Effects of altering the electronics of 2-methoxyestradiol on cell proliferation, on cytotoxicity in human cancer cell cultures, and on tubulin polymerization

Article abstract of DOI:10.1021/jm049647a

A series of new analogues of 2-methoxyestradiol (1) were synthesized to further elucidate the relationships between structure and activity. The compounds were designed to diminish the potential for metabolic deactivation at positions 2 and 17 and were analyzed as inhibitors of tubulin polymerization and for cytotoxicity. 17α-Methyl-β-estradiol (30), 2-propynyl-17α- methylestradiol (39), 2-ethoxy-17-(1′-methylene)estra-1,3,5(10)-triene-3- ol (50) and 2-ethoxy-17α-methylestradiol (51) showed similar or greater tubulin polymerization inhibition than 2-methoxyestradiol (1) and contained moieties that are expected to inhibit deactivating metabolic processes. All of the compounds tested were cytotoxic in the panel of 55 human cancer cell cultures, and generally, the derivatives that displayed the most activity against tubulin were also the most cytotoxic.

Full text of DOI:10.1021/jm049647a

5126
J . Med. Chem. 2004, 47, 5126-5139
Effects of Alter in g th e Electr on ics of 2-Meth oxyestr a d iol on Cell P r olifer a tion ,
on Cytotoxicity in Hu m a n Ca n cer Cell Cu ltu r es, a n d on Tu bu lin P olym er iza tion
Allison B. Edsall,† Arasambattu K. Mohanakrishnan,† Donglai Yang,† Philip E. Fanwick,‡ Ernest Hamel,§
Arthur D. Hanson,| Gregory E. Agoston,| and Mark Cushman*,†
Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmacal Sciences, and
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, Screening Technologies Branch,
Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick,
National Institutes of Health, Frederick, Maryland 21702, and EntreMed, Inc., Rockville, Maryland 20850
Received May 11, 2004
A series of new analogues of 2-methoxyestradiol (1) were synthesized to further elucidate the
relationships between structure and activity. The compounds were designed to diminish the
potential for metabolic deactivation at positions 2 and 17 and were analyzed as inhibitors of
tubulin polymerization and for cytotoxicity. 17R-Methyl-â-estradiol (30), 2-propynyl-17R-
methylestradiol (39), 2-ethoxy-17-(1′-methylene)estra-1,3,5(10)-triene-3-ol (50) and 2-ethoxy-
17R-methylestradiol (51) showed similar or greater tubulin polymerization inhibition than
2-methoxyestradiol (1) and contained moieties that are expected to inhibit deactivating
metabolic processes. All of the compounds tested were cytotoxic in the panel of 55 human cancer
cell cultures, and generally, the derivatives that displayed the most activity against tubulin
were also the most cytotoxic.
In tr od u ction
The endogenous estrogen metabolite 2-methoxyestra-
diol (1, 2ME2), formed by hepatic cytochrome P450
2-hydroxylation of â-estradiol and 2-O-methylation via
catechol O-methyltranseferase, has generated signifi-
cant interest as a potential anticancer agent resulting
from its potent inhibition of tumor vasculature and
tumor cell growth.1-4 Many preclinical studies have
demonstrated that solid tumor growth is dependent
upon angiogenesis, the growth of new blood vessels;
therefore, the significant antiangiogenic activity (IC50
100 nM) and tubulin polymerization inhibition of 2ME2
in vivo are of considerable therapeutic value and have
warranted further investigation in clinical trials.4-9
Numerous studies have elucidated possible mecha-
nisms responsible for the cytotoxic effects of 2ME2.
Cytotoxicity has been associated with uneven chromo-
some distribution, inhibition of mitosis, and a quantita-
tive increase in abnormal metaphases.5,6 Competitive
binding studies with [3H]colchicine indicated that the
inhibitory effect of 2ME2 on tubulin polymerization was
Various metabolic processes, similar to those that
mediated through the colchicine binding site on tubu-
metabolize â-estradiol and other steroid hormones in
vivo, are believed to generate inactive metabolites of
2ME2.13,14 Oxidation of the position 17 alcohol of estra-
diol to estrone via 17-â-hydroxysteroid dehydrogenase
and conjugation of the position 3 and 17 hydroxyl
moieties to form a sulfate or glucuronide are two main
routes for metabolic deactivation and steroid clear-
ance.13 Oxidation of the 17-hydroxyl moiety is believed
to decrease the potency of 2ME2 in vivo since 2-meth-
oxyestrone displayed 1% of the growth inhibitory po-
tency of 2ME2 in endothelial cells.4,15 Demethylation of
the 2-methyl ether could also be detrimental to the
activity of 2ME2 because the resulting 2,3-catechol may
oxidize to a quinone, a species that has been shown to
form DNA adducts.16,17 More useful analogues of 2ME2
lin.10,11 Although the antiangiogenic mechanism of ac-
tion of 2ME2 has not been thoroughly elucidated,
disruption of microtubule polymerization resulted in the
inhibition of hypoxia-inducible factor-1 (HIF) at the
posttranscriptional level.12 This dysregulation of HIF
downstream of the 2ME2/tubulin interaction inhibited
the transcriptional activation of HIF response genes
including vascular endothelial growth factor (VEGF), a
major component of angiogenesis.12
* To whom correspondence should be addressed. Tel. (765) 494-1465.
Fax (765) 494-6790. E-mail: cushman@pharmacy.purdue.edu.
† Department of Medicinal Chemistry and Molecular Pharmacology,
Purdue University.
‡ Department of Chemistry, Purdue University.
§ Screening Technologies Branch, NIH.
| EntreMed, Inc.
10.1021/jm049647a CCC: $27.50 © 2004 American Chemical Society
Published on Web 09/16/2004

Altering the Electronics of 2-Methoxyestradiol
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5127
Sch em e 1a
should retain its inhibitory effects on tubulin polymer-
ization and cell growth while addressing these metabolic
issues.
Previous investigations have elucidated structural
similarities between colchicine and 2ME2 that contrib-
ute to their ability to inhibit tubulin polymeriza-
tion.10,18,19 Most importantly, the A-ring of 2ME2 prob-
ably corresponds to the C-ring of colchicine (2), and the
C- and D-rings of 2ME2 are functionally equivalent to
the A-ring of colchicine. Researchers have focused on
changing substituents at the 2- and 6-positions of
2-methoxyestradiol to increase cytotoxicity and tubulin
polymerization inhibition, and their efforts have pro-
duced 2-((E)-1′-propenyl)estradiol (3), 2-ethoxyestradiol
(4), and 2-(1′-propynyl)estradiol (5).20-22 Additionally,
the 17-position contributes significantly to the antican-
cer activity and pharmacokinetic profile of 2ME2.4,15,23
The goal of the present research was to further elucidate
the structural and electronic requirements necessary to
inhibit tubulin polymerization, increase the cytotoxicity,
and enhance the metabolic stability of analogues of
2ME2. Specifically, we have designed and synthesized
unique analogues with modifications at positions 2 and
17 in an attempt to maintain pharmacological activity
and decrease metabolic deactivation.
Ch em istr y. Friedel-Crafts acylation of â-estradiol
(6) using aluminum chloride and acetyl chloride gave a
mixture of three products, which, when followed by
workup with potassium hydroxide and methanol, af-
forded the acetylated product 7 (Scheme 1).23 Reaction
of 7 with pyridine and acetic anhydride yielded 8.
Treatment of 8 with [bis(2-methoxyethyl)amino]sulfur
trifluoride (BEAST) and ethanol in a bomb reactor
provided the difluoroethyl analogue 9. All attempts to
generate 10 following deprotection of 9 were unsuccess-
ful and, instead, yielded the acetyl derivative 7. How-
ever, the diacetate intermediate 9 was still of interest
as a possible prodrug of compound 10. â-Estradiol was
also methylated using sodium hydride and iodomethane
to form 11. Formation of the mixed anhydride derived
from acetylation of propionic acid with trifluoroacetic
anhydride, followed by reaction with alumina and 11,
afforded the propionyl derivative 12. Deprotection of 12
with in situ-generated iodotrimethylsilane provided
compound 13.
a
Reagents and conditions: (a) (1) AcCl, AlCl3, 0 °C to room
temperature (25 h); (2) KOH, MeOH (10 h). (b) Ac2O, Pyr (20 h).
(c) BEAST, EtOH, 70 °C (11 h). (d) NaH, MeI, THF, 0 °C-rt (24
h). (e) Alumina, CH3CH2COOH, (CF3CO)2O (15 h). (f) NaI, TMSCl,
CH3CN, 0 °C (48 h).
Sch em e 2a
Ullmann coupling of 2-iodoestradiol (14),24 prepared
according to published methods, with copper and per-
fluoroethyl iodide in a bomb reactor gave the perfluo-
roethyl congener 15, a relatively stable deprotected
alternative to the unstable compound 10 (Scheme 2).
Protection of 14 with imidazole and tert-butyldimeth-
ylsilyl chloride, followed by Negishi coupling using
allylmagnesium bromide, zinc bromide, and tetrakis-
(triphenylphosphine)palladium gave 17.24 Deprotection
of 17 with dilute hydrochloric acid using sonication gave
the desired diol 18.
Compounds 19-2425-27 and 2728 were prepared ac-
cording to literature methods with the exception that
22 was synthesized using sodium hydride as the base
in tetrahydrofuran rather than potassium hydroxide in
dimethyl sulfoxide (Schemes 3 and 4). 2-Formylestradiol
(24) was reacted with cyclopropylamine in the presence
a
Reagents and conditions: (a) Cu, CF3CF2I, DMSO, 0-80 °C
(16 h). (b) TBDMSCl, DMF, imidazole, rt (16 h). (c) (1) CH2dCH-
CH2MgBr, ZnBr2, THF, rt (5 min); (2) Pd(PPh3)4, 16, rt (12 h). (d)
HCl, EtOH, 2-PrOH (3 h).
of magnesium sulfate and with 3-N,N-dimethylamino-
propylamine to provide compounds 25 and 26, respec-
tively.
Estrone (28) was reacted with BEAST to provide the
17,17-difluoro analogue 29 and with methylmagnesium

5128 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Edsall et al.
Sch em e 3a
Sch em e 5a
a
Reagents and conditions: (a) (1) sec-BuLi, THF, -78 °C (2 h);28
(2) DMF, -78 °C-rt (12 h). (b) NaBH4, MeOH, 23 °C, (2.5 h).25 (c)
NaH, MeI, THF, 0 °C-rt (7 h). (d) PPTS, MeOH, reflux (18 h). (e)
HCl, MeOH, rt.
Sch em e 4a
a
Reagents and conditions: (a) BEAST, CH2Cl2, EtOH (62 h).
(b) MeMgBr, THF, PhCH3, 0 °C-rt (8 h). (c) DIEA, MOMCl, THF,
0 °C-reflux (24 h). (d) MeMgBr, THF, 0 °C-rt (20 h). (e) (1) NaH,
DMF, 0 °C (15 min); (2) BnBr, 0 °C-rt (6 h). (f) (1) sec-BuLi, -78
to -30 °C (5 h); (2) (MeO)3B, -78 °C-rt (8 h); (3) 30% H2O2, NaOH
(aq), 0 °C (8 h). (g) K2CO3, MeI, (CH3)2CO, rt (24 h). (h) HCl,
MeOH, 2-PrOH, rt (6 h).
a
Reagents and conditions: (a) Cyclopropylamine, MgSO4, rt (8
h). (b) 3-N,N-Dimethylaminopropylamine, reflux (1 h). (c) LiAlH4,
THF, 0 °C-rt (12 h).25
coupling of 38 with tributylpropynylstannane and tet-
rakis(triphenylphosphine)palladium yielded the alkyne
39.
bromide to create 17R-methylestradiol (30, Scheme 5).
The methoxymethyl-protected derivative 31, formed by
reacting estrone with diisopropylethylamine and chlo-
romethylmethyl ether, was alkylated with methylmag-
nesium bromide to yield 32. Benzyl protection of 32
yielded 33. When 33 was reacted with sec-butyllithium
followed by addition of trimethyl borate and hydrogen
peroxide, an unexpected Wittig rearrangement occurred
to form compound 34. The phenol of compound 34 was
methylated using potassium carbonate and iodomethane.
The stereochemistry of 35, determined via X-ray crys-
tallography (Figure 1), showed that the product had
retained the original C-17 stereochemistry of 33 during
the radical anion rearrangment to form the new C-17-
C-17(1′) linkage. Removal of the methoxymethyl pro-
tecting group of 35 with concentrated hydrochloric acid
in methanol provided the phenol 36.
Compound 40 was isolated by reaction of 19 with sec-
butyllithium, followed by introduction of trimethylbo-
rate, and then hydrogen peroxide (Scheme 7). Conver-
sion of 40 to 2-methoxyestrone (41) was completed by
methylation and deprotection using known methods
followed by an Oppenauer oxidation with aluminum
isopropoxide and cyclohexanone.29 Potassium carbonate
and chloroacetone were reacted with 40 to generate,
upon stirring in hydrochloric acid, the hemiacetal 42.
Reaction of intermediate 42 with hydroxylamine hydro-
chloride at reflux yielded the oxime 43. 2-Methoxy-
estrone (41) was benzyl protected using potassium
carbonate and methanol to produce 44, which was
subsequently reacted with trifluoromethyltrimethylsi-
lane and a catalytic amount of tetrabutylammonium
fluoride generating intermediate 45. Deprotection of 45
by catalytic hydrogenation, followed by treatment with
acid, resulted in compound 46. Alkylation of 2-methoxy-
Compound 32 was selectively deprotonated with sec-
butyllithium and iodinated with iodine to provide
intermediate 37 (Scheme 6). Deprotection of 37 with
concentrated hydrochloric acid and subsequent Stille

