C under nitrogen. Triethylphosphonoa-
cetate (4.48 g, 20 mmol) was added dropwise at 0 °C, and the reaction
mixture was stirred until gas evolution ceased. Compound 13 (3.9 g, 10
mmol) in THF (10 mL) was added dropwise, and the reaction mixture
was heated to reflux for 18 h, then cooled to 0 °C, poured into water
(30 mL), and extracted with EtOAc (2 ꢁ 50 mL). The combined
organics were washed with water and brine, then dried (MgSO4)),
filtered, and concentrated in vacuo. Purification by flash column
chromatography (hexane to hexane/EtOAc 20:1) successively afforded
compounds 22 (590 mg, 13%) and 21 (3.5 g, 75%) as white powders.
Compound 21: mp 61ꢀ63 °C. δH 0.87 (3H, s), 1.23 (3H, t, J 7.4), 1.31
(3H, t, J 7.1), 1.30ꢀ1.61 (6H, m), 1.84ꢀ2.01 (3H, m), 2.19ꢀ2.30 (1H,
m), 2.41ꢀ2.48 (1H, m), 2.67 (2H, q, J 7.4), 2.77ꢀ2.96 (2H, m), 4.16
(2H, q, J 7.1), 5.04 (2H, s), 5.61 (1H, t, J 2.3), 6.64 (1H, s), 7.11 (1H, s),
þ
z found 329.2465; C22H33O2 (Mþ þ H) requires 329.2475. Anal.
(C22H32O2) C, H.
2-Ethyl-3-O-sulfamoyl-17β-methoxymethylestra-1,3,5(10)-
triene 18. Compound 17 (82 mg, 0.25 mmol) was treated with
sulfamoyl chloride (0.5 mmol) in DMA (1.0 mL) following the method
described to access compound 16. Purification by flash column chro-
matography (hexane/EtOAc 5:1) afforded compound 18 as a white
powder (80 mg, 79%), mp 132ꢀ133 °C. δH 0.65 (3H, s), 1.17ꢀ1.60
(11H, m), 1.73ꢀ2.03 (5H, m), 2.18ꢀ2.30 (2H, m), 2.68 (2H, q J 7.4),
2.80ꢀ2.87 (2H, m), 3.24ꢀ3.32 (1H, m), 3.34 (3H, s), 3.46 (1H, dd,
J 9.4, 6.9), 4.99 (2H, s), 7.06 (1H, s), 7.18 (1H, s). HRMS (ESþ): m/z
found 408.2205; C22H34NO4Sþ (Mþ þ H) requires 408.2203. Anal.
(C22H33NO4S) C, H, N.
2-Ethyl-3-hydroxy-17β-tert-butyldiphenylsilyloxymethyl-
estra-1,3,5(10)-triene 19. A solution of compound 14 (404 mg,
1 mmol), tert-butyldiphenylsilyl chloride (302 mg, 1.1 mmol), and
imidazole (82 mg, 1.2 mmol) in DMF (10 mL) was stirred at room
temperature for 4 h. Water (10 mL) was added, and the mixture was
extracted with EtOAc (2 ꢁ 30 mL). The combined organics were
washed with water and brine, then dried (MgSO4), filtered, and
concentrated in vacuo. Purification by flash column chromatography
(hexane to hexane/EtOAc 25:1) gave the silyl protected alcohol as a
colorless oil (600 mg, 93%). δH 0.67 (3H, s), 1.06 (9H, s), 1.18ꢀ1.58
(11H, m), 1.65ꢀ1.92 (4H, m), 2.12ꢀ2.36 (3H, m), 2.67 (2H, q J 7.4),
2.72ꢀ2.92 (2H, m), 3.58 (1H, dd, J 10.2, 5.7), 3.73 (1H, dd, J 10.2, 7.7),
5.04 (2H, s), 6.64 (1H, s), 7.13 (1H, s), 7.28ꢀ7.47 (11H, m), 7.65ꢀ7.73
(4H, m). LC/MS (ESꢀ): m/z 641.5 (Mꢀ ꢀ H). The above silyl
protected alcohol (570 mg, 0.89 mmol) was treated with Pd/C (10%, 50
mg) in THF (20 mL) and methanol (20 mL) following the method
described to access compound 15. Purification by flash column chro-
matography (hexane/EtOAc 25:1 to 20:1) afforded compound 19 as a
white powder (430 mg, 88%), mp 63ꢀ65 °C. δH 0.65 (3H, s), 1.04 (9H,
s), 1.17ꢀ1.51 (10H, m), 1.71ꢀ1.88 (4H, m), 2.08ꢀ2.32 (3H, m), 2.58
(2H, q, J 7.4), 2.71ꢀ2.87 (2H, m), 3.57 (1H, dd, J 10.1, 5.9), 3.71 (1H,
dd, J 10.1, 7.6), 4.44 (1H, s), 6.48 (1H, s), 7.06 (1H, s), 7.35ꢀ7.43 (6H,
m), 7.66ꢀ7.71 (4H, m). HRMS (ESꢀ): m/z found 553.3326;
C37H47O2Siꢀ (Mꢀ ꢀ H) requires 551.3340.
þ
7.28ꢀ7.46 (5H, m). HRMS (ESþ): m/z found 459.2896; C31H39O3
(Mþ þ H) requires 459.2894. Compound 22: mp 119ꢀ120 °C. δH 1.05
(3H, s), 1.22 (3H, t, J 7.4), 1.30 (3H, t, J 7.2), 1.32ꢀ1.64 (6H, m),
1.77ꢀ1.85 (1H, m), 1.88ꢀ1.96 (1H, m), 2.14ꢀ2.26 (1H, m),
2.32ꢀ2.50 (2H, m), 2.56ꢀ2.71 (3H, m), 2.76ꢀ2.89 (3H, m),
4.10ꢀ4.19 (2H, m), 5.03 (2H, s), 5.69 (1H, t, J 1.9), 6.64 (1H, s),
7.11 (1H, s), 7.27ꢀ7.46 (5H, m). HRMS (ESþ): m/z found 459.2907;
C31H39O3þ (Mþ þ H) requires 459.2894.
2-Ethyl-3-hydroxy-17β-(2-ethoxy-2-oxoethyl)estra-1,3,5(10)-
triene 23. Compounds 21 and 22 (3.4 g, 7.4 mmol) were treated with
Pd/C (10%, 250 mg) in THF (10 mL) and methanol (50 mL) as
described for the synthesis of compound 15. Purification by flash
column chromatography (hexane/EtOAc 20:1 to 10:1) afforded com-
pound 23 as a white powder (2.6 g, 95%), mp 82ꢀ84 °C. δH 0.62
(3H, s), 1.20 (3H, t, J 7.2), 1.25 (3H, t, J 7.3), 1.28ꢀ1.50 (7H, m),
1.74ꢀ1.98 (5H, m), 2.09ꢀ2.43 (4H, m), 2.58 (2H, q, J 7.3), 2.77ꢀ2.86
(2H, m), 4.12 (2H, q, J 7.2), 4.77 (1H, s), 6.49 (1H, s), 7.04 (1H, s).
HRMS (ESþ): m/z found 371.2572; C24H35O3þ (Mþ þ H) requires
371.2581. Anal. (C24H34O3) C, H.
2-Ethyl-3-O-sulfamoyl-17β-(2-ethoxy-2-oxoethyl)estra-1,
3,5(10)-triene 24. Compound 23 (100 mg, 0.27 mmol) was treated
with sulfamoyl chloride (0.54 mmol) in DMA (1.0 mL) as described for
the synthesis of compound 16. Purification by flash column chroma-
tography (hexane/EtOAc 9:1 to 4:1) afforded compound 24 as a white
powder (83 mg, 68%), mp 120ꢀ122 °C. δH 0.63 (3H, s), 1.21 (3H, t,
J 7.4), 1.27 (3H, t, J 7.0), 1.24ꢀ1.50 (7H, m), 1.74ꢀ1.98 (5H, m),
2.11ꢀ2.42 (4H, m), 2.68 (2H, q, J 7.4), 2.82ꢀ2.84 (2H, m), 4.12 (2H, q,
J 7.0), 5.08 (2H, s), 7.05 (1H, s), 7.16 (1H, s). HRMS (FABþ): m/z
found 450.2313; C24H36NO5Sþ (Mþ þ H) requires 450.2309. Anal.
(C24H35NO5S) C, H.
2-Ethyl-3-O-sulfamoyl-17β-hydroxymethylestra-1,3,5(10)-
triene 20. Sulfamoyl chloride (0.5 M in toluene, 2.2 mL, 1.1 mmol) was
added to a solution of 2,6-di-tert-butyl-4-methylpyridine (275 mg, 1.34
mmol) and compound 19 (370 mg, 0.67 mmol) in DCM (10 mL). The
reaction mixture was stirred at room temperature for 5 h. Water (10 mL)
was added, and the mixture was extracted with DCM (2 ꢁ 30 mL). The
combined organics were successively washed with water and brine, then
dried (MgSO4), filtered, and concentrated in vacuo. Purification by flash
column chromatography (hexane/EtOAc 10:1 to 5:1) afforded the
sulfamoylated compound as a white powder (340 mg, 80%), mp
79ꢀ81 °C. δH 0.66 (3H, s), 1.04 (9H, s), 1.18ꢀ1.53 (10H, m),
1.68ꢀ1.91 (4H, m), 2.13ꢀ2.32 (3H, m), 2.68 (2H, q J 7.4),
2.79ꢀ2.87 (2H, m), 3.57 (1H, dd, J 10.1, 5.9), 3.70 (1H, dd, J 10.1,
7.7), 4.94 (2H, s), 7.06 (1H, s), 7.21 (1H, s), 7.34ꢀ7.46 (6H, m),
7.66ꢀ7.70 (4H, m). HRMS (ESþ): m/z found 632.3223;
C37H50NO4SSiþ (Mþ þ H) requires 632.3224. The sulfamoylated
compound (200 mg, 0.32 mmol) was stirred in THF (10 mL), cooled to
0 °C, and treated with TBAF in THF (1M, 0.65 mL, 0.65 mmol). The
reaction mixture was stirred at room temperature for 7 h. Water (10 mL)
2-Ethyl-3-O-benzyloxy-17β-(2-hydroxyethyl)estra-1,3,5(10)-
triene 25. Compound 23 (2.6 g, 7 mmol) was stirred with potassium
carbonate (2.75 g, 20 mmol) and benzyl bromide (0.86 mL, 7.2 mmol)
in DMF (30 mL) at room temperature for 24 h. Water (50 mL) was
added, and the mixture was extracted with EtOAc (2 ꢁ 60 mL). The
combined organics were washed with water and brine, then dried
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(MgSO4), filtered, and concentrated in vacuo. Purification by flash
column chromatography (hexane/EtOAc 50:1) afforded the phenol as a
white powder (3.1 g, 91%), mp 94ꢀ95 °C. δH 0.64 (3H, s), 1.21 (3H, t, J
7.4), 1.27 (3H, t, J 7.7), 1.26ꢀ1.60 (7H, m), 1.76ꢀ2.05 (5H, m),
2.11ꢀ2.46 (4H, m), 2.68 (2H, q, J 7.7), 2.83 (2H, m), 4.14 (2H, q, J 7.4),
5.04 (2H, s), 6.63 (1H, s), 7.11 (1H, s), 7.29ꢀ7.47 (5H, m). HRMS
synthesis of compound 16. Purification by flash column chromatogra-
phy (hexane/EtOAc 10:1 to 4:1) and crystallization from hexane/
EtOAc afforded compound 29 as a white powder (260 mg, 84%), mp
168ꢀ169 °C. δH 0.61 (3H, s), 1.20 (3H, t, J 7.4), 1.22ꢀ1.54 (9H,
m), 1.70ꢀ1.95 (5H, m), 2.15ꢀ2.33 (2H, m), 2.68 (2H, q, J 7.4),
2.79ꢀ2.86 (2H, m), 3.33 (3H, s), 3.35ꢀ3.39 (2H, m), 4.95 (2H, s),
7.06 (1H, s), 7.18 (1H, s). HRMS (ESþ) m/z found 422.2360;
C23H36NO4Sþ (Mþ þ H) requires 422.2360. Anal. (C23H35NO4S)
C, H, N.
