Synthesis of analogs of 2-methoxyestradiol with enhanced inhibitory effects on tubulin polymerization and cancer cell growth

Article abstract of DOI:10.1021/jm9700833

A new series of estradiol analogs was synthesized in an attempt to improve on the anticancer activity of 2-methoxyestradiol, a naturally occurring mammalian tubulin polymerization inhibitor. The compounds were evaluated as inhibitors of tubulin polymeriza

Full text of DOI:10.1021/jm9700833

J . Med. Chem. 1997, 40, 2323-2334
2323
Syn th esis of An a logs of 2-Meth oxyestr a d iol w ith En h a n ced In h ibitor y Effects
on Tu bu lin P olym er iza tion a n d Ca n cer Cell Gr ow th
Mark Cushman,*,† Hu-Ming He,† J ohn A. Katzenellenbogen,‡ Ravi K. Varma,§ Ernest Hamel,| Chii M. Lin,|
Siya Ram, and Yesh P. Sachdeva
Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmacal Sciences,
Purdue University, West Lafayette, Indiana 47907, Department of Chemistry, University of Illinois, Urbana, Illinois 61801,
Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment, Diagnosis and
Centers, National Cancer Institute, Executive Plaza North, Suite 831, 6130 Executive Boulevard, Rockville, Maryland 20852,
Laboratory of Drug Discovery Research and Development, Developmental Therapeutics Program, Division of Cancer
Treatment, Diagnosis and Centers, National Cancer Institute, Frederick Cancer Research and Development Center,
Frederick, Maryland 21702, and Pharm-Eco Laboratories, Inc., 128 Spring Street, Lexington, Massachusetts 02173
Received February 6, 1997X
A new series of estradiol analogs was synthesized in an attempt to improve on the anticancer
activity of 2-methoxyestradiol, a naturally occurring mammalian tubulin polymerization
inhibitor. The compounds were evaluated as inhibitors of tubulin polymerization and the
binding of [3H]colchicine to tubulin, as well as for in vitro cytotoxicity in human cancer cell
cultures. Overall, the most potent of the new compounds were 2-(2′,2′,2′-trifluoroethoxy)-6-
oximinoestradiol, 2-ethoxy-6-oximinoestradiol, and 2-ethoxy-6-methoximinoestradiol. These
agents lacked significant affinity for the estrogen receptor. The cytotoxicities of the compounds
correlated in general with their abilities to inhibit tubulin polymerization, thus supporting
inhibition of tubulin polymerization as the primary mechanism causing inhibition of cell growth.
In mammals, hepatic hydroxylation of estradiol fol-
lowed by O-methylation results in the formation of
2-methoxyestradiol (1), which has very low affinity for
the estrogen receptor.1,2 General interest in 2-methoxy-
estradiol (1) has been stimulated by its cytotoxicity in
cancer cell cultures, which is associated with inhibition
of mitosis, uneven chromosome distribution, and an
increase in the number of abnormal metaphases.3,4 The
binding of 2-methoxyestradiol (1) to the colchicine
binding site of tubulin results in either inhibition of
tubulin polymerization or formation of polymer with
altered morphology and stability properties, depending
on reaction conditions.5,6 2-Methoxyestradiol (1) or an
unknown related compound might therefore be func-
tioning as a natural regulator of mammalian microtu-
bule assembly and function. Recent in vitro and in vivo
results have demonstrated that 2-methoxyestradiol (1)
also inhibits angiogenesis (the formation of new blood
vessels),7,8 required for the growth of solid tumors.9,10
After oral administration to mice, 2-methoxyestradiol
(1) acts as a potent inhibitor of neovascularization of
solid tumors and inhibits their growth at doses which
produce no apparent signs of toxicity.7,8
estradiol (1) and to exploit it as a lead compound for
the design of more potent anticancer agents, we recently
synthesized 1 and a series of structurally related
compounds.4,11 The most potent of these new analogs
proved to be 2-ethoxyestradiol (2), which was found to
be more potent than 1 as a cytotoxic agent in cancer
cell cultures as well as a tubulin polymerization inhibi-
tor.4 The present investigation was undertaken in order
to further maximize the anticancer and antitubulin
activities of compounds related structurally to 2-meth-
oxyestradiol and to obtain active analogs with enhanced
metabolic stabilities. As described below, this effort
resulted in additional potent inhibitors of cancer cell
growth, and these active analogs, like 1, interact
negligibly with the estrogen receptor.
Ch em istr y
Treatment of the protected 2-iodoestradiol derivative
312 with triethylamine and Pd(PPh3)4 in THF at room
temperature, followed by propyne and CuI, gave the
desired intermediate 4 (Scheme 1).13,14 Deprotection of
intermediate 4 with tetra-n-butylammonium fluoride in
THF at room temperature afforded the diol 5 as a minor
product in 18% yield along with the partially depro-
tected compound 6 as the major product in 38% yield.
Further treatment of 6 with tetra-n-butylammonium
fluoride in refluxing THF resulted in the formation of
compound 7, having a fused furan ring, as indicated by
the disappearance of the acetylene absorption at 2361
cm-1 in the IR spectrum of 6 and the appearance of an
additional aromatic broad singlet assigned to the furan
1
ring at δ 6.27 in the H NMR spectrum of 7.15,16 The
In an effort to investigate the structural parameters
associated with the biological activities of 2-methoxy-
conversion of 6 to 7 most likely involves deprotonation
of the phenolic hydroxyl group to generate the corre-
sponding phenoxide anion, followed by 5-endo-dig ring
closure.17 2-Methylbenzofuran itself has been prepared
in a related process involving the reaction of 1-(o-
methoxyphenyl)-1-propyne with lithium iodide in re-
fluxing 2,4,6-trimethylpyridine.18 In addition, the con-
† Purdue University.
‡ University of Illinois.
§ Drug Synthesis and Chemistry Branch.
| Laboratory of Drug Discovery Research and Development.
Pharm-Eco Laboratories, Inc.
X Abstract published in Advance ACS Abstracts, J une 15, 1997.
S0022-2623(97)00083-6 CCC: $14.00 © 1997 American Chemical Society

2324 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 15
Cushman et al.