Altering the Electronics of 2-Methoxyestradiol
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5129
F igu r e 1. Crystal structure of 17-â-(S)-(hydroxyphenylmethyl)-2-methoxy-3-O-methoxymethyl-17R-methyl-1,3,5(10)-estratriene
(35).
Sch em e 6a
The antiproliferative activities of the synthesized
compounds were assessed also in the MDA-MB-231
breast cancer cell line and the human umbilical vein
endothelial cell (HUVEC) line (Table 2). The breast
carcinoma cell line MDA-MB-231 was chosen as an
initial screen for antiproliferative activity in tumor cells
because it has been shown that breast carcinomas are
among the most sensitive cell lines to 2ME2 (NCI cell
screen data), and it is estrogen receptor negative.
Additionally, this cell line is used as an orthotopic tumor
model for lead compounds that are screened in vivo.
With the exception of compounds 30, 39, 50, and 51,
the position 2 and 17 derivatives had little or no activity
as tubulin polymerization inhibitors. The inactivity of
7, 9, 13, 25, 26, 29, 36, and 43 is reasonably consistent
with previously established data indicating that branch-
ing of the position 2 side chain and steric bulk at
position 2 and position 17 are not well tolerated.
However, the inactivity of some of the other compounds
containing favorable steric features, specifically 15, 18,
and 23, was surprising since previously published
results have indicated that straight chain substituents
at position 2 containing two or three non-hydrogen
atoms conferred maximum tubulin polymerization
inhibition.20-23,26
a
Reagents and conditions: (a) (1) sec-BuLi, THF, cyclohexane,
-78 °C (2 h); (2) I2, -78 °C-rt (6 h). (b) HCl, THF, rt (6 h). (c)
Tributylpropynylstannane, Pd(PPh3)4, THF, reflux (10 h).
estrone (41) using methylmagnesium bromide yielded
analogue 47, while fluorination of 41 with BEAST
provided the 17,17-difluoro derivative 48.
Oppenauer oxidation of 2-ethoxyestradiol (4) produced
49 (Scheme 8). Utilizing methyltriphenylphosphonium
iodide and the dimsyl anion, the Wittig reaction suc-
cessfully converted 49 to the alkene 50. A Grignard
reaction of methylmagnesium bromide with 2-ethox-
yestrone (49) provided the target compound 51.
Because difluoromethylene moieties have been used
previously as isosteric replacements for the oxygen
atoms of ethers, compounds 9 and 15 were synthesized
to inhibit the potential for oxidative O-demethylation
of 2-methoxyestradiol.31 The acetate protecting groups
of 9 could not be deprotected without reversion to
compound 7, so 15 was designed as a stable, sterically
similar alternative to 9.32 2-Perfluoroethylestradiol (15)
was also expected to be similar in size and shape to
2-methoxyestradiol.21 However, compound 15 displayed
no activity as an inhibitor of tubulin polymerization. The
surprising inactivity of this compound and the previ-
ously reported 2-cyanoestradiol22 indicate that, while
steric effects are important characteristics for inhibiting
tubulin polymerization, electronic effects are also a
component that contributes to the activity of 2-meth-
oxyestradiol analogues.
Biologica l Resu lts a n d Discu ssion
The antiproliferative activities of the new 2ME2
analogues were determined in the NCI screen, in which
the activity of each compound was examined using
approximately 55 different human cancer cell lines of
diverse tumor origins. The antiproliferative assays
provided individual cell line GI50 values and mean graph
midpoints (MGMs). The MGM values were calculated
by averaging the GI50 values for all the cell lines tested,
where the GI50 values below and above the tested
concentration range (10-4 to 10-8 M) were taken as the
minimum (10-8 M) and maximum (10-4 M) analogue
concentrations used in the screen.30
The analogues were also evaluated for their inhibitory
effects on purified bovine brain tubulin using assay
conditions as described previously.10 The tubulin po-
lymerization inhibition values, representative cell line
antiproliferative GI50 values and MGM values are
compiled in Table 1.
To illustrate the electronic differences between select
molecules, electron density contours (Figures 2 and 3)
were examined using the molecular modeling program
Sybyl (Tripos, Inc., St. Louis, MO). The contours are

5130 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Edsall et al.
Sch em e 7a
Sch em e 8a
a
Reagents and conditions: (a) Al(i-PrO)3, cyclohexanone, tolu-
ene, reflux (20 h). (b) (1) NaH, DMSO, rt - 62 °C (1.5 h); (2)
CH3P(Ph)3I, rt (30 min); (3) 49 (18 h). (c) MeMgBr, THF, rt (3 h).
of 2-(2′,2′,2′-trifluoroethoxy)estra-1,3,5(10)-triene-3,17â-
diol (MGM 2.6 µM; IC50 1.5 µM)20 was more potent than
2ME2, indicating that the electronic withdrawing effects
imposed upon the A-ring by the perfluoroethyl substitu-
ent, rather than the surface polarity, caused the inactiv-
ity of 15. The visible differences in electron density
between 2-methoxyestradiol and 15, rather than in
steric bulk, shown by the models in Figure 2 give
credence to the hypothesis that electronic effects account
for the observed disparity in biological activity.
The inactivity of compounds 18 and 23 also seems to
indicate that the activity of 2-((E)-1′-propenyl)estradiol
(3, MGM 0.14 µM; IC50 1.1 µM) and 2-ethoxyestradiol
(4, MGM 0.076 µM; IC50 0.91 µM) result from the
electronic effects afforded by the overall electronic
characteristics of the 2-positon moieties including the
π-conjugation of the 1′-alkene and electron density of
the oxygen, respectively.21 The 2-position substituent of
compound 18 possesses similar steric characteristics but
differs from the 2-(1′-propenyl) analogue (3) in that the
alkene is shifted from the 1′-2′ to the 2′-3′ position.
Additionally, the inactive methoxymethyl moiety of
compound 23 is an alternative atom arrangement of the
2-ethoxy group of 2-ethoxyestradiol (4). Analysis of the
electronic effects (Figure 2) of analogues 3 (lower left)
and 18 (lower right) show a disruption in the electron
density of 18. While the steric bulk of both molecules is
comparable and similar to other position 2 alkyl ana-
logues that have displayed at least some activity, the
electron density afforded by the π-conjugation of the
1′,2′-alkene of 3 probably accounts for its observed
inhibition of tubulin polymerization. Together, the
inactivity of 2-cyanoestradiol, 15, 18, and 23 and
analysis of the electron density maps seem to indicate
that the correct size and shape of the substituents at
position 2 are not sufficient to inhibit tubulin polymer-
ization by themselves. To maintain or improve activity,
the 2-position substituents must contribute to the
electron density or at least not withdraw electron
density from the aromatic ring in addition to maintain-
ing the critical size restrictions.
a
Reagents and conditions: (a) (1) sec-BuLi, THF, -78 °C (2 h);
(2) (MeO)3B, -78 °C-rt (5 h); (3) 30% H2O2, NaOH (aq), 0 °C (8
h). (b) K2CO3, MeI, DMF, TBAI, rt (25 h). (c) 6 N HCl, THF, rt. (6
h). (d) Al(iPrO)3, cyclohexanone, toluene, reflux (20 h). (e) (1)
K2CO3, CH3COCH2Cl, CH3CN, reflux (6 h); (2) 6 M HCl, THF, rt
(8 h). (f) NH2OH‚HCl/Pyr, 90 °C (3 h). (g) K2CO3, BnBr, DMF, rt
(19 h). (h) CF3TMS, TBAF, THF, rt (10 h). (i) (1) Pd/C, H2, MeOH,
rt (8 h); (2) dilute HCl, MeOH, rt, (20 min). (j) MeMgBr, THF, 0
°C-rt (48 h). (k) BEAST, EtOH, CH2Cl2, 0 °C-rt (48 h).
colored according to electrostatic potential (EP, kcal/mol)
ranging from regions of high electron density in blue to
regions of low electron density in red. When comparing
the electronic characteristics (Figure 2) of 2-methoxy-
estradiol (1, upper left) to the sterically similar, yet
inactive, 2-pentafluoroethyl derivative (15, upper right),
the differences between the electron densities of the
aromatic rings and position 2 side chain become obvious.
The pentafluoroethyl moiety of 15 has a dramatic effect,
lowering the overall electron density of the aromatic
ring, resulting in a green region of less electron density
than the blue aromatic portion of 2-methoxyestradiol.
Also, the 2-position ethyl moiety has lowered electron
density (represented by yellow), while the fluorines
increase the position 2 surface polarity relative to the
2-methoxy-side chain. However, the inhibitory activity
The compounds with position 17 alterations were
designed to inhibit oxidation of the 17-hydroxyl to the
less potent ketone. Oxidation of the tertiary alcohols of
compounds 30, 39, 46, 47, and 51 to a ketone would be
inhibited by the 17R-methyl or 17R-trifluoromethyl