(ESþ): m/z found 461.3057; C31H41O3 (Mþ þ H) requires
þ
461.3050. A solution of the phenol (2.8 g, 6 mmol) in THF (30 mL)
was treated with lithium aluminum hydride (0.45 g, 12 mmol) at 0 °C for
3 h. Ammonium chloride (saturated) was added, and the mixture was
extracted with EtOAc. The organic layer was washed with water and
brine, then dried (MgSO4), filtered, and concentrated in vacuo. Pur-
ification by flash column chromatography (hexane/EtOAc 15:1 to 8:1)
afforded compound 25 as a white powder (2.2 g, 88%), mp 98ꢀ99 °C.
δH 0.63 (3H, s), 1.20 (3H, t, J 7.4), 1.22ꢀ1.58 (9H, m), 1.70ꢀ1.92 (5H,
m), 2.16ꢀ2.34 (2H, m), 2.66 (2H, q, J 7.4), 2.82ꢀ2.88 (2H, m),
3.64ꢀ3.72 (2H, m), 5.03 (2H, s), 6.62 (1H, s), 7.10 (1H, s), 7.28ꢀ7.45
(5H, m). HRMS (ESþ): m/z found 419.2939; C29H39O2þ (Mþ þ H)
requires 419.2945.
2-Ethyl-3-hydroxy-17β-(2-hydroxyethyl)estra-1,3,5(10)-tri-
ene 26. Compound 25 (420 mg, 1.0 mmol) was treated with Pd/C
(10%, 40 mg) in THF (5 mL) and methanol (10 mL) as described for the
synthesis of compound 15. Purification by flash column chromatography
(hexane/EtOAc 10:1 to 3:1) and crystallization from hexane/EtOAc
afforded compound 26 as a white solid (300 mg, 91%), mp 166ꢀ167 °C.
δH 0.62 (3H, s), 1.21 (3H, t, J 7.4), 1.23ꢀ1.55 (3H, m), 1.70ꢀ1.93 (5H,
m), 2.13ꢀ2.34 (2H, m), 2.58 (2H, q, J 7.4), 2.74ꢀ2.84 (2H, m),
3.63ꢀ3.70 (2H, m), 4.57 (1H, s), 6.49 (1H, s), 7.05 (1H, s). HRMS
(ESþ): m/z found 329.2470; C22H33O2þ (Mþ þ H) requires 329.2475.
Anal. (C22H32O2) C, H.
2-Ethyl-17β-(2-sulfamoyloxyethyl)estra-3-O-sulfamoyl-1,3,
5(10)-triene 27. Compound 26 (105 mg, 0.32 mmol) was treated with
sulfamoyl chloride (0.54 mmol) in DMA (1.0 mL) as described for the
synthesis of compound 16. Purification by flash column chromatography
(hexane/EtOAc 10:1 to 4:1) and crystallization from hexane/EtOAc
affordedcompound27as a white powder (85mg, 54%), mp192ꢀ194 °C.
δH (acetone-d6): 0.69 (3H, s), 1.18 (3H, t, J 7.4), 1.29ꢀ1.62 (10H, m),
1.76ꢀ2.42 (6H, m), 2.70 (2H, q, J 7.4), 2.80ꢀ2.84 (2H, m), 4.16ꢀ4.18
(2H, m), 6.63 (2H, s), 7.09 (1H, s), 7.13 (2H, þs), 7.24 (1H, s). HRMS
(ESþ): m/z found 487.1928; C22H35O6N2S2 (Mþ þ H) requires
487.1931. Anal. (C22H34O6N2S2) C, H, N.
2-Ethyl-3-hydroxy-17β-(2-methoxyethyl)estra-1,3,5(10)-
triene 28. Compound 25 (0.42 g, 1 mmol) was treated with NaH (60%
dispersion in mineral oil, 0.06 g, 1.5 mmol) and iodomethane (0.12 mL,
2.0 mmol) in THF (30 mL) following the method described for the
synthesis of compound 17. Purification by flash column chromatogra-
phy (hexane/EtOAc 20:1) afforded the methyl ether as a white powder
(415 mg, 96%), mp 70ꢀ71 °C. δH 0.66 (3H, s), 1.25 (3H, t, J 7.4),
1.26ꢀ1.60 (9H, m), 1.76ꢀ1.96 (5H, m), 2.20ꢀ2.42 (2H, m), 2.70 (2H,
q, J 7.4), 2.82ꢀ2.89 (2H, m), 3.38 (3H, s), 3.40ꢀ3.46 (2H, m), 5.07
(2H, s), 6.66 (1H, s), 7.15 (1H, sþ), 7.31ꢀ7.50 (5H, m). HRMS (ESþ):
m/z found 433.3101; C30H41O2 (Mþ þ H) requires 433.3090. The
methyl ether (0.4 g, 0.93 mmol) was treated with Pd/C (10%, 30 mg) in
THF (5 mL) and methanol (20 mL) as described for the synthesis of
compound 15. Purification by flash column chromatography (hexane/
EtOAc 15:1 to 10:1) afforded compound 28 as a white powder (305 mg,
97%), mp 58ꢀ59 °C. δH 0.62 (3H, s), 1.19ꢀ1.56 (12H, m), 1.72ꢀ1.91
(5H, m), 2.13ꢀ2.34 (2H, m), 2.60 (2H, q, J 7.4), 2.73ꢀ2.80 (2H, m),
3.37 (3H, s), 3.42ꢀ3.48 (2H, m), 5.26 (1H, s), 6.48 (1H, s), 7.06 (1H,
s). HRMS (FABþ): m/z found 342.2555; C23H34O2þ (Mþ) requires
342.2559. Anal. (C23H34O2) C, H.
2-Ethyl-3-hydroxy-17β-(2-tert-butyldiphenylsilyloxyethyl)
estra-1,3,5(10)-triene 30. Compound 25 (419 mg, 1 mmol), imida-
zole (136 mg, 2 mmol), and tert-butyldiphenylsilyl chloride (300 mg, 1.1
mmol) were stirred in DMF (5 mL) as described for the synthesis of
compound 19. Purification by flash column chromatography (hexane/
EtOAc 25:1) afforded the silyl protected alcohol as a white powder (590
mg, 90%), mp 48ꢀ50 °C. δH 0.63 (3H, s), 1.12 (9H, s) 1.15ꢀ1.60 (11H,
m), 1.71ꢀ2.01 (5H, m), 2.20ꢀ2.44 (2H, m), 2.69 (, J 7.4), 2.84ꢀ2.91
(2H, m), 3.65ꢀ3.81 (2H, m), 5.09 (2H, s), 6.68 (1H, s), 7.20 (1H, s),
7.32ꢀ7.51 (11H, m), 7.72ꢀ7.77 (4H, m). HRMS (ESþ): m/z found
657.4119; C45H57O2Siþ (Mþ þ H) requires 657.4122. The silyl
protected alcohol (550 mg, 0.84 mmol) was treated with Pd/C (10%,
50 mg) in THF (5 mL) and methanol (15 mL) following the method
described to access compound 15. Purification by flash column chroma-
tography (hexane/EtOAc 25:1 to 15:1) afforded compound 30 as a white
powder (410 mg, 87%), mp 134ꢀ135 °C. δH 0.59 (3H, s), 1.08 (9H, s),
1.18ꢀ1.56 (12H, m), 1.68ꢀ1.89 (5H, m), 2.10ꢀ2.33 (2H, m), 2.61 (2H,
q, J 7.4), 2.73ꢀ2.82 (2H, m), 3.61ꢀ3.78 (2H, m), 4.71 (1H, s), 6.49
(1H, s), 7.07 (1H, s), 7.36ꢀ7.47 (6H, m), 7.68ꢀ7.74 (4H, m). HRMS
(ESþ):m/zfound 567.3666; C38H51O2Siþ (Mþ þ H) requires 567.3653.
2-Ethyl-3-O-sulfamoyl-17β-(hydroxyethyl)estra-1,3,5(10)-
triene 31. Compound 30 (142 mg, 0.25 mmol) was treated with
sulfamoyl chloride (0.5 mmol) in DMA (5 mL) as described for the
synthesis of compound 16. Purification by flash column chromatography
(hexane/EtOAc 20:1 to 8:1) afforded the sulfamate as a white powder
(115 mg, 70%), mp 185ꢀ186 °C. δH 0.57 (3H, s), 1.05 (9H, s),
1.18ꢀ1.55 (13H, m), 1.68ꢀ1.89 (4H, m), 2.15ꢀ2.31 (2H, m), 2.68
(2H, q, J 7.4), 2.80ꢀ2.87 (2H, m), 3.57ꢀ3.70 (2H, m), 4.91 (2H, s, br),
7.06 (1H, s), 7.19 (1H, s), 7.34ꢀ7.45 (6H, m), 7.64ꢀ7.69 (4H, m).
HRMS (FABþ): m/z found 646.3384; C38H51NO4SSiþ (Mþ þ H)
requires 646.3386. The sulfamate (110 mg, 0.17 mmol) was reacted with
TBAF (1 M in THF, 0.34 mL, 0.34 mmol) in THF (10 mL) at room
temperature for 4 h as described for the synthesis of 20. Purification by
flash column chromatography (hexane/EtOAc 10:1 to 2:1) and crystal-
lization from hexane/EtOAc afforded compound 31 as a white powder
(50 mg, 72%), mp 157ꢀ158 °C. δH: 0.62 (3H, s), 1.17ꢀ1.55 (12H, m),
1.70ꢀ1.94 (5H, m), 2.16ꢀ2.33 (2H, m), 2.68 (2H, q, J 7.4), 2.80ꢀ2.87
(2H, m), 3.60ꢀ3.76 (2H, m), 4.87 (2H, s, br), 7.06 (1H, s), 7.19 (1H, s).
HRMS (FABþ): m/z found 407.2141; C22H33NO4Sþ (Mþ) requires
407.2130. Anal. (C22H33NO4S) C, H, N.
2-Ethyl-17-(2-ethoxy-2-oxoethylidene)-3-O-(tert-butyldi-
methylsilyl)estra-1,3,5(10)-triene 33. Compound 32 (4.1 g, 10
mmol) was reacted with triethylphosphonoacetate following the meth-
od used for the synthesis of compounds 21 and 22. Purification by flash
column chromatography (hexane to hexane/EtOAc 25:1) afforded
compound 33 as a light yellow oil (4.3 g, 89%; mixture of E and Z
isomers). δH 0.22 (6H, s), 0.85 (3H, s, major) and 1.03 (3H, s, minor),
0.99 (9H, s), 1.16 (3H, t, J 7.4), 1.28 (3H, t, J 7.1), 1.31ꢀ1.59 (5H, m),
1.82ꢀ1.98 (3H,m), 2.13ꢀ2.25 (1H, m), 2.37ꢀ2.44 (1H, m), 2.55 (2H,
q, J 7.4), 2.76ꢀ2.92 (4H, m), 4.15 (2H, q, J 7.1), 5.58 (1H, t, J 2.2,
major), 5.67 (1H, t, J 1.7, minor), 6.48 (1H, s), 7.05 (1 H, s). HRMS
(ESþ): m/z found 483.3279; C30H47O3Siþ (Mþ þ H) requires
483.3289.