Sch em e 1a
Sch em e 2a
a
Reagents and conditions: (a) (1) NaH, DMF, 0 °C (15 min),
(2) C8H5CH2Br, n-Bu4NI, 2 days, 23 °C; (b) Br2, AcOH, THF, 0 to
23 °C (1.5 h); (c) (1) n-BuLi, THF, -78 °C (2 h), (2) DMF, -78 to
0 °C (1 h); (d) (1) MCPBA, TsOH, CH2Cl2, 23 °C (15 h), (2) MeOH,
H2SO4, reflux (1 h); (e) K2CO3, DMF, CF3CH2I, 110 °C (3 h), 130
°C (2 h); (f) H2, Pd(OH)2-C, THF, 23 °C (24 h).
a
Reagents and conditions: (a) CH3CtCH, Pd(PPh3)4, CuI,
Et3N, THF, 23 °C (41 h); (b) n-Bu4N+F-, THF, 23 °C (24 h); (c)
n-Bu4N+F-, THF, reflux (6.5 h).
version of 6 to 7 is in agreement with several reported
cyclizations of 2-alkynylphenols to benzofurans under
acidic19 and basic12,20-22 conditions, although the tem-
peratures utilized for these reactions (100-171 °C) are
somewhat higher than the temperature of refluxing
THF (66 °C) used here.
resulting formate, yielded the phenol 12.4 The phenolic
hydroxyl group of 12 was then alkylated with 1,1,1-
trifluoro-2-iodoethane using potassium carbonate in
anhydrous DMF as the base. The desired analog 14 was
then obtained after hydrogenolysis of the two benzyl
ether protecting groups with palladium hydroxide on
charcoal as the catalyst.
2′,2′,2′-Trifluoroethoxyestradiol (14) was prepared by
a modification of our previous synthesis of 2-alkoxy-
estradiols involving the 2-formylestradiol dibenzyl ether
intermediate 11 (Scheme 2).4,11 The synthesis of 14 was
prompted by the increased metabolic stability of tri-
fluoroethoxy ethers relative to ethyl ethers.23-25 Whereas
the prior two-step synthesis of intermediate 11 was
carried out in 10% yield by formylation of estradiol and
benzylation of the two hydroxyl groups,4 the present
three-step synthesis afforded the product in 37% overall
yield. The low yield of 11 in the prior synthesis4 was
due to the fact that the direct formylation of estradiol
gave a mixture of 2-formylestradiol and 4-formylestra-
diol, which were separated by chromatographic tech-
niques in low yield. In the present modification, the
two hydroxyl groups of estradiol were protected as
benzyl ethers in 60% yield by deprotonation with sodium
hydride in DMF followed by alkylation of the alkoxide
anions with benzyl bromide.26,27 Bromination of the
resulting intermediate 9 with bromine in acetic acid
gave the 2-bromo derivative 10 in 75% yield. Halogen-
lithium exchange was carried out on the 2-bromo
compound 10 to afford the 2-lithio derivative, which was
formylated with DMF to afford 11 in 82% yield from
10. Baeyer-Villiger oxidation of 11 with m-chloroper-
benzoic acid and a catalytic amount of p-toluenesulfonic
acid in methylene chloride, followed by hydrolysis of the
The syntheses of certain 6-keto, 6-oximino, 6-hydra-
zono, and 6-amino derivatives of 2-ethoxyestradiol and
2-(2′,2′,2′-trifluoroethoxy)estradiol are outlined in Scheme
3. Acetylation of the two hydroxyl groups of 2-ethoxy-
estradiol (2) and 2-(2′,2′,2′-trifluoroethoxy)estradiol (14)
with acetic anhydride in pyridine afforded the corre-
sponding diacetates 15 and 16, respectively. The key
oxidation at the C-6 benzylic carbons of 15 and 16, to
form 17 and 18, respectively, was accomplished with
chromium trioxide in acetic acid.28 The acetates of 17
and 18 were hydrolyzed with potassium hydroxide in
methanol to afford 19 and 20, which were converted to
the corresponding oximes 21 and 22, respectively.
Treatment of 2-ethoxy-6-ketoestradiol (19) with hydra-
zine afforded the hydrazone 23. Sequential reduction
of the oxime group of 2-ethoxy-6-oximinoestradiol (21)
with sodium borohydride in the presence of titanium
tetrachloride,29,30 followed by catalytic hydrogenolysis
over palladium on charcoal, provided the 6-amino
derivative 24.
The triacetate 25 was a minor product obtained
during the conversion of 14 to 16. It resulted from
acetylation of 2,3,17-trihydroxyestra-1,3,5(10)-triene,

Synthesis of Analogs of 2-Methoxyestradiol
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 15 2325
Sch em e 3a
Sch em e 4a
a
Reagents and conditions: (a) NaBH4, MeOH, 23 °C (6 h); (b)
TsNHNH2, AcOH, 90-95 °C, (4.5 h); (d) (1) NaBH4, MeOH, 23 °C
(3.5 h), (2) HCl, THF, 0 °C (45 min).
hydroxysteroid, the expected resonance signal due to the
axial 6â-hydrogen appeared as a broad multiplet at δ
4.60 ppm, which is approximately 0.1 ppm lower field
than the narrower equatorial 6R-hydrogen signal of the
6â-hydroxysteroid epimer. The sodium borohydride
reduction of 6-ketoestradiol derivatives to afford the
corresponding 6R-alcohols is well precedented.31-35
Treatment of 19 with tosylhydrazine in acetic acid
afforded the tosylhydrazone 27. The oxime ether 28 was
obtained by reaction of the ketone 19 with methoxy-
lamine in methanol in the presence of sodium acetate.
The 6,7-dehydro derivative 29 was obtained by sodium
borohydride reduction of the ketone 19 followed by acid-
catalyzed dehydration.
a
Reagents and conditions: (a) Ac2O, pyridine, 110 °C (1 h); (b)
CrO3, AcOH; (c) KOH, MeOH; (d) H2NOH‚HCl, AcONa, MeOH;
(e) H2NNH2, AcOH, EtOH, 95 °C (3 h); (f) (1) TiCl4, NaBH4, DME,
23 °C (3.5 days), (2) H2, Pd/C, THF, 23 °C (20 h).
The synthesis of an analog of 2-ethoxyestradiol con-
taining an additional ethoxy group in the 4-position is
portrayed in Scheme 5. Bromination of estrone (30) in
acetic acid afforded 2,4-dibromoestrone (31), which was
converted to 2,4-diethoxyestrone (32) by reacting 31
with sodium ethoxide in ethanol and DMF using CuI
as the catalyst. Sodium borohydride reduction of 32
yielded the desired 2,4-diethoxyestradiol analog 33. The
17â-configuration of the alcohol 33 is assigned based on
the known sodium borohydride reduction of estrone and
certain estrone derivatives to 17â-alcohols, which is
dictated by the approach of the hydride from the
sterically less hindered side of the carbonyl, opposite
the adjacent angular 13â-methyl group.36-39
The synthesis of an analog of 2-ethoxyestradiol in
which the 17â-hydroxyl group is replaced by a 17â-
amino group is shown in Scheme 6. The 3-hydroxyl
group of 2-ethoxyestradiol (2) was selectively benzylated
with benzyl bromide in ethanol with potassium carbon-
ate as the base to afford 34. The 17â-hydroxyl group of
34 was then oxidized to the ketone 35 with manganese
dioxide in methylene chloride. Reaction of 35 with
present as an impurity in the starting material 14 in
one experiment.