Altering the Electronics of 2-Methoxyestradiol
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5131
Ta ble 1. Cytotoxicities and Tubulin Polymerization Inhibition of 2-Methoxyestradiol and Analogues
cytotoxicity (GI50 in µM)a
inhibition of tubulin
polymerization
lung
colon
CNS
melanoma
ovarian
renal
prostate
breast
no. HOP-62 HCT-116
SF-539
UACC-62 OVCAR-3
SN12C
DU-145 MDA-MB-435
MGMb
1.3
(IC50 µM ( SD)c
1
7
9
0.70
5.4
31.6
0.47
NDd
10.7
9.3
17.8
14.1
33.1
12.3
12.6
74.1
14.5
0.70
17.0
0.32
18.6
0.36 0.21
24.5 25.7
0.95
53.7
1.8
55.0
13.8
15.5
14.5
24.0
64.6
16.6
20.9
70.8
16.6
2.0
0.080
4.3
20.9
5.3 ( 0.2
>20
17.4
20.0
11.7
18.2
21.9
33.9
17.0
18.2
38.0
17.4
12.3
11.5
13.8
13.2
24.5
16.2
13.8
23.4
14.1
1.9
4.0
27.5
13.8
16.6
18.2
56.2
70.8
25.7
63.1
18.6
10.2
14.1
11.7
12.0
18.6
18.2
38.0
16.6
20.0
34.7
16.6
2.5
14.5
3.2 ( 0.0
24.0
19.5
10.2 ( 1.0
40.7
>40
13 15.1
15 NDd
18 13.8
23 83.2
25 24.0
26 21.4
27 72.4
29 19.5
30 18.2
36 18.2
2.4
12.0
15.1
7.8
16.2
22.9
24.0
15.8
>40
13.2
21.9
40.7
14.5
19.5
31.6
11.0
>40
>40
>40
>40
>40
>40
>40
0.35
17.8
1.48
13.5
0.66
14.1
0.19
16.2
5.6 ( 0.5
>40
5.1
39 3.1 ( 0.1 4.3 ( 0.0
2.6 ( 0.2 4.0 ( 0.0
1.5 ( 0.1 7.4 ( 0.1 4.1 ( 0.1
0.3 ( 0.0
24.0
16.2
11 ( 0.5
>40
43 23.4
18.2
22.4
20.9
19.5
15.5
18.2
20.4
15.8
27.5
24.5
29.5
18.2
46 NDd
>40
47 15.2 ( 1.2 2.8 ( 0.6
6.9 ( 1.5 6.3 ( 1.1
31.6 58.9
0.12 0.54
2.8 ( 0.2 3.74 ( 0.1 2.8 ( 0.1 4.5 ( 0.3 5.2 ( 0.0
7.1 ( 0.4 7.8 ( 0.9 12.3 ( 1.6
43.7 24.5 56.2
0.28 0.19 0.47
1.4 ( 0.6
35.5
0.08
1.3 ( 0.3
>40
48 81.3
50 0.56
66.1
0.60
>40
0.79
4.7 ( 0.1
2.9 ( 0.5
4.8 ( 0.4
51 5.0 ( 0.0 3.5 ( 0.1
a
The GI50 values, denoting cytotoxicity, are the concentrations affording 50% growth inhibition of the individual human cancer cell
lines. MGM corresponds to the average concentration causing growth inhibition in all of the human cancer cell lines tested. c Reaction
conditions conducted using tubulin concentration of 1.2 mg/mL (12 µM). ND indicates that the value was not determined.
b
d
Ta ble 2. Antitumor and Antiangiogenic Activities of
2-Methoxyestradiol Analogues
Although the 17R-methyl moiety of 51 was tolerated,
the 17R-methyl of 47 and the 17R-trifluoromethyl
no.
MDA-MB-231 IC50 (µM)a
HUVEC IC50 (µM)b
moiety of 46 (IC50 > 40 µM) effectively abolished tubulin
polymerization inhibition at the concentrations tested.
Additionally, while fluorine is generally regarded as an
adequate isosteric replacement for both a hydroxyl
group and hydrogen, 17,17-difluoromethylene-2-meth-
oxyestradiol (48, IC50 > 40 µM) was completely inactive.
1
3
4
5
7
1.00 ( 0.05
0.65 ( 0.11
0.55 ( 0.33
3.99 ( 1.12
13.61 ( 0.86
95.91 ( 4.82
15.83 ( 2.52
47.49 ( 3.13
33.40 ( 8.77
53.96 ( 2.02
99.25 ( 2.66
54.43 ( 6.15
7.14 ( 0.88
47.17 ( 0.92
5.99 ( 0.32
49.27 ( 1.32
3.33 ( 0.23
70.81 ( 11.79
0.7 ( 0.8
0.84 ( 0.02
0.59 ( 0.01
0.12 ( 0.05
0.52 ( 0.14
15.64 ( 2.11
32.27 ( 13.10
22.5 ( 2.41
25.04 ( 3.55
8.51 ( 0.87
28.75 ( 4.47
7.73 ( 0.49
6.60 ( 1.96
3.09 ( 0.16
21.04 ( 0.1
1.94 ( 0.01
40.04 ( 3.56
2.40 ( 0.45
58.27 ( 11.62
0.26 ( 0.04
5.70 ( 0.61
9
13
15
18
23
27
29
30
36
39
46
47
48
50
51
The electron density contours in Figure 3 of 2-ethoxy-
estradiol (4, upper left), 2-ethoxy-17R-methyl estradiol
(51, upper right), 17,17-difluoro-2-methoxy-1,3,5(10)-
estratriene-3-ol (48, lower left), and 2-ethoxy-17-(1′-
methylene)estra-1,3,5(10)-triene-3-ol (50, lower right)
show that both steric and electronic effects of position
17 may contribute to analogue activity. Analogue 51
(upper right) has similar electronic distribution in the
C- and D-rings to the highly active 2-ethoxyestradiol (4,
upper left), but also contains additional steric bulk
afforded by the 17R-methyl substituent. This additional
bulk results in a loss of activity against tubulin.
Significantly, the 17R-methyl moiety of 2-methoxy-17R-
methylestradiol (47) rendered this congener completely
inactive. The trifluoromethyl moiety of compound 46
(not shown), while similar in steric bulk to 47, differs
in electron density at this position, but the electronic
differences were not sufficient to restore activity.
The 17,17-difluoromethylene of derivative 48 (lower
left) was sterically similar to 2-ethoxyestradiol (4, upper
left) and noticeably smaller in size than the 17R-methyl
moiety of 51 (upper right). However, the electronega-
tivity of the fluorines causes an increase in the electron
density of the R-face of the steroid. Because fluorine is
commonly used as an isosteric replacement for both
oxygen and hydrogen, the lack of activity associated
with compound 48 is likely a result of the electronic
influence exerted by the electronegative fluorines rather
than the slight increase in steric bulk.
18.35 ( 3.36
a
The IC50 values are the concentrations affording 50% growth
b
inhibition of the MDA-MB-231 cancer cell lines. The IC50 values
are the concentrations affording 50% growth inhibition of the
HUVEC lines.
substituents, while the 17-position moieties of 29, 48,
and 50 were synthesized as substitutes to inhibit the
potential for oxidation and conjugation of the position
17 hydroxyl group. Of the position 17 analogues, com-
pounds 30, 39, 50, and 51 were inhibitors of tubulin
polymerization with activity comparable to 2-methoxy-
estradiol (IC50 5.3 µM). Because estradiol (6) is not an
inhibitor of tubulin polymerization, the surprising activ-
ity of 17R-methylestradiol (30, IC50 5.6 µM) indicates
that the 17R-methyl moiety is tolerated. The active
position 2 substituents of 2-(1′-propynyl)estradiol20 (5,
IC50 4.9 µM), 2-ethoxyestradiol21 (4, IC50 0.91 µM), and
2-methoxyestradiol (1, IC50 5.3 µM) were combined with
the 17R-methyl moiety to provide the active compounds
39 (IC50 11 µM), and 51 (IC50 4.8 µM) and the inactive
compound 47 (IC50 > 40 µM), respectively.
2-Ethoxy-17-(1′-methylene)estra-1,3,5(10)-triene-3-
ol (50, IC50 2.9 µM, lower right) was overall the most
active compound of the series and, interestingly, also

5132 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Edsall et al.
F igu r e 2. Electron density contour diagram comparing the electrostatic potential (EP) of the position 2 moieties of
2-methoxyestradiol (1, upper left), 2-((E)-1′-propenyl)estradiol (3, lower left), 2-pentafluoroethyl-â-estradiol (15, upper right), and
2-allylestradiol (18, lower right). The EP range (maximum 30.0 kcal/mol and minimum -51.1 kcal/mol) was applied globally to
generate comparable regions of high (blue) and low (red) electron density. The orientation of the molecules displayed by the
electron density contours is from the â-face as shown by the capped-stick models to their immediate left.
F igu r e 3. Electron density contour diagram comparing the electrostatic potential (EP) of the position 17 moieties from the “top
view”. The diagram compares 2-ethoxyestradiol (4, upper left), 17,17-difluoro-2-methoxy-1,3,5(10)-estratrien-3-ol (48, lower left),
2-ethoxy-17R-methylestradiol (51, upper right), and 2-ethoxy-17-(1′-methylene)estra-1,3,5(10)-trien-3-ol (50, lower right). The EP
range (maximum 22.4 kcal/mol and minimum -33.4 kcal/mol) was applied globally to generate comparable regions of high (blue)
and low (red) electron density. The orientation of electron density contours is from “top view” of the steroids as shown by the line
models directly to the left of each. The plane containing the aromatic ring is perpendicular to the plane of the page with the R-
and â-faces extending above and below the plane containing the aromatic ring, respectively. The position 17 moieties are shown
nearest to the viewer at the right side of each molecule.
contained the least polar position 17 substituent (shown
in yellow). Although the steric and electronic charac-
teristics of the 17-position of 50 may not be solely
responsible for the observed activity because it also
contained a position 2 ethoxy group, the results indicate
that sterically small alternatives to the 17-hydroxyl
substituent with low levels of electron density can be
used to develop new analogues that inhibit tubulin

Altering the Electronics of 2-Methoxyestradiol
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5133
polymerization. Collectively, these analogues indicate
that small increases in steric bulk are tolerated at
position 17, but significantly more polar substituents
of comparable size and shape are not. This trend may
also account for the notable disparity between the
activities of the sterically similar 2-methoxyestrone and
2-methoxyestradiol. Additionally, the activity of com-
pounds 30, 39, 50, and 51 indicates that substitution
of 2-methoxyestradiol analogues at position 17 for the
purpose of inhibiting metabolic deactivation can result
in the retention of some tubulin polymerization inhibi-
tory activity provided appropriate substituents are also
located at position 2.
position 17 are important considerations for designing
active analogues. Compound 50 was the most cytotoxic
of the newly synthesized analogues and inhibited tu-
bulin polymerization at lower concentrations than
2-methoxyestradiol. The most promising of these ana-
logues will be further evaluated in other in vitro and in
vivo screens.
Exp er im en ta l Section
Gen er a l. Melting points were determined using capillary
tubes with a Mel-Temp apparatus and are uncorrected. The
proton nuclear magnetic resonance (1H NMR) spectra were
recorded using either an ARX300 300 MHz Bruker NMR
spectrometer or a DMX500 500 MHz Bruker spectrometer. IR
spectra were recorded using a Perkin-Elmer 1600 series FTIR
spectrometer. Flash and gravity chromatographic purification
were performed using 230-400 mesh silica gel unless other-
wise noted. Combustion microanalyses were performed at the
Purdue University Microanalysis Laboratory, and the reported
values were within 0.4% of the calculated composition.
Tu bu lin P olym er iza tion In h ibition Assa ys. Tubulin,
purified from bovine brain as described elsewhere,33 was
preincubated with various compound concentrations. The
method to determine the IC50 values for the inhibition of
purified tubulin polymerization was described previously,10 but
Beckman DU7400/7500 spectrophotometers containing “high
performance” temperature controllers were used. To enhance
cooling of the electronic elements of the temperature control
units, these were modified to include a water circuit. Software
for operating the spectrophotometers was provided by MDB
Analytical Associates, South Plainfield, NJ . After chilling the
preincubated mixtures of compound and tubulin on ice, GTP
was added and polymerization was monitored for 20 min at
26 °C by tubidimetry at 350 nm. The IC50 value was defined
as the compound concentration required to inhibit tubulin
assembly at 20 min by 50%. All compounds were examined in
at least two independent assays.
Cell Cu ltu r e. Human umbilical vein endothelial cells
(HUVEC) were obtained from Clonetics (San Diego, CA), and
MDA-MB-231 cells were obtained from ATCC. HUVEC cul-
tures were maintained for up to five passages in EGM
containing bovine brain extract (Clonetics) and 1X antibiotic-
antimycotic (BioWhittaker, Walkersville, MD). MDA-MB-231
cells were maintained in DMEM/F12 (1:1) containing 10% (v/
v) fetal bovine serum (Hyclone Laboratories, Logan, UT) and
1X antibiotic-antimycotic.
P r olifer a tion Assa ys. Proliferation was measured by cell
counting using a Coulter Z1 cell counter (Coulter Corporation,
Hialeah, FL) or by evaluation of DNA synthesis. Each condi-
tion was done in triplicate, and experiments were repeated at
least twice. To determine antitumor and antiangiogenic activi-
ties of the analogues, proliferation assays were performed by
evaluating DNA synthesis using the 5-bromo-2′-deoxyuridine
(BrdU) cell proliferation colorimetric ELISA kit from Roche
(Indianapolis, IN) according to the manufacturer’s instructions.
The cells were seeded at 1000 cells/well (MDA-MB-231,
antitumor activity) or 3000 cells/well (HUVEC, antiangiogenic
activity) in a 96 well plate, allowed to attach overnight and
then exposed to the compound for 48 h.
Regarding the cytotoxicities of the tested compounds,
each of them displayed some activity in human cancer
cell lines. As in earlier studies, the most cytotoxic
compounds also inhibited tubulin polymerization at the
lowest concentrations, and the least cytotoxic com-
pounds had no significant activity against tubulin. Of
the cytotoxic analogues, compound 50 (MGM 0.79 µM)
and compound 30 (MGM 2.5 µM) were the most cyto-
toxic followed by 39 (MGM 3.2 µM), 51 (MGM 4.8 µM),
and 47 (MGM 10.2 µM). Compound 50 was more
cytotoxic and a more potent inhibitor of tubulin polym-
erization than 2-methoxyestradiol (MGM 1.3 µM). More-
over, the cytotoxicity and antitubulin activity of 17R-
methylestradiol (30) is unprecedented and unexpected,
because previously examined estrogen derivatives that
inhibited tubulin polymerization were all substituted at
position 2.
The antiproliferitive activities of the analogues were
contrasted in epithelial and endothelial cell lines to
determine their preliminary antiangiogenic potential
(Table 2). Compounds 30, 39, 47, and 50, as well as 1,
3, 4, and 5 showed the most activity against the MDA-
MB-231 breast cancer cell line. These analogues, with
the addition of 18, 27, and 29 were also active against
the HUVEC line. Of the new analogues, as with the NCI
cell screen, only analogue 50 was more active than
2ME2. Congeners with highly polar substituents at both
position 2 and position 17, again, displayed less activity
relative to the less polar analogues. Compounds 27 and
29 showed the greatest selectivity for the endothelial
cell lines over the tumor cell lines. However, both were
much less active against the HUVEC line than 2ME2
and 3, 4, 5, and 50.
In conclusion, the electronic effects of 2- and 17-
position substituents significantly affect tubulin polym-
erization inhibition of analogues of 2-methoxyestradiol.
Of the position 2 analogues, compounds 15, 18, 23, and
27 with similar steric characteristics to 2-methoxyestra-
diol (1), 2-ethoxyestradiol (4), and 2-((E)-1′-propenyl)-
estradiol (3) were completely inactive as tubulin polym-
erization inhibitors and displayed low levels of cyto-
toxicity. The overall inactivity of these derivatives is
likely a result of the altered electronic properties of the
2-position substituents upon the A-ring of estradiol. The
17R-methyl moiety of 30, 39, and 51 is expected to
reduce the deactivating oxidative metabolism of the 17-
hydroxyl group. Some of the analogues containing this
substituent were comparable to 2ME2 in their antitu-
bulin and antiproliferative activities. The D-ring sub-
stituents in 46-48 resulted in inactive compounds,
indicating that both steric and electronic effects at
2-Acetyl-â-estr a d iol (7). Anhydrous chlorobenzene (75
mL), acetyl chloride (3.2 mL, 45.0 mmol), and aluminum
chloride (8.00 g, 60.0 mmol) were combined in a flame-dried
round-bottomed flask and cooled to 0 °C under argon. â-Es-
tradiol (6, 4.00 g, 14.68 mmol) was added slowly in four
portions over 1 h. After stirring for 25 h, 3 N HCl (60 mL)
was added dropwise to decompose the orange/red colored
material. The mixture was extracted with ethyl acetate (3 ×
200 mL), washed with water (200 mL), and dried over
anhydrous sodium sulfate. Condensation of the organic layer
gave the mixture of three products as a yellow oil. The products
were dissolved in methanol (100 mL). Methanol (10 mL)
saturated with potassium hydroxide was added to the solution
and stirred for 10 h. Following the removal of solvent, the basic