2-Ethyl-3-O-sulfamoyl-17β-(2-methoxyethyl)estra-1,3,5(10)-
triene 29. Compound 28 (250 mg, 0.73 mmol) was treated with
sulfamoyl chloride (1.5 mmol) in DMA (1.0 mL) as described for the
2-Ethyl-3-hydroxy-17-(2-ethoxy-2-oxoethylidene)estra-1,
3,5(10)-trienes 34 and 35. Compound 33 (4.0 g, 8.3 mmol) was
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treated with TBAF (1 M in THF, 9.2 mL, 9.2 mmol) in THF (50 mL) as
described for the synthesis of compound 20. Purification by flash column
chromatography (hexane to hexane/EtOAc 10:1) afforded successively
the Z-isomer 35 (380 mg, 12%) and the E-isomer 34 (2.2 g, 72%) as
white powders. Compound 34: mp 85ꢀ87 °C. δH 0.86 (3H, s), 1.23
(3H, t, J 7.4), 1.30 (3H, t, J 7.1), 1.33ꢀ1.58 (6H, m), 1.85ꢀ2.00 (3H, m),
2.18ꢀ2.25 (1H, m), 2.39ꢀ2.45 (1H, m), 2.60 (2H, q, J 7.4), 2.78ꢀ2.84
(2H, m), 2.86ꢀ2.91 (2H, m), 4.16 (2H, q, J 7.1), 4.67 (1H, s), 5.59 (1H,
t, J 2.3), 6.51 (1H, s), 7.06 (1H, s). HRMS (ESþ): m/z found 369.2416;
C24H33O3þ (Mþ þ H) requires 369.2424. Anal. (C24H32O3) C, H, N.
Compound 35: mp 157ꢀ159 °C. δH 1.04 (3H, s), 1.22 (3H, t, J 7.4), 1.29
(3H, t, J 7.2), 1.36ꢀ1.62 (6H, m), 1.78ꢀ1.84 (1H, m), 1.89ꢀ1.95 (1H,
m), 2.16ꢀ2.23 (1H, m), 2.31ꢀ2.37 (1H, m), 2.41ꢀ2.49 (1H, m),
2.57ꢀ2.65 (3H, m), 2.77ꢀ2.84 (3H, m), 4.11ꢀ4.20 (2H, m), 4.60
(1H, s, br), 5.68 (1H, t, J 1.9), 6.50 (1H, s), 7.05 (1H, s). HRMS (ESþ):
m/z found 369.2419; C24H33O3þ (Mþ þ H) requires 369.2424. Anal.
(C24H32O3) C, H, N.
4.21 (1H, dd, J 12.1, 7.4), 4.35 (1H, dd, J 12.1, 7.4), 4.57 (1H, s, br),
5.32ꢀ5.37 (1H, m), 6.50 (1H, s), 7.04 (1H, s). HRMS (ESþ): m/z
found 349.2151; C22H30O2Naþ (MþþNa) requires 349.2138. Anal.
(C22H30O2) C, H
2-Ethyl-3-O-sulfamoyl-17-vinyl-estra-1,3,5(10),16-tetraene
40. Compound 38 (80 mg, 0.24 mmol) was treated with sulfamoyl
chloride (0.5 mmol) in DMA (1.0 mL) as described for the synthesis of
compound 16. Purification by flash column chromatography (hexane/
EtOAc 10:1 to 3:1) afforded compound 40 as a colorless oil (52 mg,
56%). δH 0.91 (3H, s), 1.21 (3H, t, J 7.4), 1.27ꢀ1.55 (2H, m), 1.61ꢀ1.73
(4H, m), 1.85ꢀ2.07 (2H, m), 2.18ꢀ2.39 (4H, m), 2.69 (2H, q, J 7.4),
2.83ꢀ2.92 (2H, m), 4.88ꢀ4.97 (1H, m), 4.99 (2H, s), 5.35 (1H, d, J
18.0), 5.73 (1H, s, br), 6.32 (1H, dd, J 18.0, 11.4), 7.10 (1H, s), 7.18
(1H, s). HRMS (ESþ): m/z found 388.1937; C22H30NO3Sþ (Mþ þ H)
requires 388.1941.
Treatment of 39 (80 mg, 0.24 mmol) with sulfamoyl chloride (0.5
mmol) in DMA (1.0 mL) also yielded 40 (70 mg, 74%).
(E)-2-Ethyl-3-O-sulfamoyl-17-(2-ethoxy-2-oxoethylidene)
estra-1,3,5(10)-triene 36. Compound 34 (185 mg, 0.5 mmol) was
treated with sulfamoyl chloride (1.5 mmol) in DMA (1.0 mL) as
described for the synthesis of compound 16. Purification by flash column
chromatography (hexane/EtOAc 10:1 to 3:1) afforded compound 36 as
a white powder (195 mg, 87%), mp 116ꢀ118 °C. δH 0.86 (3H, s), 1.22
(3H, t, J 7.4), 1.29 (3H, t, J 7.1), 1.38ꢀ1.64 (6H, m), 1.76ꢀ2.02 (3H, m),
2.23ꢀ2.29 (1H, m), 2.38ꢀ2.44 (1H, m), 2.67 (2H, q, J 7.4), 2.85ꢀ2.91
(4H, m), 4.16 (2H, q, J 7.1), 4.98 (2H, s), 5.59 (1H, t, J 2.3), 7.09 (1H, s),
7.20 (1H, s). HRMS (ESþ): m/z found 448.2149; C24H34NO5Sþ
(Mþ þ H) requires 448.2152. Anal. (C24H33NO5S) C, H, N
(E)-2-Ethyl-3-hydroxy-17-(2-methoxyethylidene)estra-1,3,
5(10)-triene 41. Compound 21 (0.9 g, 1.97 mmol) was treated with
lithium aluminum hydride (0.45 g, 12 mmol) in THF (30 mL) at 0 °C as
described for the synthesis of compound 25. Purification by flash column
chromatography (hexane/EtOAc 15:1 to 6:1) afforded the allylic alcohol
as a white powder (0.75 g, 89%), mp 61ꢀ62 °C. δH 0.84 (3H, s),
1.20ꢀ1.66 (9H, m), 1.82ꢀ2.02 (3H, m), 2.21ꢀ2.51 (3H, m), 2.71 (2H,
q, J 7.4), 2.82ꢀ2.89 (2H, m), 4.10ꢀ4.21 (2H, m), 5.07 (2H, s),
5.28ꢀ5.34 (1H, m), 6.66 (1H, s), 7.15 (1H, s), 7.30ꢀ7.48 (5H, m).
HRMS (ESþ): m/z found 417.2793; C29H37O2þ (Mþ þ H) requires
417.2788. Method was as for 17 using the allylic alcohol, sodium hydride
(60% dispersion in mineral oil, 80 mg, 2 mmol), and iodomethane
(0.19 mL, 3.0 mmol) in THF (10 mL) at room temperature for 18 h.
Purification by flash column chromatography (hexane/EtOAc 20:1)
afforded the methyl ether as a colorless oil (0.59 g, 91%). δH: 0.84
(3H, s), 1.24 (3H, t, J 7.4), 1.26ꢀ1.66 (6H, m), 1.82ꢀ2.02 (3H, m),
2.20ꢀ2.51 (3H, m), 2.70 (2H, q, J 7.4), 2.82ꢀ2.89 (2H, m), 3.36 (3H, s),
3.90ꢀ3.97 (2H, m), 5.06 (2H, s), 5.24ꢀ5.30 (1H, m), 6.66 (1H, s), 7.15
(1H, s), 7.31ꢀ7.48 (5H, m). HRMS (ESþ): m/z found 431.2937;
C30H39O2þ (Mþ þ H) requires 431.2945. A solution of the benzyl ether
(180 mg, 0.42 mmol) in t-BuOH (10 g) was refluxed and treated sodium
(∼50 mg). Further chunks of sodium were added every hour until
deprotection was complete by TLC (8 h). The mixture was cooled, and
the reaction was then quenched with isopropanol. The mixture was then
diluted with water. Theorganicswere extracted with ethylacetate, andthe
organic layer was washed with water, brine and then evaporated.
Purification by flash column chromatography (hexane/EtOAc 15:1 to
8:1) afforded 41 as a white powder (95 mg, 67%), mp 133ꢀ134 °C. δH
0.79 (3H, s), 1.22 (3H, t, J 7.4), 1.28ꢀ1.60 (6H, m), 1.75ꢀ1.96 (3H, m),
2.18 (1H, m), 2.39 (3H, m), 2.59 (2H, q, J 7.4), 2.79 (2H, m), 3.35
(3H, s), 3.90ꢀ3.98 (2H, m), 5.11 (1H, s), 5.21ꢀ5.28 (1H, m), 6.4þ8
(1H, s), 7.05 (1H, s). HRMS (ESþ): m/z found 341.2472; C23H33O2
(Mþ þ H) requires 341.2475. Anal. (C23H32O2) C, H
(Z)- 2-Ethyl-3-O-sulfamoyl-17-(2-ethoxy-2-oxoethylidene)
estra-1,3,5(10)-triene 37. Compound 35 (170 mg, 0.46 mmol) was
treated with sulfamoyl chloride (1.5 mmol) in DMA (1.0 mL) as
described for the synthesis of compound 16. Purification by flash column
chromatography (hexane/EtOAc 10:1 to 3:1) afforded compound 37 as
a white powder (190 mg, 92%), mp 152ꢀ153 °C. δH 1.04 (3H, s), 1.22
(3H, t, J 7.4), 1.29 (3H, t, J 7.2), 1.34ꢀ1.64 (6H, m), 1.77ꢀ1.85 (1H, m),
1.91ꢀ1.97 (1H, m), 2.18ꢀ2.27 (1H, m), 2.31ꢀ2.38 (1H, m), 2.40ꢀ2.49
(1H, m), 2.56ꢀ2.74 (3H, m), 2.79ꢀ2.88 (3H, m), 4.10ꢀ4.18 (2H, m),
4.92 (2H, s), 5.69 (1H, t, J 2.1), 7.08 (1H, s), 7.19(1H, s). HRMS (ESþ):
m/z found 448.2143; C24H34NO5Sþ (Mþ þ H) requires 448.2152.
Anal. (C24H33NO5S) C, H, N
(E)-2-Ethyl-3-hydroxy-17-(2-hydroxyethylidene)estra-1,3,
5(10)-triene 38. A solution of 34 (370 mg, 1.0 mmol) in THF (5 mL)
stirred under nitrogen was cooled to ꢀ78 °C, and DIBAL-H (1 M in
THF, 2.2 mL, 2.2 mmol) was added dropwise. The mixture was stirred at
ꢀ78 °C for 1 h and then slowly warmed to room temperature and stirred
for 30 min. Ammonium chloride (saturated, 5 mL) was added at 0 °C,
and the mixture was extracted with EtOAc (2 ꢁ 30 mL). The combined
organics were washed with water and brine, then dried (MgSO4),
filtered, and concentrated in vacuo. Purification by flash column
chromatography (hexane to hexane/EtOAc 4:1) afforded compound
38 as a white powder (260 mg, 79%), mp 167ꢀ169 °C. δH 0.79 (3H, s),
1.21 (3H, t, J 7.4), 1.27ꢀ1.61 (6H, m), 1.74ꢀ1.97 (3H, m), 2.20ꢀ2.48
(5H, m), 2.59 (2H, q, J 7.4), 2.77ꢀ2.83 (2H, m), 4.03ꢀ4.12 (2H, m),
4.49 (1H, s), 5.26ꢀ5.37 (1H, m), 6.48 (1H, s), 7.06 (1H, s). HRMS
(ESþ): m/z found 349.2129; C22H30O2Naþ (MþþNa) requires
349.2138. Anal. (C22H30O2) C, H.
(E)-2-Ethyl-3-O-sulfamoyl-17-(2-methoxyethylidene)estra-
1,3,5(10)-triene 42. Compound 41 (170 mg, 0.5 mmol) was treated
withsulfamoylchloride(1.0 mmol) inDMA (1.0 mL) asdescribed for the
synthesis of compound 16. Purification by flash column chromatography
(hexane/EtOAc 10:1 to 5:1) afforded compound 42 as a white powder
(142 mg, 68%), mp 126ꢀ127 °C. δH 0.79 (3H, s), 1.20 (3H, t, J 7.4),
1.22ꢀ1.67 (6H, m), 1.78ꢀ1.98 (3H, m), 2.17ꢀ2.47 (4H, m), 2.68 (2H,
q, J 7.4), 2.82ꢀ2.88 (2H, m), 3.32 (3H, s), 3.87ꢀ3.93 (2H, m), 5.14
(2H, s), 5.18ꢀ5.24 (1H, m), 7.06 (1H, s), 7.19 (1H, s). HRMS (ESþ):
m/z found420.2188; C23H34O4NSþ (Mþ þ H) requires 420.2203. Anal.