The syntheses of 6-hydroxyl, 6-tosylhydrazono, 6-meth-
oximino, and 6,7-dehydro congeners are reported in
Scheme 4. The 6R-alcohol derivative 26 was obtained
by sodium borohydride reduction of the ketone 19. The
1H NMR spectrum of 26, as compared with that of
commercially available estra-1,3,5(10)-triene-3,6â,17â-
triol (STERALOIDS, Inc.), indicated the presence of
about 20% of 6â-isomer and 80% of 6R-isomer. Re-
peated recrystallization of 26 resulted in material of
90% isomeric purity (6R-hydroxysteroid), which was not
further improved by recrystallization. In the purified
sample of 26, which contained about 90% of the 6R-

2326 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 15
Cushman et al.
Sch em e 5a
a
Reagents and conditions: (a) Br2, Fe, AcOH, 17 °C (30 min);
(b) C2H5ONa, CuI, DMF, C2H5OH; (c) NaBH4, MeOH, 23 °C (5.5
h).
Sch em e 6a
in the NCI screen, in which the activity of each
compound was evaluated with approximately 55 differ-
ent cell lines of diverse tumor origins. In addition, the
new agents were examined for inhibitory effects on the
polymerization of purified bovine brain tubulin under
reaction conditions where 2-methoxyestradiol (1) had
shown maximal inhibitory effect.7 Compounds that
inhibited tubulin assembly were further evaluated for
their ability to inhibit the binding of [3H]colchicine to
tubulin. The activities against tubulin, the mean graph
midpoints (MGMs) for 50% growth inhibition of all
human cancer cell lines successfully tested, and the GI50
values obtained with selected cell lines are summarized
in Table 1. The MGM is based on a calculation of the
average GI50 for all of the cell lines tested (approxi-
mately 55) in which GI50 values below and above the
test range (10-4-10-8 M) are taken as the minimum
(10-8 M) and maximum (10-4 M) drug concentrations
used in the screening test.42 Table 1 includes published
cell line data for 2-methoxyestradiol (1) and 2-ethoxy-
estradiol (2), which was the most active of the 2-meth-
oxyestradiol analogs prepared previously,4 but the an-
titubulin data presented in the table for 1 and 2 were
obtained in experiments performed contemporaneously
with those performed with the new compounds.
a
Reagents and conditions: (a) (1) C6H5CH2Br, K2CO3, EtOH,
reflux (7.5 h); (b) MnO2, CH2Cl2, 23 °C (26 h); (c) C6H5CH2NH2,
NaCNBH3, ClCH2CH2Cl, 23 °C (96 h); (d) (1) H2, Pd(OH)2/C, 23
°C (27 h), (2) HCl, Et2O.
benzylamine afforded an intermediate Schiff base which
was reduced to the secondary amine 36 with sodium
cyanoborohydride. The H NMR spectrum of 36 shows
1
a triplet at about δ 3.0 without any additional shoulder,
suggesting the steroid is predominantly a single dia-
stereomer. We assume that the hydride attacks the
imino carbon from the less sterically hindered R-face of
the molecule, opposite the neighboring axial 13â-methyl
group, to afford the 17â-amine 36.39-41 The benzyl
groups of 36 were cleaved by hydrogenolysis with
palladium hydroxide on charcoal as the catalyst to afford
the corresponding primary amine, which was converted
to the hydrochloride salt 37.
The sulfite 38 was prepared by treatment of 2-ethoxy-
estradiol (2) with thionyl chloride in benzene. Bromi-
nation of estradiol in acetic acid afforded the 2,4-
dibromoestradiol derivative 39.
In h ibition of Tu bu lin Assem bly. Eleven of the
new analogs showed significant activity (defined as IC50
values substoichiometric to the tubulin concentration
of 12 µM) as inhibitors of tubulin polymerization. Our
experience with this assay is that a difference in IC50
Biologica l Resu lts a n d Discu ssion
The newly synthesized compounds were examined for
antiproliferative activity against human cancer cell lines

Synthesis of Analogs of 2-Methoxyestradiol
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 15 2327
Ta ble 1. Cytotoxicities and Antitubulin Activities of 2-Methoxyestradiol and Analogs
cytotoxicity (GI50, µΜ)a
inhibn of tubulin
% inhibn of
colchicine
lung
colon
CNS melanoma ovarian
renal
prostate
breast
polymnc (IC50
,
no. HOP-62 HCT-116 SF-539 UACC-62 OVCAR-3 SN12C DU-145 MDA-MB-435 MGMb
µM ( SD)
bindingd ( SD
1
2
5
0.70
0.018
1.7
19
12
20
0.47
0.026
0.48
0.51
3.2
21
0.02
3.7
0.011
0.031
2.1
40
5.1
0.35
6.7
0.049
1.1
0.80
3.2
0.49
0.32
0.014
0.95
14
1.4
23
0.71
5.1
0.013
0.021
2.8
-
3.5
-
3.5
-
2.0
-
4.0
0.36
0.016
0.50
16
2.3
18
0.01
3.3
0.017
0.015
0.88
19
2.5
0.28
-
0.052
4.8
-
3.6
1.3
0.21
0.016
0.34
18
0.46
24
0.63
3.5
0.017
0.016
5.4
45
6.4
0.35
3.2
0.026
1.9
0.95
0.039
6.9
16
4.5
20
0.18
24
1.8
0.065
3.6
17
7.0
20
0.17
15
0.080
<0.01
0.13
23
0.28
18
<0.01
27
0.010
0.010
6.5
16
0.32
5.4
0.92
0.029
0.34
0.20
1.7
1.3
0.076
1.7
14
2.6
20
0.13
9.8
0.079
0.066
4.2
40
3.7
0.83
6.2
0.27
3.8
2.7
3.3
3.1 ( 0.5
1.3 ( 0.2
4.8 ( 1
>40
19 ( 3
51 ( 6
11 ( 4
7
14
18
19
20
21
22
23
24
25
26e
27
28
29
33
37
38
39
1.5 ( 0.2
>40
11 ( 7
0.03
8.7
4.1 ( 0.8
24 ( 2
1.1 ( 0.02
2.3 ( 0.06
4.3 ( 1
>40
>40
2.9 ( 0.2
>40
1.4 ( 0.1
4.4 ( 1
8.8 ( 2
>40
5.9 ( 1
>40
19 ( 6
1.7 ( 2
63 ( 4
28 ( 3
9.6 ( 4
0.011
0.017
1.2
26
4.8
-
5.1
0.11
3.9
1.3
0.021
0.045
0.026
0.049
17
59
5.1
0.66
12
0.39
4.6
4.8
5.2
2.6
17
>100
3.2
0.71
15
12 ( 2
0.33
2.1
3.3
1.9
1.1
34 ( 1
8.6 ( 6
1.5 ( 2
0.09
1.3
0.27
11
0.83
-
0.57
21
0.22
27
1.1
25
24 ( 3
20
17
17
28
40
a
The cytotoxicity GI50 values are the concentrations corresponding to 50% growth inhibition. b Mean graph midpoint for growth inhibition
of all human cancer cell lines seccessfully tested. c Minimum of two independent determinations. Tubulin concentration was 1.2 mg/mL
(12 µM). Reaction mixtures contained 1.0 µM tubulin (0.1 mg/mL), 5.0 µM [3H]colchicine, and 50 µM inhibitor. Assays were performed
d
with triplicate samples in each experiment. e The sample tested contains 15% of the 6â-alcohol.