5134 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Edsall et al.
solution was diluted with ice-water (250 mL), extracted with
ethyl acetate (3 × 150 mL), washed with water (100 mL) and
brine (100 mL), and dried over anhydrous sodium sulfate. After
condensation, the orange oil was triturated with methanol,
resulting in precipitation of the white solid, 7 (3.73 g, 81%):
and shaken for 15 h. The red reaction mixture was extracted
with ethyl ether (3 × 50 mL), and alumina was removed via
suction filtration. The organic solution was washed with
saturated aqueous sodium bicarbonate (25 mL) and brine (25
mL) and dried over anhydrous sodium sulfate. Condensation
of the organic layer and subsequent flash chromatography
(silica gel, hexane:ethyl acetate 15:1 by volume) gave 12 (0.21
1
mp 195-196 °C (lit.23 192-195 °C). H NMR (CDCl3) δ 12.05
(s, 1 H, exchangeable with D2O), 7.61 (s, 1 H), 6.70 (s, 1 H),
3.77 (t, J ) 17.07 Hz, 1 H), 2.87 (m, 2 H), 2.74-1.32 (m, 17
H), 0.80 (s, 3 H); IR (film) 3472, 2923, 1715, 1640, 1490, 1368,
1264, 1224, 1056, and 1020 cm-1; low resolution ESIMS: m/z
(rel intensity) 313 (MH+, 100). Anal. (C20H26O3) C, H.
1
g, 36%) as a yellow solid: mp 114 °C. H NMR (CDCl3) δ 7.62
(s, 1 H), 6.63 (s, 1 H), 3.84 (s, 3 H), 3.36 (s, 3 H), 3.29 (t, J )
16.54 Hz, 1 H), 2.95 (m, J ) 11.29 Hz, 2 H), 2.84 (m, 2 H),
2.34 (m, 1 H), 2.14 (t, J ) 17.64 Hz, 1 H), 2.05 (m, 1 H), 1.66
(m, 1 H), 1.52-1.17 (m, 9 H), 1.14 (t, J ) 18.32 Hz, 3 H), 0.76
(s, 3 H); IR (film) 2930, 2362, 1664, 1605, 1406, 1260, 1129,
and 1032 cm-1; low resolution ESIMS: m/z (rel intensity) 357
(MH+, 100). Anal. (C23H32O3) C, H.
2-Acetyl-3,17â-d ih yd r oxyestr a -1,3,5(10)tr ien e 3,17-Di-
a ceta te (8). Compound 7 (1.5 g, 4.77 mmol) was dissolved in
pyridine (60 mL), and acetic anhydride (30 mL) was added.
After stirring the reaction mixture for 20 h, it was diluted with
ice-water (800 mL), acidified with 6 M HCl (100 mL),
extracted with ethyl acetate (3 × 300 mL), washed with sodium
bicarbonate (300 mL) and dried over anhydrous sodium
sulfate. After condensation, the desired product 8 (1.30 g, 68%)
was precipitated as a white solid with methanol: mp 148-
2-(1′-Ketop r op yl)-17-â-estr a d iol (13). Chlorotrimethylsi-
lane (0.22 mL, 1.75 mmol) was added to a solution of sodium
iodide (0.263 g, 1.75 mmol) and 12 (0.25 g, 0.701 mmol) in
anhydrous acetonitrile (10 mL) at 0 °C in a flame-dried round-
bottomed flask under argon. The reaction mixture was allowed
to stir at room temperature for 48 h, and it developed a deep
orange color. Methanol (15 mL) was added. The solution was
condensed, and the remaining oil was dissolved in ethyl acetate
(25 mL). The organic layer was washed with saturated aqueous
Na2S2O3 (25 mL), NaHCO3 (25 mL), and brine (25 mL) and
dried over anhydrous MgSO4. Concentration of the organic
solution yielded a yellow oil, which, upon purification via flash
chromatography (column 1: silica gel, hexanes-ethyl acetate
8:1 by volume, column 2: silica gel, chloroform), gave 13 (80
1
150 °C. H NMR (CDCl3) δ 7.74 (s, 1 H), 6.81 (s, 1 H), 4.70 (t,
J ) 16.92 Hz, 1 H), 2.90 (m, 2 H), 2.53 (s, 3 H), 2.26 (m, 4 H),
2.07 (s, 3 H), 2.05 (s, 3 H), 1.92 (d, J ) 12.75 Hz, 3 H), 1.72
(m, 2 H) 1.47-1.25 (m, 6 H), 0.84 (s, 1 H); IR (film) 2929, 1767,
1732, 1683, 1612, 1369, 1250, 1208, 1174, 1048, 1020, and 917
cm-1; low resolution (ESI): m/z (rel intensity) 399 (MH+, 75);
357 (100, MH+ - C2H3O). Anal. (C24H30O5) C, H.
2-(1′,1′-Diflu or oeth yl)-â-estr a d iol 3,17-Dia ceta te (9).
[Bis(2-methoxyethyl)amino]sulfur trifluoride (BEAST, 2.0 mL,
10.85 mmol) and 8 (1.0 g, 2.64 mmol) were dissolved in
anhydrous dichloromethane (25 mL) in a dry bomb reactor. A
drop of anhydrous ethanol (20 µL) was added to the solution,
and the sealed bomb reactor was placed in an oven and heated
at 70 °C for 11 h. After cooling, the reaction mixture was
poured into ice-water (50 mL), extracted with dichlo-
romethane (3 × 50 mL), washed with brine (50 mL), and
concentrated in vacuo. Compound 9 was isolated via flash
chromatography (silica gel, hexanes-ethyl acetate 12:1 by
volume, 0.33 g, 30%) and recrystallized from a mixture of ethyl
acetate and methanol: mp 154-156 °C. 1H NMR (CDCl3) δ
7.22 (s, 1 H), 6.60 (s, 1 H), 4.47 (t, J ) 8.52 Hz, 1 H), 2.65 (m,
2 H), 2.07 (s, 3 H), 2.04 (m, 4 H), 1.76 (t, J ) 18.42 Hz, 3 H
and m, 1 H overlapped), 1.33 (m, 5 H), 1.27-1.04 (m, 6 H),
0.61 (s, 3 H); IR (film) 2935, 2867, 1773, 1733, 1617, 1575,
1498, 1430, 1409, 1374, 1252, 1201, 1173, 1047, 1018, 925, 871,
and 801 cm-1; low resolution CIMS: m/z (rel intensity) 401.7
(M - HF+, 100). Anal. (C24H30F2O4) C, H, F.
1
mg, 35%): mp 140-146 °C. H NMR (CDCl3) δ 7.65 (s, 1 H),
6.69 (s, 1 H), 3.76 (m, 1 H), 3.02 (dq, J ) 1.24 Hz, J ) 6.41
Hz, 2 H), 2.86 (m, 2 H), 2.31-1.26 (m, 15 H), 1.21 (t, J ) 7.25
Hz, 3 H), 0.80 (s, 3 H); IR (film) 3423, 2933, 1735, 1701, 1642,
1490, 1375, 1270, 1055, and 816 cm-1; low resolution CIMS:
m/z (rel intensity) 329 (MH+, 100). Anal. (C21H28O3‚0.3 H2O)
C, H.
2-P en ta flu or oeth yl-â-estr a d iol (15). Anhydrous dimethyl
sulfoxide (5 mL), copper powder (40 mg, 0.63 mmol), and
2-iodoestradiol (14,19 100 mg, 0.25 mmol) were combined in a
Teflon bomb reactor, and the Teflon sleeve was cooled to 0 °C
in an ice bath. Perfluoroethyl iodide was cooled to -78 °C in
a round-bottomed flask, and the liquid (0.055 mL, 0.30 mmol)
was added via syringe to the bomb. After sealing the bomb
reactor, it was placed in the oven at 80 °C for 16 h. The bomb
was cooled to 0 °C before water (20 mL) and ethyl acetate (10
mL) were added. The organic mixture was washed with water
(3 × 5 mL) and dried over anhydrous magnesium sulfate. The
solvent was removed in vacuo to give a crude oil, which was
purified via flash chromatography (silica gel, 5:1 hexanes-
ethyl acetate) to give a light yellow oil. The oil was triturated
with hexane to give 15 as a white solid (40 mg, 41%): mp 120
3,17-Dim eth oxy-â-estr a -1,3,5(10)-tr ien e (11). â-Estradiol
(6, 2.00 g, 7.34 mmol) was dissolved in anhydrous THF (25
mL) in a flame-dried round-bottomed flask under argon and
cooled to 0 °C. Sodium hydride (0.40 g, 16.52 mmol) was added,
and the reaction mixture was allowed to stir for 15 min.
Iodomethane (1.03 mL, 16.52 mmol) was added, and the
reaction mixture was allowed to warm to room temperature
over 24 h. After pouring the reaction mixture into ice-water
(25 mL), the product was extracted into ethyl acetate (3 × 25
mL), washed with saturated aqueous sodium bicarbonate (25
mL), and dried over anhydrous magnesium sulfate. Purifica-
tion via flash chromatography (silica gel, hexanes-ethyl
acetate 6:1 by volume) gave 11 (0.87 g, 40%): mp 156-159
1
°C (dec). H NMR (300 MHz, CDCl3) δ 7.28 (s, 1 H), 6.69 (s, 1
H), 3.74 (t, J ) 8.6 Hz, 1 H), 2.85 (m, 2 H), 2.14-1.20 (m, 15
H), 0.79 (s, 3 H); 19F NMR (282 MHz, MeOH, TFA standard)
δ -7.25 (s, 3 F), -34.5 (s, 2 F); IR (film) 3271, 2925, 1595,
1404, 1261, 1132, 1053, 1010, and 751 cm-1; low resolution
CIMS: m/z (rel intensity) 391 (MH+, 9), 373 (MH+ - H2O).
Anal. (C20H23F5O2) C, H.
2-Allyl-3,17-bis(ter t-bu tyldim eth ylsilyl)-â-estr adiol (17).
Anhydrous zinc bromide (0.29 g, 1.28 mmol) was dissolved in
anhydrous THF (30 mL) under argon in a flame-dried round-
bottomed flask. A solution of allylmagnesium bromide (1.28
mL, 1.0 M in diethyl ether) was added via syringe, and the
resulting mixture was allowed to stir at room temperature for
approximately 10 min until it became turbid. Tetrakis(tri-
phenylphosphine)palladium(0) (0.074 g, 0.064 mmol) and
compound 1619 (400 mg, 0.64 mmol) were added, and the
mixture was allowed to stir at room temperature for 12 h. The
reaction mixture was quenched with water (100 mL), extracted
with ethyl acetate (2 × 30 mL), washed with brine (40 mL),
and dried over anhydrous sodium sulfate. Evaporation of the
solvent in vacuo and purification via gravity chromatography
(silica gel, hexane) yielded 17 (0.26 g, 75%) as a white solid:
1
°C. H NMR (CDCl3) δ 7.18 (d, J ) 8.63 Hz, 1 H), 6.68 (d, J )
8.54 Hz, 1 H), 6.60 (s, 1 H), 3.75 (s, 3 H), 3.36 (s, 3 H), 3.29 (t,
J ) 16.62 Hz, 1 H), 2.84 (m, 2 H), 2.25 (m, 1 H), 2.15 (t, J )
21.87 Hz, 1 H), 2.02 (m, 2 H), 1.84 (m, 1 H), 1.66 (m, 1 H),
1.52-1.17 (m, 7 H), 0.77 (s, 3 H); IR (film) 2945, 2914, 1608,
1504, 1449, 1234, 1104, and 1034 cm-1; low resolution CIMS:
m/z (rel intensity) 301 (MH+, 100). Anal. Calcd for C20H28O2:
C, H.
2-(1′-Ket op r op yl)-3,17-â-d im et h oxyest r a -1,3,5(10)-t r i-
en e (12). Activated alumina was prepared by heating it to 200
°C in a round-bottom flask under vacuum for 8 h and allowing
it to cool under argon. In a sealed tube, 11 (0.500 g, 1.66 mmol),
alumina (1 g), propionic acid (0.12 mL, 1.66 mmol), and
trifluoroacetic anhydride (0.23 mL, 1.66 mmol) were combined
1
mp 66 °C. H NMR (300 MHz, CDCl3) δ 7.03 (s, 1 H), 6.48 (s,