(C23H33O4NS) C, H, N
(Z)-2-Ethyl-3-hydroxy-17- (2-hydroxyethylidene)estra-1,3,
5(10)-triene 39. Compound 35 (180 mg, 0.49 mmol) was treated with
DIBAL-H (1 M in THF, 1.0 mL, 1.0 mmol) in THF (5 mL) as described
for the synthesis of 38. Purification by flash column chromatography
(hexane to hexane/EtOAc 4:1) afforded compound 39 as a white powder
(90 mg, 56%), mp 149ꢀ151 °C. δH 0.93 (3H, s), 1.23 (3H, t, J 7.4),
1.28ꢀ1.61 (6H, m), 1.72ꢀ1.82 (2H, m), 1.87ꢀ1.92 (1H, m), 2.19ꢀ2.40
(4H, m), 2.47ꢀ2.54 (1H, m), 2.60 (2H, q, J 7.4), 2.74ꢀ2.83 (2H, m),
(E)-2-Ethyl-3-O-sulfamoyl-17-(2-hydroxyethylidene)estra-1,
3,5(10)-triene 43. Compound 36 (110 mg, 0.25 mmol) was treated
with DIBAL-H in (1 M in THF, 0.5 mL, 0.5 mmol) in THF (5 mL)
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at ꢀ78 °C as described for the synthesis of compound 38. Purification by
flash column chromatography (hexane to hexane/EtOAc 1:1) afforded
compound 43 as a white powder (65 mg, 65%) with mp 145ꢀ147 °C.
δH 0.81 (3H, s), 1.22 (3H, t, J 7.1), 1.28ꢀ1.62 (7H, m), 1.78ꢀ2.01 (3H,
m), 2.20ꢀ2.27 (1H, m), 2.32ꢀ2.47 (3H, m), 2.70 (2H, q, J 7.1),
2.84ꢀ2.88 (2H, m), 4.08ꢀ4.21 (2H, m), 5.04 (1H, s, br), 5.29 (1H, t,
J 6.8), 7.08 (1H, s), 7.20 (1H, s). HRMS (ESþ): m/z found 388.1932;
C22H30NO3Sþ (MþꢀOH) requires 388.1941. Anal. (C22H31O4NS)
C, H, N
2-Ethyl-3-O-benzyl-17-(1-ethenyl)estra-1,3,5(10)-triene 45a. A
solution of ethyltriphenylphosphonium iodide (3.75 g, 6.45 mmol) in
DMSO (25 mL) was treated with sodium hydride (420 mg, 10.5 mmol,
60% dispersion in mineral oil) and then brought to 100 °C for 0.25 h.
2-Ethyl-3-O-benzylestrone (1.85 g, 4.76 mmol) in DMSO (10 mL) was
then added to the orange reaction mixture, and heating was continued for
a further 16 h. The cooled reaction mixture was then poured onto
iceꢀwater (100 mL) and extracted with ether (3 ꢁ 100 mL). The organic
layers were then washed with water (3 ꢁ 100 mL), brine (10 mL), dried,
and evaporated. The crude product was purified by column chromatog-
raphy (hexane/ethyl acetate gradient 100ꢀ97%) to give the desired
alkene 45a, as a mixture of geometric isomers, as a clear colorless oil
(1.35 g, 71%) which showed significant resonances at δH 0.91 and 0.89
(3H, 2s, 18-CH3), 1.22 (3H, t, J 7.4, CH2Me), 1.70 (app dt, J 7.2 and 1.7,:
CHMe major isomer), 2.68 (2H, q, J 7.4, CH2Me), 2.74ꢀ2.90 (2H, m,
6-CH2), 4.98ꢀ5.25 (1H, m, CH both isomers), 5.40 (2H, s, OCH2), 6.64
(1H, s, ArH), 7.12 (1H, s, ArH), and 7.27ꢀ7.48 (5H, m). m/z (APþ) 401.6
(Mþ þ H, 100%) and 309.6 (65%). HRMS (ESþ) m/z found 401.2839;
C29H36O (Mþ þ H) requires 401.2834.
synthesis of compound 15. The mixture was then filtered through a pad
of Celite and evaporated to give 48a as a white solid (180 mg, 89%)
which was then crystallized from ethyl acetate/hexane to give white
needles, mp 197ꢀ200 °C. δH 0.64 (3H, s), 1.21 (3H, t, J 7.4), 1.24ꢀ1.90
(9H, m), 2.15 (3H, s), 2.12ꢀ2.40 (4H, m), 2.58 (2H, q, J 7.4), 2.60 (1H,
app t, J 9.4), 2.74ꢀ2.86 (2H, m), 4.72 (1H, s), 6.49 (1H, s), and 7.03
(1H, s). m/z (APꢀ) 325.6 (Mþ, 100%) and 267.5 (75%).
2-Ethyl-3-O-sulfamoyl-17β-acylestra-1,3,5(10)-triene 49a.
Compound 48a (80 mg, 0.25 mmol) was treated with sulfamoyl chloride
(0.6 mmol) in DMA (2 mL) as described for the synthesis of compound
16. The desired product 2-ethyl-3-O-sulfamoyl-17β-acylestrone was
purified by column chromatography (10% acetone in chloroform) to
give a white solid (95 mg, 95%). This material was crystallized from ethyl
acetate/hexane to give 49a as fine white needles (73 mg of first crop), mp
192ꢀ194 °C. δH 0.65 (3H, s, 18-CH3), 1.21 (3H, t, J 7.4), 1.24ꢀ1.93
(9H, m), 2.15 (3H, s), 2.15ꢀ2.40 (4H, m), 2.60 (1H, dd, J 9.4, 9.0), 2.69
(2H, q, J 7.4), 2.81ꢀ2.87 (2H, m), 4.93 (2H, s), 7.07 (1H, s), and 7.17
(1H, s). m/z (APꢀ) 404.5 (Mþ, 100%). HRMS (ESþ) m/z found
423.2316; C22H31SO4Nþ þ NH4 (or C22H35SO4N2þ) (Mþ þ NH4)
requires 423.2312.
2-Methoxy-3-O-benzyl-17-(1-ethenyl)estra-1,3,5(10)-triene
45b. A solution of ethyltriphenylphosphonium iodide (1.25 g, 3 mmol)
in DMSO (20 mL) was treated with sodium hydride (120 mg, 3 mmol,
60% dispersion in mineral oil) and then brought to 100 °C for 0.25 h.
2-Methoxy-3-O-benzylestrone (390 mg, 1 mmol) was then added to the
red reaction mixture, and heating was continued for a further 16 h. The
cooled reaction mixture was then poured onto iceꢀwater (50 mL) and
extracted with ether (3 ꢁ 30 mL). The organic layers were then washed
with water (3 ꢁ 30 mL), brine (10 mL), dried, and evaporated. The
crude product was purified by column chromatography (hexane/ethyl
acetate gradient 100ꢀ94%) to give the desired alkene 45b as a mixture of
geometric isomers (∼9:1) as a clear colorless oil (200 mg, 57%). δH 0.93
and 0.80 (3H, 2s), 1.25ꢀ2.54 (14 H, m including 1.70 (app dt, J 7.2,
1.7)), 2.68ꢀ2.84 (2H, m), 3.88 (3H, s), 5.08ꢀ5.23 (1H, m), 5.12
(2H, s), 6.64 (1H, s), 6.87 (1H, s), 7.26ꢀ7.48 (5H, m). m/z (ESþ)
425.2 (Mþ þ Na, 100%).
2-Ethyl-3-O-benzyl-17β-(1-hydroxyethyl) estra-1,3,5(10)-
triene 46a. To a room temperature solution of 45a (700 mg, 1.74
mmol, as a mixture of geometric isomers) was added borane THF
(16 mL, 1M). The mixture was stirred for 14 h at room temperature and
then treated with sodium hydroxide (20 mL, 10% aqueous) (causing
vigorous gas evolution) and then hydrogen peroxide (60 mL, 27.5%
aqueous). After 2 h of further stirring the THF was removed on a rotary
evaporator and the resultant mixture was extracted into ether (2 ꢁ
100 mL). The combined organic layers were then washed with water (2 ꢁ
100 mL) and brine (75 mL), dried, and evaporated to give a colorless oil.
The crude product was purified by column chromatography to give two
fractions. The second fraction (Rf = 0.22 in 15% ethyl acetate/hexane), a
clear colorless oil (350 mg, 48%), proved to be the desiredalcohol 46a as a
single diastereoisomer with 17β configuration at C-17 (likely (S)-config-
uration at C-20). δH 0.65 (3H, s), 1.21 (3H, t, J 7.4), 1.18ꢀ2.34 (21H, m,
including 1.26 (3H, d, J 6.2) and 1.20 (3H, t, J 7.4)), 2.66 (2H, q, J 7.4),
2.76ꢀ2.92 (2H, m), 3.69ꢀ3.79 (1H, m), 5.04 (2H, s), 6.63 (1H, s), 7.09
(1H, s), and 7.29ꢀ7.44 (5H, m). m/z (APþ) 419.6 (Mþ þ H, 100%)
and 401.6 (75%). HRMS (ESþ) m/z found 419.2945; C29H38O2 (Mþ þ
H) requires 419.2950.
2-Methoxy-3-O-benzyl-17β-(1-hydroxyethyl)estra-1,3,5(10)-
triene 46b. To an ice cold solution of 45b (160 mg, 0.39 mmol) in
THF (5 mL) was added boraneꢀTHF (5 mL, 5 mmol). The mixture
was stirred for 14 h at 0 °C, then treated with sodium hydroxide (5 mL,
10% aqueous) and hydrogen peroxide (5 mL, 30% aq). After the mixture
was further stirred for 1 h, the THF was removed on a rotary evaporator
and the resultant mixture was extracted into ether (2 ꢁ 30 mL). The
combined organic layers were then washed with water (2 ꢁ 20 mL) and
brine (25 mL), dried, and evaporated to give a colorless oil. The crude
product was purified by column chromatography (4:1 hexane/ethyl
acetate) to give the desired alcohol 46b as a white solid (98 mg, 59%). δH
0.66 (3H, s), 1.15ꢀ2.30 (17H, m including 1.24 (3H, s)), 2.68ꢀ2.80
(2H, m), 3.70ꢀ3.83 (1H, m), 3.85 (3H, s), 5.09 (2H, s), 6.61 (1H, s),
6.82 (1H, s), 7.26ꢀ7.45 (5H, m). m/z (ESþ) 443.6 (Mþ þ Na, 100%).
2-Methoxy-3-O-benzyl-17β-(acyl)estra-1,3,5(10)-triene 47b.
To a stirred, 0 °C solution of 46b (90 mg, 0.21 mmol) in dichloro-
methane (5 mL) was added DessꢀMartin periodinane (109 mg, 1.2
equiv, 0.26 mmol) in one portion. The mixture was stirred for 16 h and
then diluted with ether (75 mL) and sodium hydroxide (1 mL, 1 M
aqueous) and then stirred for a further 0.5 h prior to washing with water
(100 mL) and brine (100 mL), drying, and evaporating. The desired
ketone 47b was obtained as a white solid (70 mg, 80%). δH 0.64 (3H, s,
18-CH3), 1.20ꢀ1.95 (10H, m), 2.08ꢀ2.34 (6H, m including 2.14
(3H, s)), 2.60 (1H, dd, J 9.1, 8.6), 2.70ꢀ2.80 (2H, m), 3.85 (3H, s),
5.09 (2H, s), 6.61 (1H, s), 6.82 (1H, s), 7.26ꢀ7.45 (5H, s). m/z (ESþ)
441.6 (Mþ þ Na, 100%).