values greater than 20% is probably significant. Three
compounds (14, 21, and 28), with IC50 values from 1.1
to 1.5 µM, had activities comparable to that of 2-ethoxy-
estradiol (2) (1.3 µM), and two additional compounds
(22 and 26) had activities between those of 2 and 1 (IC50
value, 3.1 µM). An additional seven compounds (in
order of activity 19, 23, 29, 5, 38, 33, and 20) were less
active than 2-methoxyestradiol (1) as inhibitors of
tubulin polymerization (IC50 values ranging from 4.1 to
24 µM), and another seven were essentially inactive (7,
18, 24, 25, 27, 37, and 39) with IC50 values over 40 µM.
In h ibition of Colch icin e Bin d in g. In the inhibi-
tion of colchicine binding assay, in which compound 2
was significantly more active than 1 in both the previ-
ous4 and current experiments, only compound 21 had
activity greater than that of compound 2. The other two
F igu r e 1. Activities of 2-methoxyestradiol analogs as inhibi-
tors of tubulin assembly compared to their activities as
strongest inhibitors of tubulin assembly had signifi-
cantly weaker inhibitory effects on colchicine binding.
inhibitors of cancer cell growth. The solid symbols represent
Compound 28 had activity intermediate between the
compounds shown in Table 1; the open symbols, compounds
described in ref 4. The upright triangles represent the data
activities of 1 and 2, while 14 had less inhibitory activity
than 2-methoxyestradiol (1). Three additional com-
pounds (19, 22, and 38) had inhibitory effects on
colchicine binding comparable to that of 1, while the
remainder of those examined had minimal or negligible
inhibitory effects.
In h ibition of Ca n cer Cell Gr ow th . Selected an-
tiproliferative data obtained in the in vitro cancer cell
screen42 of the NCI Developmental Therapeutics Pro-
gram are summarized in Table 1 [GI50 results with
HOP-62 (non-small-cell lung), HCT-116 (colon), SF-59
(central nervous system), UACC-62 (melanoma),
OVCAR-3 (ovarian), SN12C (renal), DU-145 (prostate),
and MDA-MB-435 (breast), together with the MGM
values for 50% inhibition of cell growth of all of the cell
lines tested].
Two of the new compounds had antiproliferative
activity comparable to that of 2-ethoxyestradiol (2),
which was the most active of the analogs prepared
previously (MGM, 76 nM).4 These were the two 6-ox-
imino derivatives 21 and 22. MGM values intermediate
between those of 2 and 2-methoxyestradiol (1) (1.3 µM)
obtained with 2-methoxyestradiol (1); the inverted triangles,
the data obtained with 2-ethoxyestradiol (2).
were obtained with compounds 19, 26, 28, and 38. The
remaining compounds were less inhibitory for cell
growth than 2-methoxyestradiol. The highest MGM
value was 40 µM for compound 24.
Cor r ela tion of Cell Gr ow th Effects w ith An titu -
bu lin Activity. There appears to be a rough correla-
tion between the inhibition of tubulin polymerization
and cytotoxicity in the 2-methoxyestradiol series of
compounds. Figure 1 displays this graphically. All of
the compounds having MGM values less than 1 µM had
IC50 values for inhibition of tubulin polymerization of
4.1 µM or less. On the other hand, all of the compounds
with IC50 values greater than 40 µM for inhibition of
tubulin polymerization had MGM values of 3.3 µM or
greater. A low IC50 value for inhibition of tubulin
polymerization seems to be a necessary, but not suf-
ficient condition for potent cytotoxicity. The chief
problem with the assembly assay data is that it fails to

2328 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 15
Cushman et al.
Ta ble 2. Relative Estrogen Receptor Binding Affinities of
Selected 2-Substituted Estradiol Analog Tubulin
Polymerization Inhibitors
distinguish agents with nanomolar effects on cell growth
(MGM values < 0.5 µM) from those that require low
micromolar concentrations to inhibit cell growth.
compd
RBAa (0 °C)
There appears to be less correlation in the current
study than previously between inhibition of colchicine
binding and inhibition of either tubulin polymerization
or cell growth. In the earlier study,4 all three com-
pounds more active than 1 as polymerization inhibitors
also inhibited colchicine binding more strongly than 1.
With the preparation of five additional agents that
inhibit assembly better than 1 (compounds 14, 21, 22,
26 and 28), this observation no longer holds. Only
compounds 21 and 28, and perhaps 22 as well, are more
active than 1 as inhibitors of colchicine binding.
1
2
21
22
28
0.245b
0.011b
<0.001
0.007
<0.001
a
Relative binding affinity determined at 0 °C in a competitive
radiometric assay, estradiol ) 100; details are given in the
Experimental Section and in reference 29. b Data published previ-
ously in ref 4.
roethoxy analog (14), the latter differed little in its effect
on tubulin assembly, was significantly less potent as an
inhibitor of colchicine binding, and was substantially
(over 30-fold) less cytotoxic than 2. Finally, comparing
the 6-keto derivatives 19 (2-ethoxy) and 20 [2-(2′,2′,2′-
trifluoroethoxy)], the 2-ethoxy compound was substan-
tially more active than 20 in both tubulin and cell-based
assays.
Str u ctu r e-Activity Cor r ela tion . The synthetic
chemistry effort summarized here has yielded two
additional compounds comparable to 2-ethoxyestradiol
as inhibitors of the growth of human tumor cells. These
are the two 6-oximino derivatives 21 and 22, which are
also among the most potent inhibitors of tubulin as-
sembly. On the basis of the polymerization assay and
also the colchicine binding assay, 21 has a higher
affinity for tubulin than does either 22 or 2. Compounds
21 and 22 differ structurally only in the C-2 substituent,
an ethoxy group in the former and a 1,1,1-trifluoro-
ethoxy group in the latter.