Altering the Electronics of 2-Methoxyestradiol
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5135
1 H), 5.96 (m, 1 H), 5.00 (m, 2 H), 4.13 (t, J ) 8.03 Hz, 1 H),
3.31 (d, J ) 5.74 Hz, 2 H), 2.78 (m, 2 H), 2.29-1.04 (m, 13 H),
0.99 (s, 9 H), 0.88 (s, 9 H), 0.73 (s, 3 H), 0.21 (s, 6 H), 0.03 (s,
3 H), 0.01 (s, 3 H); IR (neat) 2929, 2858, 1501, 1256, 1118,
1100, 895, 806, and 777 cm-1; low resolution CIMS: m/z (rel
intensity) 541 (MH+, 100). Anal. (C33H56O2Si2) C, H.
2-Cyclop r op ylim in om eth ylestr a d iol (25). Cyclopropyl-
amine (1 mL, 14.4 mmol) and anhydrous magnesium sulfate
(1 g) were added to substrate 2418 (0.3 g, 1 mmol), and the
reaction mixture was stirred at room temperature for 8 h. The
mixture was diluted with ethyl acetate (40 mL) and filtered.
The filtrate was evaporated to give the pure imine 25 as a
pale yellow solid (0.32 g, 94%): mp 188-190 °C. 1H NMR
(CDCl3) δ 8.43 (s, 1 H), 7.11 (s, 1 H), 6.64 (s, 1 H), 3.74 (m, 1
H), 2.95-2.82 (m, 3 H), 2.35-1.12 (m, 15 H), 0.96-0.91 (m, 4
2-Allyl-â-estr a d iol (18). Compound 17 (100 mg, 0.185
mmol) was dissolved in a solution of 2% hydrochloric acid in
95% ethanol (5 mL) and 2-propanol (5 mL). The reaction
mixture was sonicated for 3 h. The volume of the reaction
mixture was reduced in volume by ∼80%, and water (5 mL)
was added. The aqueous mixture was extracted with ethyl
acetate (3 × 10 mL), and the combined organic extracts were
dried over anhydrous sodium sulfate. The organic solvent was
removed in vacuo, and the crude yellow oil was purified by
flash chromatography (silica gel: 1% MeOH in CHCl3) yielding
18. Compound 18 was recrystallized from dichloromethane and
hexane to afford a solid (20 mg, 35%): mp 84-85 °C (lit.34 82-
H), 0.78 (s, 3 H); IR (KBr) 3475, 2920, 2862, and 1624 cm-1
;
low resolution CIMS: m/z (rel intensity) 340 (MH+, 100). Anal.
(C22H29NO2) C, H, N.
2-(3-N ,N -Dim e t h yla m in op r op yl)im in om e t h yle st r a -
d iol (26). 3-N,N-Dimethylaminopropylamine (1.675 mL, 13.3
mmol) was added to substrate 2418 (0.4 g, 1.33 mmol), and the
suspension was heated at reflux for 1 h. The excess amine was
removed in vacuo, and the crude product was purified by
passing it through a column of silica gel (230-400 mesh;
methanol) to give 26 as a pale yellow solid (0.42 g, 82%): mp
1
84 °C). H NMR (300 MHz, DMSO) δ 9.02 (s, 1 H), 6.88 (s, 1
1
104 °C. H NMR (CDCl3) δ 8.30 (s, 1 H), 7.13 (s, 1 H), 6.68 (s,
H), 6.48 (s, 1 H), 5.89 (m, 1 H), 4.96 (m, 2 H), 3.50 (t, J ) 8.28
Hz, 1 H), 3.19 (d, J ) 6.37 Hz, 2 H), 2.66 (m, 2 H), 2.20-0.69
(m, 14 H), 0.64 (s, 3 H).
1 H), 3.75-3.69 (t, J ) 9.00 Hz, 1 H), 2.87-2.83 (m, 2 H), 2.34-
1.10 (m, 27 H), 0.78 (s, 3 H); IR (KBr) 3397, 2926, 2865,
1634 cm-1; CIMS m/z (rel intensity) 385 (MH+, 100). Anal.
(C24H36N2O2‚0.5 H2O) C, H, N.
2-(1′-H yd r oxym et h yl)-3,17-â-O-b is(m et h oxym et h yl)-
est r a d iol (21).25 Compound 2028 (500 mg, 1.28 mmol) was
dissolved in a solution of NaBH4 (0.010 g, 2.60 mmol) in
methanol (65 mL), and the mixture was stirred at room
temperature for 2.5 h. The reaction mixture was quenched
with water (50 mL) and acidified to pH 1 with 3 N HCl. The
reaction mixture was extracted into dichloromethane (3 × 100
mL), washed with water (50 mL), dried over anhydrous sodium
sulfate, and concentrated to yield 21 as a clear oil (0.500 g,
17,17-Diflu or o-â-est r a -1,3,5(10)-t r ien -3-ol (29). Com-
pound 28 (500 mg, 1.85 mmol) and [bis(2-methoxyethyl)amino]-
sulfur trifluoride (1.70 mL, 9.25 mmol) were dissolved in
dichloromethane (15 mL) under argon in a plastic bottle. The
reaction mixture was stirred for 48 h. The argon inlet was
removed, one drop of ethanol was added to the reaction
mixture, and the reaction mixture was allowed to stir under
nonanhydrous conditions for an additional 24 h. The reaction
mixture was poured into water (10 mL). The aqueous layer
was extracted with dichloromethane (2 × 15 mL), washed with
brine (15 mL), and dried over anhydrous MgSO4. Evaporation
of the solvent gave an impure yellow oil, which upon flash
chromatography (silica gel, hexanes-ethyl acetate 8:1 by
volume) yielded the yellow solid 29 (160 mg, 30%): mp 156-
1
100%). H NMR (CDCl3) δ 7.04 (s, 1 H), 6.61 (s, 1 H), 5.17 (s,
2 H), 4.49 (s, 2 H), 4.48 (s, 2 H), 3.44 (t, J ) 8.36 Hz, 1 H),
3.31 (s, 3 H), 3.19 (s, 3 H), 2.66 (m, 2 H), 2.34 (m, 1 H), 2.14 (t,
J ) 17.64 Hz, 1 H), 2.05 (m, 1 H), 2.16-0.87 (m, 11 H), 0.63
(s, 3 H); IR (film) 3422, 2930, 2362, 1616, 1500, 1467, 1397
1341, 1257, 1206, 1150, 1117, 1056, 1022, 1001, 918 and 868
cm-1; low resolution CIMS: m/z (rel intensity) 373 (MH+, 100).
Anal. (C23H34O5‚0.4 H2O) C, H.
1
158 °C. H NMR (300 MHz, CDCl3) δ 7.13 (d, J ) 8.43 Hz, 1
H), 6.62 (dd, J ) 8.32 and 2.60 Hz, 1 H), 6.55 (bs, 1 H), 4.43
(s, 1 H), 2.80 (m, 2 H), 2.37-1.24 (m, 13 H), 0.89 (d, J ) 1.71,
3 H); IR (film) 3331, 2942, 2357, 1614, 1588, 1501, 1454, 1384,
1342, 1316, 1289, 1233, 1166, 1103, 1067, 372, 929 cm-1; low
resolution CIMS: m/z (rel intensity) 293 (MH+, 100) Anal.
(C18H22F2O) C, H,
2-(1′-Met h oxym et h yl)-3,17-â-O-b is(m et h oxym et h yl)-
estr a d iol (22).26 Compound 21 (500 mg, 1.28 mmol) was
dissolved in anhydrous THF (10 mL) in a flame-dried round-
bottomed flask under argon and cooled to 0 °C. Sodium hydride
(0.048 g, 2.0 mmol) was added, followed by addition of
iodomethane (0.125 mL, 2.0 mmol) after hydrogen gas had
ceased to evolve. The reaction mixture was allowed to warm
to room temperature and stir for 7 h. The solution was poured
into ice-water (15 mL), extracted into ethyl acetate (3 × 20
mL), washed with saturated aqueous sodium bicarbonate (20
mL), and dried over anhydrous magnesium sulfate. Condensa-
tion of the organic layer gave the desired product 22 (450 mg,
17r-Meth yl-â-estr a d iol (30). A solution of estrone (28, 500
mg, 1.85 mmol) in anhydrous THF (50 mL) was cooled to 0 °C
in flame-dried round-bottomed flask under argon. A 1.4 M
solution of methylmagnesium bromide (36.99 mmol) in toluene
(19.8 mL), and THF (6.6 mL) was added slowly to the cold
solution via syringe. The reaction mixture was allowed to stir
at room temperature for 8 h before it was quenched with
saturated aqueous ammonium chloride (20 mL). The aqueous
layer was extracted with ethyl acetate (2 × 50 mL), and the
combined organic layers were dried over anhydrous sodium
sulfate. Condensation and purification via flash chromatog-
raphy (silica gel, hexanes-ethyl acetate 10:1 by volume) of the
crude material gave the white solid 30 (428 mg, 81%): mp
1
87%) as an oil. H NMR (CDCl3) δ 7.24 (s, 1 H), 6.78 (s, 1 H),
5.15 (s, 2 H), 4.63 (s, 2 H), 4.45 (s, 2 H), 3.59 (t, J ) 12.39 Hz,
1 H), 3.45 (s, 3 H), 3.38 (s, 3 H), 3.35 (s, 3 H), 2.80 (m, 2 H),
2.30 (m, 1 H), 2.15 (s, 2 H), 2.01-1.13 (m, 13 H), 0.78 (s, 3 H);
IR (film) 3365, 2928, 1684, 1611, 1501, 1447, 1382, 1260, 1150,
1058, and 919 cm-1; low resolution CIMS: m/z (rel intensity)
405 (1, M+), 373 (MH+ - CH3OH, 100).
2-Meth oxym eth yl-â-estr a d iol (23).26 Compound 22 (150
mg, 0.37 mmol) and pyridinium p-toluenesulfonate (0.93 g, 3.7
mmol) were dissolved in methanol (30 mL), and the reaction
mixture was heated at reflux for 18 h. The organic material
was extracted into ethyl acetate (3 × 50 mL), washed with
brine (2 × 50 mL), and dried over anhydrous sodium sulfate
to yield impure 23 as a brown oil. Concentration and purifica-
tion via flash chromatography (silica gel, hexane:ethyl acetate
2:1 by volume) gave a yellow solid which was recrystallized
from ethyl acetate to provide pure 23 (60 mg, 51%) as a white
solid: mp 171-173 °C (lit.26 169 °C). 1H NMR (300 MHz,
CDCl3) δ 7.19 (s, 1 H), 6.92 (s, 1 H), 6.69 (s, 1 H), 4.65 (d, J )
12.28 Hz, 1 H), 4.59 (d, J ) 12.31 Hz, 1 H), 3.73 (m, 1 H), 3.43
(s, 3 H), 2.80 (m, 2 H), 2.31-1.15 (m, 15 H), 0.78 (s, 3 H).
1
215-216 °C (lit.35 191-193 °C). H NMR (300 MHz, CDCl3) δ
7.13 (d, J ) 8.53 Hz, 1 H), 6.60 (d, J ) 8.42 Hz, 1 H), 6.54 (s,
1 H), 4.64 (s, 1 H), 2.81 (m, 2 H), 2.29-1.30 (m, 12 H), 1.25 (s,
3 H), 0.87 (s, 3 H).
3-O-Meth oxym eth ylestr on e (31). Diisopropylethylamine
(4.83 mL, 27.7 mmol) and estrone (28, 5 g, 18.5 mmol) were
dissolved in anhydrous THF (50 mL), and the mixture was
stirred at 0 °C under argon for 0.5 h. Chloromethylmethyl
ether (2.11 mL, 27.7 mmol) was added dropwise at 0 °C. The
solution was allowed to warm to room temperature for 1 h and
was stirred at reflux for 24 h. Following addition of saturated
ammonium chloride (100 mL), the solution was extracted with
anhydrous diethyl ether (3 × 100 mL), washed with brine (100
mL), and dried over anhydrous magnesium sulfate. Concen-
tration of the product gave a crude orange oil which upon
purification via flash chromatography (silica gel, 6:1 hexanes-