2-Ethyl-3-O-benzyl-17β-(acyl)estra-1,3,5(10)-triene 47a. To
a stirred, 0 °C solution of 46a (330 mg, 0.77 mmol) in dichloromethane
(20 mL) was added DessꢀMartin periodinane (392 mg, 1.2 equiv, 0.92
mmol) inone portion. Themixture was stirredovernight and then diluted
with ether (100 mL) and sodium hydroxide (2 mL, 1 M aqueous) and
then stirred for a further 0.5 h prior to washing with water (100 mL) and
brine (100 mL), drying, and evaporating. The desired ketone 47a was
isolated by adding hexane to the resultant oil which caused white needles
to form (280 mg, 87%), mp 134ꢀ135 °C (Rf = 0.45 in 4:1 hexane/ethyl
acetate). δH 0.65 (3H, s), 1.21 (3H, t, J 7.4), 1.25ꢀ2.40 (13H, m), 2.15
(3H, s), 2.66 (2H, q, J 7.4), 2.59ꢀ2.71 (1H, m), 2.76ꢀ2.92 (2H, m), 5.04
(2H, s), 6.63 (1H, s), 7.10 (1H, s), and 7.28ꢀ7.45 (5H, m). m/z (APþ)
417.6 (Mþ þ H, 100%).
2-Ethyl-3-O-hydroxy-17β-acylestra-1,3,5(10)-triene 48a. A
solution of 47a (260 mg, 0.62 mmol) in THF (3 mL) and methanol
(20 mL) was treated with Pd/C (10%, 50 mg) as described for the
2-Methoxy-3-O-hydroxy-17β-acylestra-1,3,5(10)-triene 48b.
Compound 47b (55 mg, 0.13 mmol) in THF (1 mL) and methanol
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(5 mL) was treated with Pd/C (10%, 15 mg) as described for the
synthesis of compound 15. The resultant solid was washed with a small
volume of ethyl acetate to remove colored impurities, and the resultant
white solidwas collectedby filtration anddried togive 48bas a white solid
(35 mg, 82%), mp 217ꢀ219 °C. δH 0.65 (3H, s), 1.20ꢀ1.91 (10H, m),
2.10ꢀ2.34 (6H, m including 2.14 (3H, s)), 2.60 (1H, dd, J 8.9, 8.1),
2.72ꢀ2.82 (2H, m), 3.89 (3H, s), 5.41 (1H, s, OH), 6.63 (1H, s), 6.77
(1H, s). m/z (ESꢀ) 327.6 (Mþ ꢀ H), 100%). HRMS (ESþ) m/z found
329.2122; C21H29O3þ (Mþ þ H) requires 329.2117.
compound 52 as a white powder (300 mg, 69%), mp 77ꢀ80 °C. δH 0.61
and 0.62 (3H, 2s), 1.18ꢀ1.60 (13H, m), 1.68ꢀ2.00 (4H, m), 2.12ꢀ2.28
(2H, m), 2.65ꢀ2.83 (2H, m), 3.78ꢀ3.87 (3H, m), 5.10 (2H, s), 6.61
(1H, s), 6.84 (1H, s), 7.26ꢀ7.45 (5H, m). HRMS (FABþ): m/z found
435.2880; C29H39O3þ (Mþ þ H) requires 435.2894.
2-Ethyl-3-benzyloxy-17β-(2-hydroxypropyl)estra-1,3,5(10)-
triene 58. Compound 5724 (390 mg, 0.94 mmol) was treated with
methylmagnesium bromide (3 M in diethyl ether, 0.5 mL, 1.5 mmol) in
THF (10 mL) at 0 °C as described for the synthesis of 52. Purification by
flash column chromatography (hexane/EtOAc 20:1 to 10:1) afforded
compound 58 as a white powder (310 mg, 77%), mp 66ꢀ69 °C. δH 0.61
and 0.63 (3H, s), 1.18ꢀ1.59 (17H, m), 1.73ꢀ1.96 (3H, m), 2.16ꢀ2.35
(2H, m), 2.66 (2H, q, J 7.4), 2.83 (2H, m), 3.84 (1H, m), 5.03 (2H, s), 6.63
(1H, s), 7.11 (1H, s), 7.27ꢀ7.46 (5H, m). HRMS (ESþ): m/z found
433.3093; C30H41O2þ (Mþ þ H) requires 433.3101.
2-Methoxy-3-O-sulfamoyl-17β-acylestra-1,3,5(10)-triene 49b.
Compound 48b (22 mg, 0.093 mmol) in DMA (1.5 mL) was added
treated with sulfamoyl chloride (0.65 mmol) as described for the
synthesis of compound 16. The desired sulfamate 49b was crystallized
from ethyl acetate/hexane to give fine white needles (15 mg, 40% first
crop), mp 178ꢀ180 °C. δH 0.66 (3H, s, 18-CH3), 1.30ꢀ2.35 (H, m
including 2.14 (3H, s)), 2.60 (1H, dd, J 9.0, 9.0), 2.82ꢀ2.90 (2H, m), 3.87
(3H, s), 4.97 (2H, s), 6.91 (1H, s), 7.02 (1H, s). m/z (ESꢀ) 406.6 (Mþ,
100%). HRMS (ESþ) m/z found 425.2105; C21H29SO5Nþ þ NH4 (or
C21H33SO5N2þ) (Mþ þ NH4) requires 425.2105.
2-Methoxy-3-benzyloxy-17β-(2-oxopropyl)estra-1,3,5(10)-
triene 53. A solution of 52 (250 mg, 0.58 mmol) in DCM (10 mL) was
stirred at 0 °C, and DessꢀMartin periodinane (293 mg, 0.69 mmol) was
added portionwise. The reaction mixture was stirred at 0 °C for 4 h. Then
diethyl ether (50 mL) and sodium hydroxide (1 M, 5 mL) were added,
and the solution was stirred at 0 °C for 1 h. The organic layer was
separated, washed with water and brine, then dried (MgSO4), filtered, and
concentrated in vacuo. Purification by flash column chromatography
(hexane/EtOAc 10:1 to 5:1) afforded compound 53 as a white powder
(165 mg, 66%), mp 120ꢀ121 °C. δH 0.62 (3H, s), 1.20ꢀ1.52 (7H, m),
1.73ꢀ2.02 (5H, m), 2.16 (3H, s), 2.20ꢀ2.31 (3H, m), 2.52 (1H, dd,
J 15.8, 3.6), 2.76ꢀ2.82 (2H, m), 3.85 (3H, s), 5.09 (2H, s), 6.61 (1H, s),
6.83 (1H, s), 7.28ꢀ7.45 (5H, m). HRMS (ESþ): m/z found 433.2731;
C29H37O3þ (Mþ þ H) requires 433.2737.
2-Methoxy-3-benzyloxy-17β-cyanomethylestra-1,3,5(10)-
triene 50. Diethyl cyanomethylphosphonate (3.55 g, 20 mmol) in THF
(60 mL) was treated with NaH (60% dispersion in mineral oil, 0.4 g, 20
mmol). 2-Methoxy-3-O-benzylestrone (3.94 g, 10 mmol) in THF
(20 mL) was added dropwise at room temperature, and the solution
was stirred for 18 h. The reaction mixture was cooled to 0 °C, and water
(30 mL) was added. The mixture was extracted with EtOAc. The organic
layer was washed with water and brine, then dried (MgSO4), filtered, and
concentrated in vacuo. Purification by flash column chromatography
(hexane/EtOAc 25:1 to 6:1) afforded the olefin (mixture of Z and E
isomers). The olefin was treated with Pd/C (10%, 200 mg) in THF
(15 mL) and methanol (50 mL) as described for the synthesis of
compound 15. The resultant suspension was filtered through Celite,
washed with EtOAc, and concentrated in vacuo. The residue was stirred
with potassium carbonate (2.8 g, 20 mmol) and benzyl bromide
(1.18 mL, 10 mmol) in DMF (20 mL) for 24 h at room temperature.
Water (50 mL) was added, and the mixture was extracted with EtOAc
(2 ꢁ 80 mL). The combined organics were washed with water and brine,
then dried (MgSO4), filtered, and concentrated in vacuo. Purification by
flash column chromatography (hexane/EtOAc 20:1 to 5:1) afforded
compound 50 as a white solid (3.45 g, 83% over three steps), mp
175ꢀ176 °C. δH 0.67 (3H, s), 1.21 (3H, t, J 7.4), 1.21ꢀ1.52 (7H, m),
1.75ꢀ1.88 (3H, m), 1.96ꢀ2.11 (2H, m), 2.17ꢀ2.41 (4H, m), 2.71ꢀ2.77
(2H, m), 3.85 (3H, s), 5.10 (2H, s), 6.61 (1H, s), 6.82 (1H, s), 7.25ꢀ7.45
(5H, m). LC/MS (ESþ): m/z 416.2 (Mþ þ H).
2-Ethyl-3-benzyloxy-17β-(2-oxopropyl)estra-1,3,5(10)-tri-
ene 59. Compound 58 (290 mg, 0.67 mmol) was treated with
DessꢀMartin periodinane (355 mg, 0.83 mmol) in DCM (20 mL) at
0 °C as described for the synthesis of compound 53. Purification by flash
column chromatography (hexane/EtOAc 10:1 to 5:1) afforded com-
pound 59 as a white powder (220 mg, 76%), mp 46ꢀ47 °C. δH 0.65
(3H, s), 1.23 (3H, t, J 7.3), 1.26ꢀ1.58 (7H, m), 1.65ꢀ2.05 (5H, m), 2.18
(3H, s), 2.20ꢀ2.41 (3H, m), 2.52ꢀ2.59 (1H, m), 2.69 (2H, q, J 7.3),
2.83ꢀ2.88 (2H, m), 5.06 (2H, s), 6.63 (1H, s), 7.13 (1H, s), 7.30ꢀ7.48
(5H, m). HRMS (ESþ): m/z found 431.2937; C30H39O2þ (Mþ þ H)
requires 431.2945.
2-Methoxy-3-hydroxy-17β-(2-oxopropyl)estra-1,3,5(10)-
triene 54. Compound 53 (155 mg, 0.36 mmol) was treated with Pd/C
(10%, 20 mg) in THF (5 mL) and methanol (15 mL) as described for
the synthesis of compound 15. Purification by flash column chroma-
tography (hexane/EtOAc 6:1) afforded compound 54 as a white powder
(105 mg, 86%), mp 177ꢀ178 °C. δH 0.62 (3H, s), 1.20ꢀ1.58 (7H, m),
1.72ꢀ2.02 (5H, m), 2.16 (3H, s), 2.19ꢀ2.32 (3H, m), 2.52 (1H,dd,
J 15.7, 3.9), 2.72ꢀ2.79 (2H, m), 3.84 (3H, s), 5.42 (1H, s), 6.63 (1H, s),
2-Methoxy-3-benzyloxyestra-1,3,5(10)-triene-17β-acetal-
dehyde 51. Compound 50 (1.5 g, 3.6 mmol) was treated with DIBAL
(1.5 M in THF, 7.2 mL, 10.8 mmol) in THF (30 mL) at 0 °C as
described for the synthesis of compound 38. After addition of HCl (2 M,
20 mL) the mixture was stirred for 0.5 h and then extracted with EtOAc
(3 ꢁ 30 mL). The combined organics were washed with water and brine,
dried (MgSO4), filtered, and concentrated in vacuo to afford compound
51 as a white powder (1.15 g, 77%), mp 142ꢀ144 °C. δH 0.64 (3H, s),
1.21ꢀ1.52 (7H, m), 1.75ꢀ2.05 (5H, m), 2.15ꢀ2.35 (3H, m),
2.49ꢀ2.57 (1H, m), 2.66ꢀ2.78 (2H, m), 3.85 (3H, s), 5.10 (2H, s),
6.61 (1H, s), 6.83 (1H, s), 7.26ꢀ7.45 (5H, m), 9.78 (1H, t, J 2.2).