Compound 5 represents an additional extension of the
earlier study,4 which primarily examined the effects of
C-2 substituents. At that time, we found that the most
active agents were 2-ethoxyestradiol (2) and 2-[(E)-1′-
propenyl]estradiol (41), both of which have three-atom
side chains. In the prior investigation, the 2-[(E)-1′-
propenyl] analog 41 displayed an IC50 value of 1.1 µM
for inhibition of tubulin polymerization.4 The 1′-pro-
pynyl substituent at C-2 in 5 resulted in decreased
interaction with tubulin and decreased cytotoxicity
relative to the 2-ethoxy and 2-[(E)-1′-propenyl] com-
pounds. Compound 5 and the corresponding 2-n-propyl
analog4 had nearly identical, diminished effects on
tubulin assembly and cell growth relative to the more
active 2-[(E)-1′-propenyl] analog 41.4 It is interesting to
note that the conformationally restricted analog 7 of the
2-[(E)-1′-propenyl] analog 41 was inactive. The inactiv-
ity of 7 could be due to the effect of restricted rotation
of the conformationally free C-2-C-1′ single bond
present in 41. This would indicate that the biological
activity of the 2-[(E)-1′-propenyl] analog 41 is due to a
specific conformation about the C-2 to C-1′ single bond.
On the other hand, the inactivity of 7 might simply
indicate a requirement for a free hydroxyl group at C-3
for activity.
Finally, compound 38 deserves mention. Although
modification at C-17 has generally resulted in substan-
tially reduced activity,4,5 compound 38, a dimer of
2-ethoxyestradiol (2), was comparable to 2-methoxy-
estradiol (1) as an inhibitor of cancer cell growth and
of colchicine binding to tubulin and half as active as an
inhibitor of assembly. These activities may result from
hydrolysis of 38 to 2, but the compound or a related
dimer may be worth considering as a possible strategy
for cross-linking tubulin molecules.
Affin ities for Estr ogen Recep tor . One potential
concern about the present series of tubulin polymeri-
zation inhibitors is that they might have some affinity
for estrogen receptors and therefore possess either
estrogenic or antiestrogenic activity or be estrogen
antagonists, thus compromising their use as anticancer
drugs with selective actions. We had previously shown4
that 1 and 2 had limited affinity for the estrogen
receptor, and the data shown in Table 2 extends this
observation to the most potent compounds in the present
series, namely analogs 21, 22, and 28. The previously
The effect of the oximino substituent at C-6 can be
evaluated by comparison of 2 vs 21 and 14 vs 22. The
effect on tubulin binding and polymerization is small,
but the increase in cytotoxicity resulting from oxime
incorporation at C-6 is dramatic in the case of 14 vs 22.
The reason for the increase in cytotoxicity in this case
is not known, but it does not appear to result from a
direct effect on tubulin polymerization.
Considering only C-6 modifications with a C-2 ethoxy
group, replacing the oximino group of 21 with a meth-
oximino group in 28 or a keto group in 19 only modestly
reduced inhibitory effects in both the cell growth and
tubulin assays. Comparing a C-6 R-hydroxy group in
26 with the C-6 keto group in 19, the former was slightly
more potent as an inhibitor of assembly and the latter
as an inhibitor of cell growth. Replacement of the
oximino group of 21 with either a hydrazone group in
23 or a C-6/C-7 double bond with no C-6 substituent in
29 yielded compounds with inhibitory effects on tubulin
similar to that of the 6-keto derivative 19, but with over
a 30-fold reduction in antiproliferative activity relative
to 19. Compounds with a bulky C-6 substituent (the
tosylhydrazone 27) or a C-6 amino group (the amine 24)
had little activity as inhibitors either of cell growth or
tubulin polymerization. This variability of the C-6
effects may support the hypothesis5 that the A ring of
methoxyestradiol (1) interacts with the same portion
of the tubulin molecule as the C ring of colchicine (40).
The C-6 substituents in 2-methoxyestradiol analogs are
probably analogous to C-7 side chains in colchicinoids.
The effect of the 2′,2′,2′-trifluoroethoxy group replac-
ing the ethoxy group was somewhat unpredictable.
Analogs with this substituent were therefore prepared
in order to investigate whether they would retain
activity against cancer cells and tubulin. Comparing
the 6-oximino derivatives 21 (ethoxy) and 22 (trifluo-
roethoxy), the ethoxy compound 21 had a moderately
higher affinity for tubulin (2-fold lower IC50 value in
assembly), while both compounds had similar cytotox-
icity. Comparing 2-ethoxyestradiol (2) with the trifluo-

Synthesis of Analogs of 2-Methoxyestradiol
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 15 2329
were extracted with ether (2 × 20 mL). The combined ether
layer was washed with aqueous saturated NaCl solution (20
mL) and dried over anhydrous Na2SO4. Evaporation of the
filtrate gave the crude product as an oil, which was subjected
to preparative TLC purification (silica gel uniplate 1000
microns, ether-hexane, 1:3, by volume) to give compound 5
(20 mg, 17%) as a yellow solid: mp 96-98 °C. Analytical
HPLC (C18 reverse phase; solvent A, 0.1% TFA in water;
solvent B, 0.1% TFA in acetonitrile; gradient, 0-90% B in 30
min at a flow rate of 1 mL/min) indicated that compound 5
was pure: 1H NMR (300 MHz, CDCl3) δ 7.21 (s, 1 H), 6.62 (s,
1 H), 5.59 (s, 1 H, exchangeable), 3.72 (t, J ) 8.6 Hz, 1 H),
2.81 (m, 2 H), 2.27 (m, 1 H), 2.11 (s, 3 H), 2.10 (m, 2 H), 1.90
(m, 2 H), 1.68 (m, 1 H), 1.60-1.10 (m, 7 H), 0.90 (m, 1 H),
0.77 (s, 3 H); LRCIMS (isobutane) m/ z (rel intensity) 311
(MH+, 84); HRCIMS (isobutane) calcd C21H27O2 MH+ m/ z
311.2011, found 311.1998.
obtained relative estrogen receptor binding affinities of
2-methoxyestradiol (1) and 2-ethoxyestradiol (2) are
also presented in Table 2. To obtain these data,
comparative radiometric binding assays were employed
using rat uterine cytosol at 0 °C to determine the
relative binding affinities (RBA) as compared to estra-
diol (RBA ) 100, Kd ) 0.3 nM). Both 2-ethoxy-6-
oximinoestradiol (21) and 2-ethoxy-6-methoximinoestra-
diol (28) had no affinity for the estrogen receptor within
the limits of the assay (RBA < 0.001), while 2-(2′,2′,2′-
trifluoroethoxy)-6-oximinoestradiol (22) displayed very
weak affinity for the receptor (RBA ) 0.007). These new
analogs had even lower affinity for the estrogen receptor
than either of the very weak binders 2-methoxyestradiol
(1) (RBA ) 0.245) or 2-ethoxyestradiol (2) (RBA )
0.011). These results make it clear that effects due to
estrogen receptor binding should not be a factor in the
therapeutic application of compounds 21, 22, and 28.