5136 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Edsall et al.
ethyl acetate) gave 31 (5.2 g, 89%) as a white solid: mp 93-
95 °C. 1H NMR (300 MHz, CDCl3) δ 7.20 (d, J ) 8.54 Hz, 1
H), 6.82 (dd, J ) 8.53 and 2.47 Hz, 1 H), 6.78 (bs, 1 H), 5.13
(s, 2 H), 3.46 (s, 3 H), 2.87 (m, 2 H), 2.53-1.42 (m, 13 H), 0.89
2934, 1591, 1504, 1278, 1152, 1064, 1002, 984, and 710 cm-1
;
low resolution CIMS: m/z (rel intensity) 437 (55, MH+), 419
(MH+ - H2O, 100). Anal. (C28H36O4) C, H.
17-â-(S)-(Hyd r oxyp h en ylm eth yl)-2-m eth oxy-3-O-m eth -
oxym et h yl-17r-m et h yl-1,3,5(10)-est r a t r ien e (35). Com-
pound 34 (1.0 g, 2.29 mmol) was dissolved in acetone (10 mL)
and potassium carbonate (1.58 g, 11.45 mmol) was added.
Iodomethane (0.71 mL, 11.45 mmol) was added via syringe,
and the reaction mixture was allowed to stir at room temper-
ature for 24 h. Water (20 mL) was added, and the aqueous
mixture was extracted with ethyl acetate (2 × 30 mL). The
combined organic layers were washed with brine (25 mL),
dried over anhydrous magnesium sulfate, and concentrated
in vacuo to give a brown oil. Purification via flash chromatog-
raphy (silica gel, 8:1 hexanes-ethyl acetate) and recrystalli-
zation from ethyl acetate and hexane gave colorless crystals
of 35 (0.90 g, 87%), whose molecular connectivity was con-
firmed by X-ray analysis: mp 181-182 °C. 1H NMR (300 MHz,
CDCl3) δ 7.37-7.26 (m, 5 H), 6.87 (s, 1 H), 6.86 (s, 1 H), 5.20
(s, 2 H), 4.97 (s, 1 H), 3.87 (s, 3 H), 3.52 (s, 3 H), 2.78 (m, 2 H),
2.05-1.26 (m, 14 H), 1.08 (s, 3 H), 0.95 (s, 3 H); IR (film) 3526,
(s, 3 H); IR (film) 2928, 1739, 1607, 1498, 1152, and 1005 cm-1
;
low resolution CIMS: m/z (rel intensity) 315 (MH+, 100). Anal.
(C20H26O3‚0.3H2O) C, H.
3-O-Meth oxym eth yl-17r-m eth yl-â-estr a d iol (32). A so-
lution of 31 (3 g, 9.54 mmol) in anhydrous THF (50 mL) was
cooled to 0 °C in a flame-dried round-bottomed flask under
an argon atmosphere. A 3.0 M solution of methylmagnesium
bromide (16 mL, 47.7 mmol) in diethyl ether was added slowly
to the cold solution via syringe. The reaction mixture was
allowed to stir at room temperature for 20 h and was quenched
with saturated aqueous ammonium chloride (50 mL). The
aqueous layer was acidified to pH 7 and extracted with ethyl
acetate (3 × 100 mL). The combined organic layers were dried
over anhydrous sodium sulfate. Evaporation of the solvent and
purification of the crude material via flash chromatography
(silica gel, hexanes-ethyl acetate 6:1 by volume) gave 32 as a
white solid (2.3 g, 73%): mp 86-89 °C. 1H NMR (300 MHz,
CDCl3) δ 7.19 (d, J ) 8.46 Hz, 1 H), 6.81 (dd, J ) 8.46 and
2.58 Hz, 1 H), 6.75 (d, J ) 2.44 Hz, 1 H), 5.13 (s, 2 H), 3.46 (s,
3 H), 2.83 (m, 2 H), 2.31-1.29 (m, 14 H), 1.26 (s, 3 H), 0.89 (s,
3 H); IR (film) 3435, 2932, 1609, 1497, 1370, 1241, 1151, and
1016 cm-1; low resolution CIMS: m/z (rel intensity) 313 ( MH+
- H2O, 100). Anal. (C21H30O3‚0.23H2O) C, H.
2934, 1607, 1507, 1262, 1152, 1069, 1022, 980, and 710 cm-1
;
low resolution CIMS: m/z (rel intensity) 451 (MH+, 55). Anal.
(C29H38O4) C, H.
17â-(S)-(Hyd r oxyp h en ylm eth yl)-2-m eth oxy-17r-m eth -
yl-1,3,5(10)-estr a tr ien -3-ol (36). Compound 35 (80 mg, 0.18
mmol) was dissolved in methanol (10 mL), and 2-propanol (10
mL) and 6 N HCl (10 mL) were added. The reaction mixture
was allowed to stir at room temperature for 6 h, and water
(20 mL) was added. The aqueous mixture was reduced in
volume by evaporation, yielding a turbid white aqueous
mixture, which was extracted with ethyl acetate (2 × 20 mL).
The combined organic layers were dried over anhydrous
magnesium sulfate and concentrated in vacuo. Purification via
flash chromatography (silica gel, hexane:ethyl acetate 5:1 by
volume) yielded 36 (50 mg, 69%) as a white solid: mp 183-
185 °C. 1H NMR (300 MHz, CDCl3) δ 7.40-7.24 (m, 5 H), 6.81
(s, 1 H), 6.65 (s, 1 H), 5.41 (s, 1 H), 4.98 (s, 1 H), 3.87 (s, 3 H),
2.76 (m, 2 H), 2.23-1.27 (m, 14 H), 1.08 (s, 3 H), 0.95 (s, 3 H);
IR (film) 3436, 2933, 1506, 1450, 1276, 1242, 1122, 1023, 873,
and 710 cm-1; low resolution (CI): m/z (rel intensity) 407
(MH+, 100). Anal. (C27H34O3) C, H.
17â-O-Ben zyl-3-O-m eth oxym eth yl-17r-m eth ylestr a d i-
ol (33). Compound 32 (3.25 g, 9.84 mmol) was dissolved in
anhydrous DMF (30 mL) in a flame-dried round-bottomed flask
under argon. The reaction mixture was cooled to 0 °C, and
sodium hydride (0.71 g, 29.5 mmol) was added. After allowing
the reaction mixture to stir at this temperature for 15 min,
benzyl bromide (5.9 mL, 49.2 mmol) was added via syringe,
and the mixture was allowed to stir for 6 h. The reaction
mixture was cooled to 0 °C, quenched with 50% aqueous
ethanol (20 mL), acidified with 3 N HCl to pH 5, and extracted
with diethyl ether (3 × 50 mL). The combined organic layers
were washed with water (50 mL) and brine (40 mL) and dried
over anhydrous magnesium sulfate. The organic solution was
concentrated in vacuo to give a yellow oil, which, upon
purification via flash chromatography (silica gel, 20:1 hex-
anes-ethyl acetate by volume), gave 33 as a clear oil (2.9 g,
2-Iod o-3-O-(m eth oxym eth yl)-17r-m eth ylestr a d iol (37).
sec-Butyllithium (1.3 M in cyclohexane, 9.9 mL, 12.9 mmol)
was added to a solution of 32 (0.85 g, 2.57 mmol) in anhydrous
THF (30 mL) at -78 °C under argon, and the reaction mixture
was stirred at the same temperature for 2 h. A solution of
iodine (3.27 g, 12.9 mmol) in dry THF (30 mL) was added to
the resultant pale yellow-colored solution of the anion, and
the reaction mixture was slowly raised to room temperature
over 6 h. It was poured into ice-water (100 mL). The crude
product was extracted into ethyl acetate (2 × 40 mL), washed
with a solution of saturated sodium thiosulfate (2 × 20 mL),
and dried (Na2SO4). The removal of the solvent gave the crude
compound 37 as a brown oil. Purification by column chroma-
tography (silica gel, 15:1 hexanes-ethyl acetate) yielded 37
(1.0 g, 85%) as an off-white solid; mp 54-57 °C. 1H NMR
(CDCl3) δ 7.65 (s, 1 H), 6.79 (s, 1 H), 5.19 (s, 2 H), 3.51 (s, 3
H), 2.83-2.79 (m, 2 H), 2.42-1.33 (m, 14 H), 1.28 (s, 3 H),
0.88 (s, 3 H); IR (film) 3436, 2934, 1482, 1380, 1239, 1152,
1086, 1016, 1000, 925, and 736 cm-1; low resolution (CI): m/z
1
70%). H NMR (300 MHz, CDCl3) δ 7.31-7.14 (m, 6 H), 6.77
(dd, J ) 8.49 and 2.60 Hz, 1 H), 6.71 (d, J ) 2.47 Hz, 1 H),
5.09 (s, 2 H), 4.70 (d, J ) 11.76 Hz, 1 H), 4.43 (d, J ) 11.77
Hz, 1 H), 3.41 (s, 3 H), 2.80 (m, 2 H), 2.26-1.27 (m, 13 H),
1.26 (s, 3 H), 0.94 (s, 3 H); IR (film) 2932, 1608, 1242, 1152,
1017, 922, and 734 cm-1; low resolution ESIMS: m/z (rel
intensity) 313 (MH+ - C7H8O, 100). Anal. (C28H36O3) C, H.
2-Hyd r oxy-17-â-(S)-(h yd r oxyp h en ylm eth yl)-3-O-m eth -
oxym eth yl-17r-m eth yl-1,3,5(10)-estr a tr ien -2-ol (34). Com-
pound 33 (2.1 g, 4.99 mmol) was dissolved in anhydrous THF
(50 mL) in a flame-dried round-bottomed flask under argon.
After cooling to -78 °C, sec-butyllithium (1.3 M in cyclohex-
anes, 19.2 mL, 25 mmol) was added. The reaction mixture was
allowed to warm to -30 °C over 5 h before trimethyl borate
(5.59 mL, 49.9 mmol) was added via syringe. The reaction
mixture was allowed to warm to room temperature and stir
for 8 h. After the reaction mixture was cooled to 0 °C, 30%
H2O2 (5.65 mL, 49.9 mmol) and a solution of sodium hydroxide
(200 mg, 4.99 mmol) in water (5.4 mL) was slowly added. The
reaction mixture was allowed to warm to room temperature
and stir for 8 h before it was once again cooled to 0 °C. The
peroxide was quenched with saturated aqueous sodium thio-
sulfate. The aqueous mixture was extracted with ethyl acetate
(3 × 50 mL), and the combined organic layers were dried over
anhydrous magnesium sulfate and concentrated in vacuo to
give a colorless oil. Purification via flash chromatography
(silica gel, hexanes-ethyl acetate 10:1 by volume) gave 34 (1.5
g, 70%) as a white solid: mp 150-153 °C. 1H NMR (300 MHz,
CDCl3) δ 7.37-7.26 (m, 5 H), 6.92 (s, 1 H), 6.80 (s, 1 H), 5.73
(s, 1 H), 5.16 (s, 2 H), 4.97 (s, 1 H), 3.51 (s, 3 H), 2.78 (m, 2 H),
2.21-1.26 (m, 14 H), 1.07 (s, 3 H), 0.90 (s, 3 H); IR (film) 3498,
(rel intensity) 457 (MH+, 5), 330 (MH+ - I, 100). Anal. (C21H29
IO3‚0.45 C6H14) C, H.
-
2-Iod o-17r-m eth ylestr a d iol (38). Hydrochloric acid (6 M,
30 mL) was added to a solution of 37 (1.2 g, 2.6 mmol) in THF
(30 mL), and the reaction mixture was stirred at room
temperature for 6 h. The reaction mixture was poured into
ice-water (100 mL) and extracted into ethyl acetate (3 × 20
mL). The combined organic phases were dried (Na2SO4).
Removal of solvent followed by column chromatographic
purification (silica gel, 1:5 ethyl acetate-hexane) gave 38 (0.55
g, 52%) as a colorless solid: mp 140-142 °C. 1H NMR (CDCl3)
δ 7.52 (s, 1 H), 6.71 (s, 1 H), 2.81-2.76 (dd, J ) 6.0 Hz, 2 H),
2.26-2.23 (m, 1 H), 2.18-2.10 (m, 1 H), 1.89-1.31 (m, 13 H),