2-Methoxy-3-benzyloxy-17β-(2-hydroxypropyl)estra-1,3,
5(10)-triene 52. A solution of 51 (420 mg, 1.0 mmol) in THF
(10 mL) was stirred under nitrogen, cooled to 0 °C, and treated with
methylmagnesium bromide (3 M in diethyl ether, 0.50 mL, 1.5 mmol).
The reaction mixture was stirred at 0 °C for 4 h. Ammonium chloride
(saturated, 5 mL) was added, and the mixture was extracted with EtOAc
(2 ꢁ 30 mL). The combined organics were washed with water and brine,
then dried (MgSO4), filtered, and concentrated in vacuo. Purification by
flash column chromatography (hexane/EtOAc 20:1 to 3:1) afforded
þ
6.77 (1H, s). HRMS (FABþ): m/z found 343.2264; C22H31O3
(Mþ þ H) requires 343.2268. Anal. (C23H30O3) C, H.
2-Ethyl-3-hydroxy-17β-(2-oxopropyl)estra-1,3,5(10)-triene
60. Compound 59 (200 mg, 0.46 mmol) was treated with Pd/C (10%, 30
mg) in THF (5 mL) and methanol (15 mL) as described for the synthesis
of compound 15. Purification by flash column chromatography (hexane/
EtOAc 5:1) afforded compound 60 as a white powder (145 mg, 92%), mp
125ꢀ126 °C. δH 0.63 (3H, s), 1.23 (2H, t, J 7.3), 1.27ꢀ1.53 (7H, m),
1.74ꢀ1.81 (2H, m), 1.84ꢀ1.93 (2H, m), 1.95ꢀ2.03 (1H, m), 2.18 (3H,
s), 2.16ꢀ2.24 (1H, m), 2.26ꢀ2.33 (1H, m), 2.52ꢀ2.58 (1H, m), 2.60
(2H, q, J 7.3), 2.74ꢀ2.82 (2H, m), 4.99 (1H, s), 6.51 (1þH, s), 7.05 (1H, s).
HRMS (FABþ): m/z found 340.2394; C23H32O2 (Mþ) requires
340.2402. Anal. (C23H32O2) C, H.
2-Methoxy-3-O-sulfamoyl-17β-(2-oxopropyl)estra-1,3,5(10)-
triene 55. Compound 54 (70 mg, 0.2 mmol) was treated with sulfamoyl
chloride (0.6 mmol) in DMA (1.0 mL) as described for the synthesis of
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ARTICLE
compound 16. Purification by flash column chromatography (hexane/
EtOAc 5:1 to 3:1) afforded compound 55 as a white powder (70 mg,
66%), mp 186ꢀ187 °C. δH (acetone-d6): 0.66 (3H, s), 1.22ꢀ1.56 (7H, m),
1.70ꢀ1.98 (5H, m), 2.10 (3H, s), 2.22ꢀ2.40 (3H, m), 2.57 (1H, dd, J 15.9,
3.7), 2.72ꢀ2.79 (2H, m), 3.82 (3H, s), 6.90 (2H, s, br), 6.90 (1H, s), 7.01
(1H, s). HRMS (ESþ): m/z found 422.2002; C22H32NO5Sþ (Mþ þ H)
requires 422.1996. Anal. (C22H31NO5S) C, H, N.
2-Methoxy-3-hydroxy-17β-(2-oxopropyl)estra-1,3,5(10)-
triene 54. Compound 64 (1.993 g, 4.0 mmol) was treated with TBAF
(1 M in THF, 4.4 mL, 4.4 mmol) in THF (7.6 mL) as described for the
synthesis of compound 20. Purification by flash column chromatogra-
phy (SiO2, 50 g, hexane/EtOAc 9:1 to 4:1) afforded compound 54 as
white solid (1.12 g, 82%). Data are as above.
2-Methoxy-3-O-sulfamoyl-17β-(2-oxopropyl)estra-1,3,5(10)-
triene 55. By use of the method described for the synthesis of 16,
compound 54 (1.028 g, 3.0 mmol) was reacted with sulfamoyl chloride
(9.0 mmol) in DMA (9.0 mL) at room temperature for 2 h. Purification
by flash column chromatography (SiO2, 60 g, chloroform/acetone 9:1)
and crystallization from diethyl ether afforded compound 55 as white
solid (1.026 g, 81%). Data are as above.
17β-(2-Hydroxypropyl)-2-methoxy-3-O-sulfamoylestra-1,
3,5(10)-triene 65. Sodium borohydride (15.5 mg, 0.4 mmol) was added
to 2-propanol (4.0 mL) under stirring. A solution of compound 55 (84 mg,
0.2 mmol) in THF (2.0 mL) was added dropwise, and the reaction mixture
was stirred at room temperature for 2 h. The mixture was then poured into
water (40 mL) and ammonium chloride (saturated, 10 mL) and extracted
with EtOAc (3 ꢁ 30 mL). The combined organics were dried (MgSO4),
filtered, and concentrated in vacuo. Purification by flash column chromatog-
raphy (SiO2, 10 g, chloroform/acetone 9:1) afforded compound 65 as a fluffy
white solid (63 mg, 75%), mp 170ꢀ172 °C. δH 0.51 (3H, s), 1.02ꢀ1.56
(15H, m), 1.59ꢀ1.70 (1H, m), 1.72ꢀ1.89 (3H, m), 2.59ꢀ2.73 (2H, m),
3.74 (3H, s), 6.26 (2H, s, br), 6.81 (1H, s), 6.93 (1H, s). LC/MS(ESꢀ):m/
z 422.43 (Mꢀ ꢀ H) and 422.50 (Mꢀ ꢀ H). HRMS (ESꢀ): m/z found
422.2013; C22H32NO5Sꢀ (Mꢀ ꢀ H) requires 422.2006.
2-Ethyl-3-O-sulfamoyl-17β-(2-oxopropyl)estra-1,3,5(10)-tri-
ene 61. Compound 60 (100 mg, 0.29 mmol) was treated with sulfamoyl
chloride (0.6 mmol) in DMA (1.0 mL) as described for the synthesis of
compound 16. Purification by flash column chromatography (hexane/
EtOAc 5:1 to 3:1) afforded compound 61 as a white powder (100 mg,
81%), mp 204ꢀ205 °C. δH (acetone-d6): 0.40 (3H, s), 0.94 (3H, t, J 7.3),
0.95ꢀ1.33 (7H, m), 1.51ꢀ1.77 (5H, m), 1.91 (3H, s), 1.99ꢀ2.12 (3H, m),
2.28ꢀ2.34 (1H, m), 2.45 (2H, q, J 7.3), 2.56ꢀ2.63 (2H, m), 6.27 (2H, s),
6.84 (1H, s), 6.92 (1H, s). HRMS (FABþ): m/z found 419.2123;
C23H33NO4Sþ (Mþ) requires 419.2130. Anal. (C23H33NO4S) C, H, N.
2-Methoxy-3-(triisopropylsilyloxy)estrone 62. 2-Methoxyes-
trone (1.801 g, 6.0 mmol) was treated with chlorotriisopropylsilane (1.389
g, 7.2 mmol) and imidazole (1.225 g, 18.0 mmol) in DMF (18 mL) as
described for the synthesis of compound 19. Compound 62 was isolated as
a pale yellow oil (3.31 g, >99%). δH (0.91 (3H, s), 1.03ꢀ1.10 (18H, m),
1.14ꢀ1.31 (3H, m), 1.32ꢀ1.69 (6H, m), 1.90ꢀ2.30 (5H, m), 2.31ꢀ2.39
(1H, m), 2.49 (1H, dd, J 18.2, 8.3), 2.68ꢀ2.87 (2H, m), 3.75 (3H, s), 6.56
(1H, s), 6.74 (1H, s). HRMS (ESþ): m/z found 457.3125; C28H45O3Siþ
(Mþ ꢀ H) requires 457.3133.
2-Methoxy-3-(triisopropylsilyloxy)-17β-(2-ethoxy-2-ox-
oethyl)estra-1,3,5(10)-triene 63. Triethyl phosphonoacetate
(3.624 g, 16.2 mmol) in THF (10 mL) was treated with sodium hydride
(60%, 661 mg, 16.5 mmol). Compound 62 (3.20 g; 5.8 mmol) in THF
(20 mL) was added dropwise, and the mixture was refluxed for 60 h. The
reaction mixture was cooled to 0 °C, poured into water (30 mL), and
extracted with DCM (4 ꢁ 50 mL). The combined organics were washed
with ammonium chloride (saturated, 50 mL) and brine, then dried
(MgSO4)), filtered, and concentrated in vacuo. Purification by flash
column chromatography (hexane to hexane/EtOAc 20:1) gave the
olefin as a pale yellow oil (4.68 g, >99%). δH 0.85 (3H, s), 1.02ꢀ1.09
(18H, m), 1.12ꢀ1.63 (12H, m), 1.74ꢀ2.04 (3H, m), 2.13ꢀ2.39 (2H,
m), 2.64ꢀ3.03 (4H, m), 3.74 (3H, s), 4.06ꢀ4.23 (2H, m), 5.57 (1H ꢁ
0.85, t, J 2.5, E-isomer), 5.66 (1H ꢁ 0.15, t, J 2.1, Z-isomer), 6.55 (1H, s),
6.74 (1H, s). HRMS (ESþ): m/z found 527.3540; C32H51O4Siþ
(Mþ ꢀ H) requires 527.3551. The above olefin (4.50 g, 5.6 mmol)
was treated with Pd/C (10%, 458 mg) in THF (18 mL) and MeOH
(6 mL) asdescribed forthe synthesis ofcompound 15. Compound 63was
obtained as a pale brown oil (4.31 g, 95%). δH 0.63 (3H, s), 1.02ꢀ1.10
(18H, m), 1.14ꢀ1.51 (12H, m), 1.59ꢀ2.02 (6H, m), 2.05ꢀ2.28 (3H, m),
2.39 (1H, dd, J 14.6, 4.7), 2.61ꢀ2.78 (2H, m), 3.74 (3H, s), 4.11 (2H, q, J
7.2), 6.54 (1H, s), 6.74 (1H, s). HRMS (ESþ): m/z found 529.3699;
C32H53O4Siþ (Mþ ꢀ H) requires 529.3708.
17β-(2-Hydroxyiminopropyl)-2-methoxy-3-O-sulfamoyles-
tra-1,3,5(10)-triene 66. Compound 55 (84 mg, 0.2 mmol), sodium
acetate (165 mg, 2.0 mmol), and hydroxylamine hydrochloride (141 mg,
2.0 mmol) were mixed together and dissolved in methanol (3.6 mL) and
water (0.4 mL). The reaction mixture was stirred at room temperature for
2 h and then concentrated in vacuo. Water (20 mL) and ammonium
chloride (saturated, 20 mL) were added, and the mixture was extracted
with EtOAc (2 ꢁ 30 mL). The combined organics were dried (MgSO4),
filtered, and concentrated in vacuo. Purification by flash column chroma-
tography (SiO2, 10 g, DCM/acetone 4:1) afforded compound 66 as a
white wax (mixture of Z and E isomers) (44 mg, 51%). δH (CDCl3/
DMSO-d6) 0.57 (3H ꢁ 0.75, s, E-isomer), 0.60 (3H ꢁ 0.25, s, Z-isomer),
1.09ꢀ1.51 (7H, m), 1.54ꢀ1.85 (6H, m), 1.77 (3H ꢁ 0.25, s, Z-isomer),
1.79 (3H ꢁ 0.75, s, E-isomer), 2.04ꢀ2.26 (3H, m), 2.63ꢀ2.74 (2H, m),
3.74 (3H, s), 6.22 (2H, s, br), 6.81 (1H, s), 6.94 (1H, s), 8.30 (1H, s, br).
LC/MS (ESꢀ): m/z 435.51 (Mꢀ ꢀ H). HRMS (ESꢀ): m/z found
435.1952; C22H31N2O5Sꢀ (Mꢀ ꢀ H) requires 435.1959.