Con clu sion . An array of new estradiol analogs has
been synthesized in an effort to improve on the anti-
cancer and antitubulin activities of 2-methoxyestradiol
(1) and 2-ethoxyestradiol (2) and to further decrease
their affinity for the estrogen receptor. These objectives
have resulted in compounds 21, 22, and 28, which have
negligible estrogen receptor affinities. These com-
pounds, along with 2-ethoxyestradiol (2), are being
investigated in vivo as anticancer agents.
The above preparative TLC also gave compound 6 (33.4 mg,
38%) as a pale yellow solid: mp 168-170 °C; IR (neat) 2927,
2361, 1725, 1676, 1604, 1492, 1460, 1256, 1094, 888, 836, 776
cm-1; 1H NMR (300 MHz, CDCl3) δ 7.20 (s, 1 H), 6.63 (s, 1 H),
5.58 (s, 1 H), 3.62 (t, J ) 8.6 Hz, 1 H), 2.79 (m, 2 H), 2.15 (m,
2 H), 2.12 (s, 3 H), 1.85 (m, 4 H), 1.65-1.05 (m, 7 H), 0.89 (s,
9 H), 0.72 (s, 3 H), 0.05 (s, 3 H), 0.04 (s, 3 H); LRCIMS
(isobutane) m/ z 424 (M+, minute), 367 (M+ - Me3C, minute),
t
291 (M+ - BuMe2SiOH, 2).
2′-Meth ylfu r a n o[2,3-b]-1,3,5(10)-estr a tr ien -17â-ol (7). A
solution of 6 (60.0 mg, 0.14 mmol) in THF (10 mL) containing
TBAF (1.0 M in THF, 0.3 mL, 2.1 equiv) was heated at reflux
under Ar for 6.5 h. The reaction mixture was diluted with
aqueous saturated NaCl solution (10 mL) and extracted twice
with ether (30, 20 mL). The combined ether layer was washed
with aqueous saturated NaCl solution (2 × 20 mL) and dried
over anhydrous Na2SO4. Evaporation of the filtrate gave the
crude product as an oil. Preparative TLC purification (silica
gel uniplate 1000 µm, ether-hexane, 1:2 by volume) gave
compound 7 (40 mg, 92%) as a yellow solid after recrystalli-
zation from CHCl3-ether-hexane: mp 126-128 °C; IR (neat)
Exp er im en ta l Section
Melting points were determined in capillary tubes on a Mel-
Temp apparatus and are uncorrected. Spectra were obtained
as follows: CI mass spectra on a Finnegan 4000 spectrometer;
FAB mass spectra and EI mass spectra on a Kratos MS50
spectrometer; 1H NMR spectra on a Varian VXR-500S or XL-
200A spectrometer or on a Bruker AC 300 spectrometer; IR
spectra on a Beckman IR-33 spectrophotometer, a Perkin-
Elmer 1600 series FTIR spectrophotometer, or a Nicolet FT-
IR Impact 410 spectrophotometer. Microanalyses were per-
formed at the Purdue Microanalysis Laboratory or by Micro-
Atlantic Laboratories, Atlanta, and all values were within
(0.4% of the calculated compositions.
3,17â-Bis[(ter t-bu tyld im eth ylsilyl)oxy]-2-(1-p r op yn yl)-
1,3,5(10)-estr a tr ien e (4). To a solution of 2-iodoestradiol
derivative 3 (3.04 g, 4.85 mmol) in anhydrous THF (80 mL)
were added Et3N (0.85 mL, 6.06 mmol, 1.25 equiv) and Pd-
(PPh3)4 (566 mg, 99%, 0.49 mmol, 0.1 equiv). The reaction
mixture was stirred at room temperature under Ar for 0.5 h.
A dark yellow solution developed. Propyne gas (25 g, 620
mmol, 128 equiv) was bubbled into the reaction mixture at
room temperature. CuI (745.4 mg, 3.9 mmol, 0.8 equiv) was
added, and the reaction mixture was stirred at room temper-
ature under Ar for 41 h. The reaction mixture was diluted
with saturated aqueous NaCl solution (50 mL) and extracted
twice with ether (100, 50 mL). The combined ether layer was
washed with aqueous saturated NaCl solution (2 × 30 mL)
and dried over anhydrous Na2SO4. Evaporation of the filtrate
gave the crude product as a black solid. Flash chromatography
(silica gel; ether-hexane, 1:60 to 1:30 by volume) gave
compound 4 (0.31 g, 12%) as a pale yellow solid: mp 56 °C; IR
(neat) 3419, 2363, 1654, 999 cm-1; 1H NMR (300 MHz, CDCl3)
δ 7.22 (s, 1 H), 6.48 (s, 1 H), 3.62 (t, J ) 8.6 Hz, 1 H), 2.76 (m,
2 H), 2.25 (m, 1 H), 2.10 (m, 1 H), 2.04 (s, 3 H), 1.86 (m, 3 H),
1.70-1.10 (m, 8 H), 1.02 (s, 9 H), 0.89 (s, 9 H), 0.73 (s, 3 H),
0.20 (s, 6 H), 0.03 (s, 3 H), 0.02 (s, 3 H); LRCIMS (isobutane)
m/ z (rel intensity) 539 (MH+, 56).
1
2924, 2867, 1605, 1467, 1433, 1264, 1054, 873, 732 cm-1; H
NMR (300 MHz, CDCl3) δ 7.37 (s, 1 H), 7.10 (s, 1 H), 6.27 (s,
1 H), 3.73 (t, J ) 8.2 Hz, 1 H), 2.95 (m, 2 H), 2.41 (s, 3 H), 2.30
(m, 2 H), 2.11 (m, 1 H), 1.95 (m, 2 H), 1.75-1.15 (m, 9 H),
0.78 (s, 3 H); LRCIMS (isobutane) m/ z 311 (MH+, 20), 293
(MH - H2O, 100). Anal. (C21H26O2) C, H.
â-Estr a d iol Diben zyl Eth er (9). Sodium hydride (2.6 g,
65 mmol) was added in four portions to a solution of â-estradiol
(8, 5.0 g, 18 mmol) in anhydrous DMF (150 mL) under Ar at
0 °C, and the resulting mixture was stirred at 0 °C for 15 min.