Altering the Electronics of 2-Methoxyestradiol
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5137
1.27 (s, 3 H), 0.89 (s, 3 H); IR (film) 3235, 2932, 1657, 1594,
1449, 1150, 1088, and 955 cm-1; low resolution EIMS: m/z
(rel intensity) 412 (M+, 100). Anal. (C19H25IO2‚0.7C6H14) C, H.
g, 31.8 mmol) and chloroacetone (1.27 mL, 15. 9 mmol) were
added to a solution of 40 (1.2 g, 3.18 mmol) in acetonitrile (40
mL), and the reaction mixture was heated at reflux under
argon for 6 h. After in vacuo removal of the solvent, the crude
product was extracted into ethyl acetate (3 × 30 mL), washed
with water (50 mL), and dried (Na2SO4). The organic solvent
was removed, and the sticky residue was dissolved in THF
(50 mL). Following addition of 6 M hydrochloric acid (50 mL),
the reaction mixture was stirred at room temperature for 8 h.
The reaction mixture was poured into ice-water (50 mL),
extracted into ethyl acetate (4 × 20 mL), and dried (Na2SO4).
The removal of the solvent followed by trituration with
dichloromethane gave the pure product 42 (0.62 g, 56%) as a
colorless solid: mp 190-192 °C. 1H NMR (DMSO-d6) δ 6.80
(s, 1 H), 6.71 (s, 1 H), 6.45 (s, 1 H), 4.45 (s, 1 H), 3.90-3.87 (d,
J ) 9.0 Hz, 1 H), 3.79-3.76 (d, J ) 9.0 Hz, 1 H), 3.53-3.48 (t,
J ) 9.0 Hz, 1 H), 2.68-2.66 (m, 2 H), 2.19-1.53 (m, 5 H), 1.39
(s, 3 H), 1.36-1.07 (m, 8 H), 0.78 (s, 3 H); IR (KBr) 3396, 3224,
2932, 2872, 1586, and 1501 cm-1; low resolution CIMS: m/z
(rel intensity) 345 (MH+, 100). Anal. (C21H28O4‚0.2CH3CO2Et)
C, H.
2-(2′-Hyd r oxyim in op r op yloxy)est r a d iol (43). Hydrox-
ylamine hydrochloride (0.374 g, 5.0 mmol) was added to a
solution of compound 42 (0.344 g, 1.0 mmol) in pyridine (20
mL). The resulting mixture was stirred at 90 °C for 3 h. The
pyridine was removed, and the residue was dissolved in ethyl
acetate (100 mL) and water (50 mL). The organic layer was
washed with brine (2 × 50 mL) and dried (Na2SO4). Removal
of solvent followed by column chromatography (silica gel, 10:1
ethyl acetate-methanol) gave pure oxime 43 as a fluffy solid
(0.3 g, 84%): mp 160-162 °C. 1H NMR (CDCl3) δ 6.89 (s, 1
H), 6.66 (s, 1 H), 4.64 (s, 2 H), 3.76-3.70 (t, J ) 9.0 Hz, 1 H),
2.79-2.74 (m, 2 H), 2.27-1.15 (m, 19 H), 0.78 (s, 3 H); IR (KBr)
3327, 2925, 1590, and 1504 cm-1; low resolution CIMS: m/z
(rel intensity) 360 (MH+, 100). Anal. (C21H29NO4‚0.6H2O) C,
H, N.
2-P r op yn yl-17r-m eth ylestr a d iol (39). Tributylpropynyl-
stannane (0.48 g, 1.45 mmol) was added to a solution of 38
(0.3 g, 0.73 mmol) in dry THF (40 mL). Tetrakis(triphen-
ylphosphine)palladium (0.084 g, 0.073 mmol) was added, and
the reaction mixture was heated at reflux under argon for 10
h. Removal of the solvent followed by chromatographic puri-
fication (silica gel, 6:1 hexanes-ethyl acetate) of the crude
product gave pure 39 (0.18 g, 76%) as a colorless solid: mp
188-190 °C. 1H NMR (CDCl3) δ 7.21 (s, 1 H), 6.64 (s, 1 H),
5.59 (s, 1 H, exchangeable with D2O), 2.83-2.79 (dd, J ) 6.0
Hz, 2 H), 2.32-2.24 (m, 1 H), 2.12 (s, 3 H), 1.86-1.30 (m, 13
H), 1.27 (s, 3 H), 0.88 (s, 3 H); IR (film) 3401, 2933, 1495, 1372,
1344, 1241, 1086, and 931 cm-1; low resolution CIMS: m/z
(rel intensity) 307 (MH+ - H2O, 100), 325 (MH+, 88). Anal.
(C22H28O2) C, H.
2-Hyd r oxy-3,17-O-bis(m eth oxym eth yl)estr a d iol (40).
Compound 19 (4.0 g, 11.1 mmol) was dissolved in anhydrous
THF (150 mL) in a flame-dried round-bottomed flask under
argon. After cooling to -78 °C, sec-BuLi (1.3 M in cyclohexane,
26 mL, 33.3 mmol) was added. The reaction mixture was
allowed to stir at this temperature for 2 h before trimethyl
borate (12.4 mL, 110.9 mmol) was added via syringe. The
reaction mixture was allowed to warm to room temperature
and stir for 5 h. After the reaction mixture was cooled to 0 °C,
30% H2O2 (6.4 mL, 110.9 mmol) and a solution of 1 M aqueous
sodium hydroxide (1 mL) were slowly added. The reaction
mixture was allowed to warm to room temperature over 8 h.
It was cooled to 0 °C, and the peroxides were quenched with
saturated aqueous sodium thiosulfate. The aqueous mixture
was extracted with ethyl acetate (4 × 50 mL), and the
combined organic layers were washed with water (1 × 100
mL). The organic material was dried over anhydrous magne-
sium sulfate and concentrated in vacuo to give a colorless oil.
Purification via flash chromatography (silica gel, hexanes-
ethyl acetate 8:1 by volume) gave 40 (3.96 mg, 95%) as a clear
oil: 1H NMR (CDCl3) δ 6.89 (s, 1 H), 6.79 (s, 1 H), 5.75 (s, 1
H), 5.16 (s, 2 H), 4.66 (s, 2 H), 3.61 (t, J ) 8.24 Hz, 1 H), 3.51
(s, 3 H), 3.37 (s, 3 H), 2.77 (m, 2 H), 2.26-1.18 (m, 13 H), 0.81
(s, 3 H); low resolution CIMS: m/z (rel intensity) 377 (MH+,
40), 345 (MH+ - OCH3, 60), 315 (MH+ - OCH2OCH3, 100).
Anal. (C22H32O5) C, H.
2-Meth oxyestr on e (41). 2-Methoxyestradiol (1,29 1.50 g,
4.96 mmol) was placed in a 250 mL round-bottomed flask that
was equipped with a 25 mL Dean-Stark trap and a reflux
condenser. The entire apparatus was flame-dried under an
argon atmosphere. Toluene (60 mL) was added to dissolve the
starting material. Aluminum isopropoxide (5.1 g, 24.8 mmol)
and cyclohexanone (20.5 mL, 198.4 mmol) were added, and
the entire reaction mixture was heated at reflux (145-150 °C)
for 20 h. The reaction mixture was allowed to cool to room
temperature and saturated aqueous sodium bicarbonate solu-
tion (100 mL) was added. The organic material was extracted
with dichloromethane (3 × 150 mL). The aqueous emulsion
was acidified with 3 N HCl (∼20 mL) until the emulsion
separated. The aqueous layer was again extracted with dichlo-
romethane (100 mL). The combined organic extracts were dried
over anhydrous magnesium sulfate, concentrated in vacuo, and
purified via flash chromatography (silica gel, hexanes-ethyl
acetate 10:1 by volume) to yield the desired product 41.
Fractions containing 41, contaminated with cyclohexanone,
were combined and concentrated in vacuo. The resulting oil
was triturated with hexane to give additional pure product
(1.32 g, 89%): mp 187-190 °C (lit.36 190-192 °C). 1H NMR
(300 MHz, CDCl3) δ 6.79 (s, 1 H), 6.66 (s, 1 H), 3.86 (s, 3 H),
2.82 (m, 2 H), 2.53-1.37 (m, 14 H), 0.92 (s, 3 H); IR (neat)
3333, 2927, 2854, 1720, 1587, 1515, 1504, 1447, 1431, 1357,
1326, 1291, 1268, 1201, 1026, 873, 827, 581 cm-1; low resolu-
tion CIMS: m/z (rel intensity) 300 (M+, 100). Anal. (C19H24O3)
C, H.
2-Meth oxyestr on e 3-Ben zyl Eth er (44). 2-Methoxyestro-
ne (41, 100 mg, 0.33 mmol) and potassium carbonate (68 mg,
0.495 mmol) were dissolved in anhydrous DMF (5 mL) in a
flame-dried round-bottomed flask under argon. Benzyl bromide
(0.39 mL, 3.3 mmol) was added, and the reaction mixture was
allowed to warm to room temperature and stir for 19 h. The
reaction was quenched with water (5 mL), acidified with 3 N
HCl and extracted with diethyl ether (3 × 15 mL). The
combined organic layers were washed with water (5 mL) and
brine (5 mL) and dried over anhydrous magnesium sulfate.
After condensation of the solvent in vacuo, purification by flash
chromatography (silica gel: 10:1 hexanes-ethyl acetate by
volume) yielded the desired product 44 (113 mg, 88%): mp
89-92 °C (lit.23 154-156 °C). 1H NMR (300 MHz, CDCl3) δ
7.50-7.23 (m, 5 H), 6.87 (s, 1 H), 6.67 (s, 1 H), 5.15 (s, 2 H),
3.91 (s, 3 H), 3.21-1.27 (m, 16 H), 1.01 (s, 1 H); IR (film) 2929,
2364, 2735, 1513, 1453, 1262, 1215, 1015, and 697 cm-1
.
3-Ben zyloxy-2-m eth oxy-17r-tr iflu or om eth yl-1,3,5(10)-
estr a tr ien e-17â-tr im eth ylsila n e (45). Compound 44 (130
mg, 0.33 mmol) was dissolved in anhydrous THF (3 mL) in a
flame-dried round-bottomed flask under argon and trifluo-
romethyltrimethylsilane (0.074 mL, 0.50 mmol) was added,
followed by one drop of tetrabutylammonium fluoride (1.0 M
in THF). The reaction mixture was allowed to stir at room
temperature for 10 h, and water (5 mL) was added. The
aqueous mixture was extracted with ether (3 × 10 mL), and
the combined organic layers were washed with brine (10 mL)
and dried over anhydrous magnesium sulfate. After condens-
ing the mixture in vacuo, the crude solid was recrystallized
from ethyl acetate to give 45 (110 mg, 70%) as yellow
1
crystals: mp 44-46 °C. H NMR (300 MHz, CDCl3) δ 7.31-
7.15 (m, 5 H), 6.69 (s, 1 H), 6.48 (s, 1 H), 4.95 (s, 2 H), 3.71 (s,
3 H), 2.59 (m, 2 H), 2.08-1.09 (m, 13 H), 0.74 (s, 3 H), 0.01 (s,
9 H); IR (film) 2953, 1514, 1255, 1168, 1144, 1028, 841, and
756 cm-1; low resolution CIMS m/z (rel intensity) 533 (MH+,
100). Anal. (C30H39F3O3Si) C, H.
5′,6′-Dih yd r oestr a -1,3,5(10)-tr ien e-[2,3-â]-1′,4′-d ioxin -3′-
m eth yl-3′,17â-d iol (42). Anhydrous potassium carbonate (4.4
2-Meth oxy-17r-tr iflu or om eth yl-â-estr a d iol (46). Com-
pound 45 (100 mg, 0.188 mmol) and palladium on carbon (10