2-Ethyl-3-benzyloxy-17β-(2-fluoroethyl)estra-1,3,5(10)-tri-
ene 67. A solution of compound 25 (0.84 g, 2 mmol) in THF (20 mL)
was cooled to 0 °C under nitrogen and treated with diethylaminosulfur
trifluoride (DAST) (0.40 mL, 3.0 mmol). The reactionmixture was stirred
at 0 °C for 4 h. Sodium bicarbonate (saturated, 10 mL) was added, and the
mixture was extracted with EtOAc (2 ꢁ 30 mL). The combined organics
were washed with water and brine, then dried (MgSO4), filtered, and
concentrated in vacuo. Purification by flash column chromatography
(hexane/EtOAc 50:1) afforded compound 67 as a white powder (0.42
g, 50%), mp 113ꢀ114 °C. δH 0.66 (3H, s), 1.30 (3H, t, J 7.4), 1.32ꢀ1.66
(9H, m), 1.81ꢀ1.87 (1H, m), 1.92ꢀ2.04 (4H, m), 2.25ꢀ2.33 (1H, m),
2.39ꢀ2.45 (1H, m), 2.76 (2H, q, J 7.4), 2.84ꢀ2.95 (2H, m), 4.45ꢀ4.53
(1H, m), 4.58ꢀ4.66 (1H, m), 5.12 (2H, s), 6.72 (1H, s), 7.20 (1H, s),
7.37ꢀ7.54 (5H, s). LC/MS (APCIþ): m/z 421.3 (Mþ þ H).
2-Methoxy-3-(triisopropylsilyloxy-17β-(2-oxopropyl)estra-
1,3,5(10)-triene 64. Tebbe reagent (0.5 M in toluene, 12.6 mL, 6.3
mmol) was added dropwise over 15 min into a solution of compound 63
(4.20 g, 5.2 mmol) in toluene (17.4 mL), THF (10 mL) and pyridine
(0.84 mL) at ꢀ78 °C. The reaction mixture was stirred at ꢀ78 °C for 1 h,
then warmed to 0 °C within 2 h. The reaction mixture was poured into HCl
(2 M, 400 mL), stirred vigorously at room temperature for 16 h, and
extracted with diethyl ether (3 ꢁ 100 mL). The combined organics were
dried (MgSO4), filtered, and concentrated in vacuo. Purification by flash
column chromatography (SiO2, 80 g, hexane/diethyl ether 9:1) afforded
compound 64 as a light brownish oil (2.504 g, 92% overall from 2-meth-
oxyestrone). δH 0.61 (3H, s), 1.02ꢀ1.10 (18H, m), 1.12ꢀ1.55 (11H, m),
1.71ꢀ2.04 (4H, m), 2.15 (3H, s), 2.17ꢀ2.35 (3H, m), 2.52 (1H, dd, J 15.7,
3.8), 2.62ꢀ2.82 (2H, m), 3.74 (3H, s), 6.54 (1H, s), 6.73 (1H, s). HRMS
(ESþ): m/z found 499.3607; C31H51O3Siþ (Mþ ꢀ H) requires 499.3602.
2-Ethyl-3-hydroxy-17β-(2-fluoroethyl)estra-1,3,5(10)-tri-
ene 68. Compound 67 (420 mg, 1.0 mmol) was treated with Pd/C
(10%, 30 mg) in THF (10 mL) and methanol (30 mL) as described for
the synthesis of compound 15. Purification by flash column chroma-
tography (hexane/EtOAc 20:1 to 15:1) afforded compound 68 as a
white powder (250 mg 76%), mp 138ꢀ139 °C. δH 0.63 (3H, s), 1.22
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Journal of Medicinal Chemistry
ARTICLE
(3H, t, J 7.4), 1.25ꢀ1.61 (10H, m), 1.73ꢀ1.98 (4H, m), 2.14ꢀ2.35 (2H,
m), 2.59 (2H, q, J 7.4), 2.77ꢀ2.88 (2H, m), 4.34ꢀ4.44 (1H, m),
4.50ꢀ4.60 (2H, m), 6.49 (1H, s), 7.05 (1H, s). HRMS (ESþ): m/z
found 331.2432; C22H32FOþ (Mþ þ H) requires 331.2437. Anal.
(C22H31FO) C, H.
’ ABBREVIATIONS USED
DAST, diethylaminosulfur trifluoride; VEGF, vascular endothe-
lial growth factor; E2bisMATE, estradiol-3,17-O,O-bis-sulfamate
2-Ethyl-3-O-sulfamoyl-17β-(2-fluoroethyl)estra-1,3,5(10)-
triene 69. Compound 68 (132 mg, 0.4 mmol) was treated with
sulfamoyl chloride (0.5 mmol) in DMA (1.0 mL) as described for the
synthesis of compound 16. Purification by flash column chromatogra-
phy (hexane/EtOAc 20:1 to 15:1) afforded compound 69 as a white
powder (110 mg, 66%), mp 152ꢀ153 °C. δH 0.63 (3H, s), 1.21 (3H, t,
J 7.4), 1.23ꢀ1.59 (10H, m), 1.73ꢀ1.97 (4H, m), 2.16ꢀ2.35 (2H, m),
2.68 (2H, q, J 7.4), 2.79ꢀ2.88 (2H, m), 4.34ꢀ4.42 (1H, m), 4.51ꢀ4.59
(1H, m), 4.90 (2H, s), 7.11 (1H, s), 7.24 (1H, s). HRMS (ESþ): m/z
found 410.2163; C22H33FNO3Sþ (Mþ þ H) requires 410.2165. Anal.
(C22H32FNO3S) C, H, N.
2-Ethyl-3-benzyloxy-17β-(2,2-difluoroethyl)estra-1,3,5(10)-
triene 70. Compound 57 (0.86 g, 2.0 mmol) was treated with diethy-
laminosulfur trifluoride (DAST) (0.80 mL, 6.0 mmol) in THF (20 mL) as
described for the synthesis of compound 67. Purification by flash column
chromatography (hexane/EtOAc 100:1) affordedcompound70 as a white
powder (0.42 g, 48%), mp 114ꢀ115 °C. δH 0.64 (3H, s), 1.22 (3H, t,
J 7.3), 1.26ꢀ2.09 (14H, m), 2.20ꢀ2.29 (1H, m), 2.31ꢀ2.40 (1H, m), 2.68
(2H, q, J 7.3), 2.78ꢀ2.89 (2H, m), 5.05 (2H, s), 5.84 (1H, ddt, J 57.3,
5.0, 3.7), 6.64 (1H, s), 7.12 (1H, s), 7.29ꢀ7.46 (5H, m). LC/MS
(APCIþ): m/z 439.1 (Mþ þ H).
’ REFERENCES
(1) Folkman, J.; Bach, M.; Rowe, J. W.; Davidoff, F.; Lambert, P.;
Hirsch, C.; Goldberg, A.; Hiatt, H. H.; Glass, J.; Henshaw, E. Tumor
angiogenesis—therapeutic implications. N. Engl. J. Med. 1971, 285 (21),
1182–1186.
(2) (a) Ferrara, N.; Hillan, K. J.; Gerber, H. P.; Novotny, W.
Discovery and development of bevacizumab, an anti-VEGF antibody
for treating cancer. Nat. Rev. Drug Discovery 2004, 3 (5), 391–400. (b)
Panares, R. L.; Garcia, A. A. Bevacizumab in the management of solid
tumors. Expert Rev. Anticancer Ther. 2007, 7 (4), 433–445.
(3) Wilhelm, S.; Carter, C.; Lynch, M.; Lowinger, T.; Dumas, J.;
Smith, R. A.; Schwartz, B.; Simantov, R.; Kelley, S. Discovery and
development of sorafenib: a multikinase inhibitor for treating cancer.
Nat. Rev. Drug Discovery 2006, 5 (10), 835–844.
(4) Faivre, S.; Demetri, G.; Sargent, W.; Raymond, E. Molecular
basis for sunitinib efficacy and future clinical development. Nat. Rev.
Drug Discovery 2007, 6 (9), 734–745.
(5) Kohne, C. H.; Lenz, H. J. Chemotherapy with targeted agents for
the treatment of metastatic colorectal cancer. Oncologist 2009, 14 (5),
478–488.
(6) Jain, R. K. Taming vessels to treat cancer. Sci. Am. 2008, 298 (1),
56–63.
2-Ethyl-3-hydroxy-17β-(2,2-difluoroethyl)estra-1,3,5(10)-
triene 71. Compound 70 (350 mg, 0.8 mmol) was treated with Pd/C
(10%, 40 mg) in THF (10 mL) and methanol (30 mL) as described for
the synthesis of compound 15. Purification by flash column chroma-
tography (hexane/EtOAc 50:1 to 40:1) afforded compound 71 as white
powder (240 mg, 86%), mp 84ꢀ86 °C. δH 0.62 (3H, s), 1.21 (3H, t,
J 7.3), 1.23ꢀ2.07 (14H, m), 2.16ꢀ2.24 (1H, m), 2.27ꢀ2.36 (1H, m),
2.59 (2H, q, J 7.3), 2.78 (2H, m), 4.58 (1H, s), 5.84 (1H, ddt, J 57.2, 9.2,
5.2), 6.49 (1H, s), 7.05 (1H, s). HRMS (ESþ): m/z found 349.2340;
C22H31F2Oþ (Mþ þ H) requires 349.2337. Anal. (C22H30F2O) C, H, N.
2-Ethyl-3-O-sulfamoyl-17β-(2,2-difluoroethyl)estra-1,3,5(10)-
triene 72. Compound 71 (175 mg, 0.5 mmol) was treated with
sulfamoyl chloride (0.5 mmol) in DMA (1.0 mL) as described for the
synthesis of compound 16. Purification by flash column chromatogra-
phy (hexane/EtOAc 15:1 to 10:1) afforded compound 72 as a white
powder (155 mg, 72%), mp 164ꢀ165 °C. δH 0.62 (3H, s), 1.20 (3H, t,
J 7.3), 1.23ꢀ2.08 (17H, m), 2.18ꢀ2.35 (2H, m), 2.68 (2H, q, J 7.3), 2.82
(2H, m), 4.93 (2H, s, br), 5.82 (1H, tt, J 57.0, 4.7), 7.06 (1H, s), 7.18
(1H, s). HRMS (ESþ): m/z found 428.2068; C22H32F2NO3Sþ (Mþ þ
H) requires 428.2065. Anal. (C22H31F2NO3S) C, H, N.
(7) Casanovas, O.; Hicklin, D. J.; Bergers, G.; Hanahan, D. Drug
resistance by evasion of antiangiogenic targeting of VEGF signaling in
late-stage pancreatic islet tumors. Cancer Cell 2005, 8 (4), 299–309.
(8) (a) Ebos, J. M. L.; Lee, C. R.; Cruz-Munoz, W.; Bjarnason, G. A.;
Christensen, J. G.; Kerbel, R. S. Accelerated metastasis after short-term
treatment with a potent inhibitor of tumor angiogenesis. Cancer Cell
2009, 15 (3), 232–239. (b) Paez-Ribes, M.; Allen, E.; Hudock, J.;
Takeda, T.; Okuyama, H.; Vinals, F.; Inoue, M.; Bergers, G.; Hanahan,
D.; Casanovas, O. Antiangiogenic therapy elicits malignant progression
of tumors to increased local invasion and distant metastasis. Cancer Cell
2009, 15 (3), 220–231.
(9) (a) Vredenburgh, J. J.; Desjardins, A.; Herndon, J. E.; Marcello,
J.; Reardon, D. A.; Quinn, J. A.; Rich, J. N.; Sathornsumetee, S.;
Gururangan, S.; Sampson, J.; Wagner, M.; Bailey, L.; Bigner, D. D.;
Friedman, A. H.; Friedman, H. S. Bevacizumab plus irinotecan in
recurrent glioblastoma multiforme. J. Clin. Oncol. 2007, 25 (30),
4722–4729. (b) Burger, R. A.; Sill, M. W.; Monk, B. J.; Greer, B. E.;
Sorosky, J. I. Phase II trial of bevacizumab in persistent or recurrent
epithelial ovarian cancer or primary peritoneal cancer: a gynecologic
oncology group study. J. Clin. Oncol. 2007, 25 (33), 5165–5171.