Benzyl bromide (12.5 mL, 105 mmol) was added, followed by
tetrabutylammonium iodide (0.50 g, 1.35 mmol). Stirring was
continued for 2 days at room temperature. The reaction
mixture was cooled to 0 °C, and 50% aqueous ethanol (20 mL)
was added slowly, followed by dropwise addition of 3 N HCl
(18 mL) at 0-5 °C. The compound was extracted into ether
(3 × 200 mL). The ether layer was washed with water (100
mL) and brine (100 mL) and dried over anhydrous sodium
sulfate. Evaporation of the ether layer under reduced pressure
gave a viscous oil. Trituration with hexane afforded a white
solid (5.0 g, 60%), which was collected by filtration and dried
under vacuum: mp 74-78 °C [lit.26 mp 81-82 °C (ethanol,
acetone); lit.27 mp 62 °C (aqueous methanol)]; Rf 0.334 [hex-
ane-ethyl acetate (95:5), silica gel]; IR (KBr) 2910, 2850, 1600,
1
745, 685 cm-1; H NMR (CDCl3) δ 0.9 (s, 3 H), 1.05-2.45 (m,
14 H), 2.83 (m, 1 H), 3.5 (t, J ) 8.5 Hz, 1 H), 4.57 (s, 2 H), 5.05
(s, 2 H), 6.67-6.82 (m, 2 H), 7.12-7.57 (m, 11 H). Anal.
(C32H36O2) C, H.
2-Br om o-â-estr a d iol Diben zyl Eth er (10). Bromine (0.58
mL, 11.3 mmol) was added by syringe to an ice-cold, stirred
solution of 8 (4.5 g, 10 mmol) in acetic acid-THF mixture (3:
2, 45 mL). The resulting reaction mixture was allowed to
warm to room temperature with stirring. After 1.5 h, the
mixture was poured onto an ice-water mixture (200 mL), and
compound was extracted with dichloromethane (3 × 200 mL).
The combined organic layer was washed with water (200 mL),
saturated aqueous sodium bicarbonate (200 mL), 10% aqueous
sodium thiosulfate (150 mL), water (100 mL), and brine (100
mL) and dried over anhydrous sodium sulfate. The organic
2-(1-P r op yn yl)-1,3,5(10)-estr a tr ien e-3,17â-d iol (5) a n d
17â-[(ter t-b u t yld im et h ylsilyl)oxy]-2-(1-p r op yn yl)-1,3,5-
(10)-estr a tr ien -3-ol (6). A solution of the silyl ether 4 (200
mg, 0.37 mmol) in THF (20 mL) containing TBAF (1.0 M in
THF, 1.5 mL, 2 equiv) was stirred at room temperature for 24
h. The reaction mixture was acidified to pH 5 with 2 N HCl,
saturated NaCl solution (20 mL) was added, and the products

2330 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 15
Cushman et al.
layer, on evaporation under vacuum, followed by trituration
with hexane-ethyl acetate mixture, gave the product 10 (3.9
g, 75%). An analytical sample was prepared by crystallization
from ethyl acetate: mp 156-158 °C; IR (KBr) 2960-2840,
(CDCl3) δ 0.9 (s, 3 H), 1.1-2.4 (m, 13 H), 2.65-2.85 (m, 2 H),
3.50 (t, J ) 8.5 Hz, 1 H), 4.40 (q, J ) 8.5 Hz, 2 H), 4.56 (s, 2
H), 5.05 (s, 2 H), 6.70 (s, 1 H), 6.98 (s, 1 H), 7.2-7.55 (m, 10
H). Anal. (C34H37 F3O3) C, H.
1
1600, 1590, 730-720, 685 cm-1; H NMR (CDCl3) δ 0.9 (s, 3
2-(2′,2′,2′-Tr iflu or oeth oxy)estr a -1,3,5(10)-tr ien e-3,17â-
d iol (14). Pd(OH)2-C (20%, 1.0 g) was added carefully under
Ar to a solution of 13 (1.0 g, 1.8 mmol) in anhydrous THF (50
mL). The resulting mixture was hydrogenated at 45-50 psi
on a Parr apparatus for 24 h. The catalyst was removed by
filtration using a Celite pad under Ar, and the pad was washed
with dichloromethane (200 mL). Evaporation of the filtrate
under reduced pressure, followed by purification on a silica
gel column, gave compound 14 (0.5 g, 75%): mp 167-168 °C;
H), 1.10-2.40 (m, 13 H), 2.75-2.85 (m, 2 H), 3.5 (t, J ) 8.5
Hz, 1 H), 4.6 (s, 2 H), 5.63 (s, 2 H), 6.67 (s, 1 H), 7.25-7.60
(m, 11 H). Anal. (C32H35BrO2) C, H.
2-For m yl-3,17â-bis(ben zyloxy)estr a-1,3,5(10)-tr ien e (11).
A 1.6 M solution of n-BuLi (17.5 mL) in hexanes was added
dropwise by syringe under Ar at -78 °C to a solution of
compound 10 (5.88 g, 11.2 mmol) in anhydrous THF (150 mL),
and the resulting reaction mixture was stirred at -78 °C for
2 h. Anhydrous DMF (5.5 mL) was added, and stirring was
continued for 1 h at -78 °C. The mixture was warmed to 0
°C, stirred for 1 h, and poured onto an ice-cold solution of 3 N
HCl (100 mL). The aqueous layer was extracted with ether
(3 × 300 mL). The combined organic layer was washed with
50% brine-water (100 mL) and brine (150 mL) and dried over
anhydrous sodium sulfate. The organic layer was filtered and
evaporated under reduced pressure to afford impure 11, which
was purified over a silica gel column using hexane-ethyl
acetate as eluant. Appropriate fractions were combined and
evaporated under reduced pressure to afford compound 11
(4.36 g, 82%): mp 145-147 °C; IR (KBr) 2920, 2820, 1675,
1600,728, 685 cm-1; 1H NMR (CDCl3) δ 0.88 (s, 3 H), 1.1-2.5
(m, 13 H) , 2.80-2.97 (m, 2 H), 3.50 (t, J ) 8.5 Hz, 1 H), 4.56
(s, 2 H), 5.65 (s, 2 H), 6.75 (s, 1 H), 7.20-7.50 (m, 10 H), 7.78
(s, 1 H), 10.50 (s, 1 H). Anal. (C33H36O3) C, H.