5138 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Edsall et al.
wt % on carbon, 0.01 mmol) were combined in a round-
bottomed flask under argon and covered with methanol (2 mL).
The reaction flask atmosphere was filled with argon and
evacuated three times before it was evacuated and filled with
hydrogen gas. The reaction mixture was allowed to stir for 8
h. After filtering through a pad of Celite and washing the pad
with methanol, dilute hydrochloric acid (10%, 1 mL) was
added, and the reaction mixture was allowed to stir for 20 min.
The reaction mixture was concentrated in vacuo and purified
via flash chromatography (silica gel, hexanes-ethyl acetate
5:1) to give pure 46 as a white solid (70 mg, 100%): mp 58-
63 °C. 1H NMR (300 MHz, CDCl3) δ 6.79 (s, 1 H), 6.66 (s, 1
H), 5.49 (s, 1 H), 3.86 (s, 3 H), 2.80 (m, 2 H), 2.49-1.26 (m, 14
H), 0.92 (s, 3 H); IR (film) 3436, 2930, 1624, 1508, 1447, 1276,
1167, 1131, 1107, 1034, and 875 cm-1; low resolution CIMS:
m/z (rel intensity) 371 (MH+, 7), 301 (MH+ - HCF3, 100). Anal.
(C20H25F3O3‚0.16C6H14) C, H.
3436, 2930, 1738, 1506, 1276, 1199, 1051, and 875 cm-1; low
resolution (CI): m/z (rel intensity) 315 (MH+, 100). Anal.
(C20H26O3‚0.27H2O) C, H.
2-Eth oxy-17-(1′-m eth ylen e)estr a-1,3,5(10)-tr ien -3-ol (50).
Anhydrous DMSO (10 mL) was added via syringe to a flame-
dried round-bottomed flask containing sodium hydride (130
mg, 5.12 mmol) under argon. The reaction mixture was heated
to 62 °C behind a blast shield for 1.5 h until a dark green color
was observed. (Warning: the dimsyl anion is potentially
explosive at temperatures above 60 °C.) The reaction mixture
was allowed to cool to room temperature, and methyltriphen-
ylphosphonium iodide (2.07 g, 5.12 mmol) was added. The
reaction mixture was allowed to stir for 30 min while a red/
brown color emerged. A solution of 49 (200 mg, 0.64 mmol) in
anhydrous DMSO (5 mL) was added via syringe, and the
reaction mixture was allowed to stir at room temperature for
18 h. After cooling to 0 °C, ice-water (20 mL) was added, and
the aqueous mixture was extracted with diethyl ether (3 × 50
mL). The combined ether phases were washed with water (2
× 25 mL) and concentrated in vacuo to give an orange oil.
Purification via flash chromatography (silica gel, hexanes-
ethyl acetate 10:1 by volume) gave 50 as an orange oil (35 mg,
18%): 1H NMR (300 MHz, CDCl3) δ 6.83 (s, 1 H), 6.67 (s, 1
H), 5.55 (s, 1 H), 4.71 [d (overlapped), J ) 2.16 Hz, 1 H], 4.70
[d (overlapped), J ) 2.14 Hz, 1 H], 4.11 (m, 2 H), 2.84-1.26
(m, 15 H), 1.45 (t, J ) 7.0 Hz, 3 H), 0.85 (s, 3 H); IR (film)
3548, 2928, 1654, 1592, 1505, 1274, 1197, 1097, 1041, and 875
cm-1; low resolution CIMS: m/z (rel intensity) 313 (MH+, 100).
Anal. (C21H28O2‚0.6H2O) C, H.
2-Meth oxy-17r-m eth yl-â-estr a d iol (47). Methylmagne-
sium bromide (1.4 M in toluene, 6.0 mL, 8.4 mmol) was added
dropwise to a solution of 2-methoxyestrone (41, 0.30 g, 1.0
mmol) in dry THF (40 mL) at 0 °C. The reaction mixture was
stirred at room temperature for 48 h. The reaction mixture
was quenched with ammonium chloride solution (10 mL). The
mixture was extracted into ethyl acetate (3 × 20 mL), dried
(Na2SO4), and purified by column chromatography on silica
gel (15 g, ethyl acetate-hexane 1:2) to afford the product 47
1
(0.22 g, 70%) as a white solid: mp 158-160 °C. H NMR (300
MHz, CDCl3) δ 6.80 (s, 1 H), 6.64 (s, 1 H), 5.44 (s, 1 H), 3.86
(s, 3 H), 2.77 (m, 2 H), 1.84-1.21 (m, 14 H), 1.28 (s, overlapped,
3 H), 0.92 (s, 3 H); IR (film) 3542, 2931, 1507, 1273, 1121 and
1085 cm-1; low resolution CIMS: m/z (rel intensity) 316 (M+,
100). Anal. (C20H28O3) C, H.
2-E t h oxy-17r-m et h ylest r a d iol (51). Methylmagnesium
bromide (1.4 M in toluene, 6.83 mL, 9.6 mmol) was added to
a solution of 49 (0.3 g, 0.95 mmol) in anhydrous THF (30 mL)
under argon. The reaction mixture was stirred at room
temperature for 3 h, and the excess Grignard reagent was
carefully quenched with a saturated aqueous solution of
ammonium chloride (10 mL). The crude product was extracted
into ethyl acetate (3 × 20 mL). The combined organic phases
were dried (Na2SO4) and purified by column chromatography
on silica gel (230-400 mesh, 1:2 ethyl acetate-hexane) to give
17,17-Diflu or o-2-m eth oxy-1,3,5(10)-estr a tr ien -3-ol (48).
BEAST (0.46 mL, 2.50 mmol) and 2-methoxyestrone (41, 150
mg, 0.50 mmol) were dissolved in anhydrous dichloromethane
(10 mL) in a plastic bottle under argon. One drop of anhydrous
ethanol (∼20 µL) was added to the solution, and the reaction
mixture was allowed to stir at room temperature for 2 days.
The reaction mixture was cooled to 0 °C, and water (20 mL)
was added. The reaction mixture was extracted with dichlo-
romethane (3 × 30 mL), and the combined organic phases were
dried over anhydrous magnesium sulfate and concentrated in
vacuo to give a crude brown oil. Compound 48 was isolated as
a yellow oil via flash chromatography (silica gel, hexanes-
ethyl acetate 15:1 by volume, 30 mg, 19%): 1H NMR (500 MHz,
CDCl3) δ 6.79 (s, 1 H), 6.65 (s, 1 H), 5.45 (s, 1 H), 3.87 (s, 3 H),
2.78 (m, 2 H), 2.35-1.36 (m, 13 H), 0.93 (s, 3 H); 19F NMR
[282 MHz, CDCl3 (TFA standard)] δ -24.14 (dt, J FF ) 217.0
Hz, J HF ) 25.2 Hz, 1 F), -36.58 (dd, J FF ) 218.0 Hz, J HF ) 11
Hz, 1 F); IR (film) 3544, 2936, 1508, 1463, 1317, 1277, 1236,
1165, 1127, 1020, and 875 cm-1; low resolution CIMS: m/z
(rel intensity) 322 (M+, 100). Anal. (C19H24F2O2‚0.6H2O) C, H.
1
51 (0.24 g, 76%) as a white solid: mp 139-143 °C. H NMR
(300 MHz, CDCl3) δ 6.79 (s, 1 H), 6.65 (s, 1 H), 4.11-4.05 (q,
J ) 6 Hz, 2 H), 2.80-2.74 (m, 2 H), 2.27-1.30 (m, 18 H), 1.25
(s, 3 H), 0.90 (s, 3 H); IR (film) 3420, 2930, and 1507 cm-1
;
low resolution CIMS: m/z (rel intensity) 313 (MH+, 100). Anal.
(C21H30O3‚0.7EtOAc) C, H.
X-r a y Cr ysta llogr a p h ic An a lysis of 35. A colorless chunk
of 35, C29H38O4 (0.48 mm × 0.44 mm × 0.35 mm), was mounted
on a glass fiber in a random orientation. Preliminary examina-
tion and data collection were performed using Mo KR radiation
(λ ) 0.71073 Å) on a Nonius KappaCCD diffractometer. The
cell constants and an orientation matrix for data collection
were obtained from least-squares refinement, using the setting
angles of 9815 reflections in the range of 1° < θ < 27°. The
data were collected at a temperature of 150 ( 1 K. A total of
9815 reflections were collected, of which 4581 were unique.
The structure was solved by direct methods using SIR2002.37
The remaining atoms were located in succeeding difference
Fourier syntheses. Hydrogen atoms were included in the
refinement but restrained to ride on the atom to which they
were bonded. The structure was refined in full-matrix least
2-Eth oxyestr on e (49). 2-Ethoxyestradiol (4, 1.88 g, 5.67
mmol) was placed in a 250 mL round-bottomed flask that was
equipped with
a 25 mL Dean-Stark trap and a reflux
condenser. The entire apparatus had been flame dried under
an argon atmosphere. Toluene (50 mL) was added to dissolve
the starting material. Aluminum isopropoxide (5.7 g, 28.4
mmol) and cyclohexanone (23.5 mL, 226.8 mmol) were added,
and the entire reaction mixture was heated at reflux (145-
150 °C) for 20 h. The reaction mixture was cooled to room
temperature and saturated aqueous sodium bicarbonate solu-
tion (100 mL) was added. The organic material was extracted
with dichloromethane (3 × 150 mL). The aqueous emulsion
was acidified with 3 N HCl (∼20 mL) until it separated, and
again the aqueous layer was again extracted with ethyl acetate
(2 × 75 mL). The combined organic extracts were dried over
anhydrous magnesium sulfate and concentrated in vacuo. The
crude product was purified via flash chromatography (silica
gel, hexanes-ethyl acetate 8:1 by volume) to yield the 49 as a
white solid (1.7 g, 94%): mp 140-142 °C. 1H NMR (300 MHz,
CDCl3) δ 6.78 (s, 1 H), 6.66 (s, 1 H), 5.49 (s, 1 H), 4.07 (dq, J
) 6.9 and 1.6 Hz, 2 H), 2.83 (m, 2 H), 2.52 (m, 2 H), 2.36-1.37
(m, 11 H), 1.42 (t, J ) 7.0 Hz, 3 H), 0.92 (s, 3 H); IR (film)
2
2
squares where the function minimized was Σw(|Fo| - |Fc| )2.
Refinement was performed on an AlphaServer 2100 using
SHELX-97. Crystallographic drawings were done using pro-
grams ORTEP and PLUTON.
Ack n ow led gm en t. We are thankful for the gener-
ous supply of 2-methoxyestradiol and expert advice
provided by EntreMed, Inc. We are also grateful for the
research funding provided by EntreMed, Inc., the Pur-
due University Cancer Center, and the Purdue Research
Foundation.
Su p p or tin g In for m a tion Ava ila ble: Table of elemental
analyses.This material is available free of charge via the
Internet at http://pubs.acs.org.

Altering the Electronics of 2-Methoxyestradiol
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5139
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