(10) Leese, M. P.; Leblond, B.; Smith, A.; Newman, S. P.; Di Fiore,
A.; De Simone, G.; Supuran, C. T.; Purohit, A.; Reed, M. J.; Potter,
B. V. L. 2-Substituted estradiol bis-sulfamates, multitargeted antitumor
agents: synthesis, in vitro SAR, protein crystallography, and in vivo
activity. J. Med. Chem. 2006, No. 49, 7683–7696.
’ ASSOCIATED CONTENT
S
Supporting Information. Elemental analysis results and
b
13C NMR data. This material is available free of charge via the
Internet at http://pubs.acs.org.
(11) Leese, M. P.; Hejaz, H. A. M.; Mahon, M. F.; Newman, S. P.;
Purohit, A.; Reed, M. J.; Potter, B. V. L. A-Ring-substituted estrogen-3-
O-sulfamates: potent multitargeted anticancer agents. J. Med. Chem.
2005, 48 (16), 5243–5256.
(12) Ireson, C. R.; Chander, S. K.; Purohit, A.; Perera, S.; Newman,
S. P.; Parish, D.; Leese, M. P.; Smith, A. C.; Potter, B. V. L.; Reed, R. J.
Pharmacokinetics and efficacy of 2-methoxyoestradiol and 2-methox-
yoestradiol-bis-sulphamate in vivo in rodents. Br. J. Cancer 2004, 90 (4),
932–937.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: þ44 1225 386639. Fax: þ44 1225 386114. E-mail:
B.V.L.Potter@bath.ac.uk.
(13) (a) Ho, Y. T.; Purohit, A.; Vicker, N.; Newman, S. P.; Robinson,
J. J.; Leese, M. P.; Ganeshapillai, D.; Woo, L. W. L.; Potter, B. V. L.; Reed,
M. J. Inhibition of carbonic anhydrase II by steroidal and non-steroidal
sulphamates. Biochem. Biophys. Res. Commun. 2003, 305 (4), 909–914.
(b) Newman, S. P.; Ireson, C. R.; Tutill, H. J.; Day, J. M.; Parsons,
M. F. C.; Leese, M. P.; Potter, B. V. L.; Reed, M. J.; Purohit, A. The role of
’ 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.
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Journal of Medicinal Chemistry
ARTICLE
17 beta-hydroxysteroid dehydrogenases in modulating the activity of
2-methoxyestradiol in breast cancer cells. Cancer Res. 2006, 66 (1),
324–330.
(28) Hartley, R. C.; Li, J. F.; Main, C. A.; McKiernan, G. J. Titanium
carbenoid reagents for converting carbonyl groups into alkenes. Tetra-
hedron 2007, 63 (23), 4825–4864.
(14) Elger, W.; Schwarz, S.; Hedden, A. M.; Reddersen, G.; Schneider,
B. Sulfamates of various estrogens are prodrugs with increased systemic
and reduced hepatic estrogenicity at oral application. J. Steroid Biochem.
Mol. Biol. 1995, 55 (3ꢀ4), 395–403.
(29) Tsuchiya, T. Chemistry and developments of fluorinated
carbohydrates. Adv. Carbohydr. Chem. Biochem. 1990, 48, 91–277.
(30) Chander, S. K.; Foster, P. A.; Leese, M. P.; Newman, S. P.;
Potter, B. V. L.; Purohit, A.; Reed, M. J. In vivo inhibition of angiogenesis
by sulphamoylated derivatives of 2-methoxyoestradiol. Br. J. Cancer
2007, 96 (9), 1368–1376.
(31) Laurence, C.; Brameld, K. A.; Graton, J.; Le Questel, J. Y.;
Renault, E. The pK(BHX) database: toward a better understanding of
hydrogen-bond basicity for medicinal chemists. J. Med. Chem. 2009, 52
(14), 4073–4086.
(15) Reed, M. J.; Purohit, A.; Newman, S. P.; Potter, B. V. L. Steroid
sulfatase: molecular biology, regulation and inhibition. Endocr. Rev.
2005, 26 (2), 171–202.
(16) Jourdan, F.; Leese, M. P.; Dohle, W.; Hamel, E.; Ferrandis, E.;
Newman, S. P.; Purohit, A.; Reed, M. J.; Potter, B. V. L. Synthesis,
antitubulin, and antiproliferative SAR of analogues of 2-methoxyestra-
diol-3,17-O,O-bis-sulfamate. J. Med. Chem. 2010, 53 (7), 2942–2951.
(17) (a) Newman, S. P.; Foster, P. A.; Ho, Y. T.; Day, J. M.;
Raobaikady, B.; Kasprzyk, P. G.; Leese, M. P.; Potter, B. V. L.; Reed,
M. J.; Purohit, A. The therapeutic potential of a series of orally bioavailable
antiangiogenic microtubule disruptors as therapy for hormone-indepen-
dent prostate and breast cancers. Br. J. Cancer 2007, 97 (12), 1673–1682.
(b) Newman, S. P.; Foster, P. A.; Stengel, C.; Day, J. M.; Ho, Y. T.; Judde,
J. G.; Lassalle, M.; Prevost, G.; Leese, M. P.; Potter, B. V. L.; Reed, M. J.;
Purohit, A. STX140 is efficacious in vitro and in vivo in taxane-resistant
breast carcinoma cells. Clin. Cancer Res. 2008, 14 (2), 597–606.
(18) MacCarthy-Morrogh, L.; Townsend, P. A.; Purohit, A.; Hejaz,
H. A. M.; Potter, B. V. L.; Reed, M. J.; Packham, G. Differential effects of
estrone and estrone-3-O-sulfamate derivatives on mitotic arrest, apop-
tosis, and microtubule assembly in human breast cancer cells. Cancer Res.
2000, 60 (19), 5441–5450.
(32) Hollingshead, M.; Alley, M. C.; Camalier, R. F.; Abbott, B. J.;
Mayo, J. G.; Malspeis, L.; Grever, M. R. In vivo cultivation of tumor cells
in hollow fibres. Life Sci. 1995, 57, 131–141.
(33) (a) Passaniti, A.; Taylor, R. M.; Pili, R.; Guo, Y.; Long, P. V.;
Haney, J. A.; Pauly, R. R.; Grant, D. S.; Martin, G. R. Methods in
laboratory investigation—a simple, quantitative method fot assessing
angiogenesis and antiangiogenic agents using reconstituted base-mem-
brane, heparin, and fibroblast growth-factor. Lab. Invest. 1992, 67 (4),
519–528. (b) Prewett, M.; Huber, J.; Li, Y. W.; Santiago, A.; O’Connor,
W.; King, K.; Overholser, J.; Hooper, A.; Pytowski, B.; Witte, L.; Bohlen,
P.; Hicklin, D. J. Antivascular endothelial growth factor receptor (fetal
liver kinase 1) monoclonal antibody inhibits tumor angiogenesis and
growth of several mouse and human tumors. Cancer Res. 1999, 59 (20),
5209–5218.
(34) Workman, P.; Twentyman, P.; Balkwill, F.; Balmain, A.;
Chaplin, D.; Double, J.; Embleton, J.; Newell, D.; Raymond, R.; Stables,
J.; Stephens, T.; Wallace, J. United Kingdom Co-Ordinating Committee
on Cancer Research (UKCCCR) guidelines for the welfare of animals in
experimental neoplasia (second edition). Br. J. Cancer 1998, 77 (1),
1–10.
(19) Purohit, A.; Vernon, K. A.; Hummelinck, A. E. W.; Woo,
L. W. L.; Hejaz, H. A. M.; Potter, B. V. L.; Reed, M. J. The development
of A-ring modified analogues of oestrone-3-O-sulphamate as potent
steroid sulphatase inhibitors with reduced oestrogenicity. J. Steroid
Biochem. Mol. Biol. 1998, 64 (5ꢀ6), 269–275.
(20) Purohit, A.; Hejaz, H. A. M.; Walden, L.; MacCarthy-Morrogh,
L.; Packham, G.; Potter, B. V. L.; Reed, M. J. The effect of 2-methox-
yoestrone-3-O-sulphamate on the growth of breast cancer cells and
induced mammary tumours. Int. J. Cancer 2000, 85 (4), 584–589.
(21) Leese, M. P.; Newman, S. P.; Purohit, A.; Reed, M. J.; Potter,
B. V. L. 2-Alkylsulfanyl estrogen derivatives: synthesis of a novel
class of multi-targeted anti-tumour agents. Bioorg. Med. Chem. Lett.
2004, 14 (12), 3135–3138.
(35) Appel, R.; Berger, G. Uber das hydrazidosulfamid (on
hydrazidosulfamide). Chem. Ber. 1958, 91, 1339–1341.
(36) Woo, L. W. L.; Lightowler, M.; Purohit, A.; Reed, M. J.; Potter,
B. V. L. Heteroatom-substituted analogues of the active-site directed
inhibitor estra-1,3,5(10)-trien-17-one-3-sulphamate inhibit estrone sul-
phatase by a different mechanism. J. Steroid Biochem. Mol. Biol. 1996, 57
(1ꢀ2), 79–88.
(22) Leese, M. P.; Leblond, B.; Newman, S. P.; Purohit, A.; Reed,
M. J.; Potter, B. V. L. Anti-cancer activities of novel D-ring modified
2-substituted estrogen-3-O-sulfamates. J. Steroid Biochem. Mol. Biol.
2005, 94 (1ꢀ3), 239–251.
(23) Leese, M. P.; Jourdan, F. L.; Gaukroger, K.; Mahon, M. F.;
Newman, S. P.; Foster, P. A.; Stengel, C.; Regis-Lydi, S.; Ferrandis, E.; Di
Fiore, A.; De Simone, G.; Supuran, C. T.; Purohit, A.; Reed, M. J.; Potter,
B. V. L. Structureꢀactivity relationships of C-17 cyano-substituted
estratrienes as anticancer agents. J. Med. Chem. 2008, 51 (5), 1295–1308.
(24) Jourdan, F.; Bubert, C.; Leese, M. P.; Smith, A.; Ferrandis, E.;
Regis-Lydi, S.; Newman, S. P.; Purohit, A.; Reed, M. J.; Potter, B. V. L.
Effects of C-17 heterocyclic substituents on the anticancer activity of
2-ethylestra-1,3,5(10)-triene-3-O-sulfamates: synthesis, in vitro evalua-
tion and computational modelling. Org. Biomol. Chem. 2008, 6 (22),
4108–4119.
(25) Bubert, C.; Leese, M. P.; Mahon, M. F.; Ferrandis, E.; Regis-
Lydi, S.; Kasprzyk, P. G.; Newman, S. P.; Ho, Y. T.; Purohit, A.; Reed,
M. J.; Potter, B. V. L. 3,17-Disubstituted 2-alkylestra-1,3,5(10)-trien-3-ol
derivatives: synthesis, in vitro and in vivo anticancer activity. J. Med.
Chem. 2007, 50, 4431–4443.
(26) Yan, T. H.; Tsai, C. C.; Chien, C. T.; Cho, C. C.; Huang, P. C.
Dichloromethane activation. Direct methylenation of ketones and
aldehydes with CH2Cl2 promoted by Mg/TiCl4/THF. Org. Lett.
2004, 6 (26), 4961–4963.
(27) Okada, M.; Iwashita, S.; Koizumi, N. Efficient general method
for sulfamoylation of a hydroxyl group. Tetrahedron Lett. 2000, 41 (36),
7047–7051.
4879
dx.doi.org/10.1021/jm200483x |J. Med. Chem. 2011, 54, 4863–4879