2-H yd r oxy-3,17â-b is(b en zyloxy)est r a -1,3,5(10)-t r ien e
(12). MCPBA (3.3 g, 19.1 mmol) and p-toluenesulfonic acid
monohydrate (0.16 g, 0.84 mmol) were added successively to
a solution of compound 11 (5.8 g, 12.1 mmol) in anhydrous
dichloromethane (150 mL). The resulting mixture was stirred
at room temperature, and the reaction was followed by TLC
(dichloromethane, silica gel). After 10 h, additional amounts
of MCPBA (1.0 g) and p-toluenesulfonic acid (0.040 g) were
added, and stirring was continued for 5 h. The reaction
mixture was diluted with dichloromethane (300 mL), and the
organic layer was washed with 10% sodium sulfite solution
(100 mL), water (100 mL), and brine (100 mL) and dried over
anhydrous sodium sulfate. The organic layer on evaporation
under reduced pressure afforded crude compound 12 (4.85 g).
This was suspended in anhydrous methanol (250 mL), four
drops of concentrated H2SO4 were added, and the resulting
mixture was stirred at reflux for 1 h. The methanol was
removed under reduced pressure at 50 °C, and the resulting
residue was dissolved in dichloromethane (400 mL). The
organic layer was washed with water (100 mL), saturated
sodium bicarbonate solution (100 mL), water (100 mL), and
brine (100 mL) and dried over anhydrous sodium sulfate. The
dichloromethane layer on evaporation under reduced pressure
gave crude compound 12 (4.15 g) as a gummy solid, which on
purification over a silica gel column using hexane-dichlo-
romethane mixture as an eluant provided pure product 12 (3.8
g, 67%) as a white solid: mp 117-118 °C; Rf 0.364 (dichlo-
romethane, silica gel); IR (KBr) 3540, 2960-2840, 1510, 1450,
735-720, 685 cm-1; 1H NMR (CDCl3) δ 0.9 (s, 3 H), 1.05-2.40
(m, 14 H), 2.80 (m, 1 H), 3.50 (t, J ) 8.5 Hz, 1 H), 4.60 (s, 2
H), 5.08 (s, 2 H), 5.5 (s, 1 H), 6.65 (s, 1 H), 6.82 (s, 1 H), 7.20-
7.50 (m, 10 H). Anal. (C32H36O3) C, H.
IR (KBr) 3550, 2960-2840, 1590, 1510, 870 cm-1 1H NMR
;
(CDCl3) δ 0.80 (s, 3 H), 1.10-2.40 (m, 13 H), 2.70-2.85 (m, 2
H), 3.75 (m, 1 H), 4.40 (q, J ) 8.5 Hz, 2 H), 5.35 (s, 1 H, OH),
5.5 (s, 1 H), 6.70 (s, 1 H), 6.80 (s, 1 H); MS m/ z (relative
intensity) 370 (100), 311 (13), 270 (8), 244 (8), 205 (6). Anal.
(C20H25O3‚0.7H2O) C, H.
2-Eth oxy-3,17â-bis(acetyloxy)estr a-1,3,5(10)-tr ien e (15).
Acetic anhydride (1.7 mL, 18 mmol) was added dropwise under
Ar at 0 °C to a solution of 2-ethoxyestra-1,3,5(10)-triene-3,17â-
diol (2) (0.44 g, 1.4 mmol) in anhydrous pyridine (6 mL). The
resulting mixture was stirred at 110 °C for 1 h. The reaction
mixture was cooled to room temperature and poured onto ice
cold 3 N HCl (50 mL). The compound was extracted with ethyl
acetate (150 mL × 2). The combined organic layer was washed
with water (80 mL), sodium bicarbonate (100 mL), water (80
mL), and brine (100 mL) and dried over anhydrous sodium
sulfate. The ethyl acetate layer, on evaporation under reduced
pressure, gave compound 15 (0.50 g, 90%): mp 135-136 °C;
Rf 0.66 [hexane-ethyl acetate (7:3), silica gel]; IR (KBr) 2970-
2860, 1765, 1725, 1505, 920, 885 cm-1; 1H NMR (CDCl3) δ 0.87
(s, 3 H), 1.15-2.00 (m, 13 H), 2.10 (s, 3 H), 2.12-2.38 (m, 3
H), 2.35 (s, 3 H), 2.70-2.90 (m, 2 H), 4.05 (q, J ) 6.4 Hz, 2 H),
4.71 (t, J ) 8.5 Hz, 1 H), 6.75 (s, 1 H), 6.9 (s, 1 H). Anal.
(C24H32O5) C, H.
3,17â-Bis(a cet yloxy)-2-(2′,2′,2′-t r iflu or oet h oxy)est r a -
1,3,5(10)-tr ien e (16). Acetic anhydride (10 mL) was added
dropwise under Ar at 0 °C to a solution of 2-(trifluoroethoxy)-
estra-1,3,5(10)-triene-3,17â-diol (14) (2.50 g, 6.7 mmol) in
anhydrous pyridine (20 mL). The resulting mixture was
stirred at 110 °C for 1 h. The reaction mixture was cooled to
room temperature and poured onto ice cold 3 N HCl (100 mL).
The compound was extracted into ether (2 × 250 mL). The
combined organic layer was washed with water (60 mL),
sodium bicarbonate (60 mL), water (60 mL), and brine (80 mL)
and dried over anhydrous magnesium sulfate. The ether layer,
on evaporation under reduced pressure, gave an almost
quantitative yield (2.5 g) of compound 16. An analytical
sample was prepared by purification by column chromatog-
raphy: mp 116-118 °C; Rf 0.63 [hexane-ethyl acetate (8:2),
silica gel]; IR (KBr) 2960-2840, 1755, 1705, 1490, 950 cm-1
;
1H NMR (CDCl3) δ 0.85 (s, 3 H), 1.15-2.00 (m, 10 H), 2.05 (s,
3 H), 2.15-2.40 (m, 6 H), 2.70-2.90 (m, 2 H), 4.30 (q, J ) 8.5
Hz, 2 H), 4.70 (t, J ) 8.5 Hz, 1 H), 6.80 (s, 1 H), 6.95 (s, 1 H).
Anal. (C24H29F3O5) C, H.
2-E t h oxy-3,17â-d ia cet oxy-6-oxoest r a -1,3,5(10)-t r ien e
(17). A solution of chromium trioxide (1.6 g, 16 mmol) in 90%
glacial acetic acid (7.5 mL) was added dropwise at 15 °C to a
solution of 2-ethoxy-3,17â-diacetyl derivative 15 (1.5 g, 3.7
mmol) in glacial acetic acid (12 mL). The resulting mixture
was stirred at 15 °C for 35 min. The mixture was poured onto
an ice-water mixture (150 mL), and the compound was
extracted into ethyl acetate (3 × 180 mL). The combined
organic layer was washed with water (100 mL), NaHCO3
solution (100 mL), water (100 mL), and brine (100 mL) and
dried over anhydrous sodium sulfate. The organic layer, upon
evaporation under reduced pressure, provided a crude com-
pound, which on purification on a silica gel column using
hexane-ethyl acetate mixture as eluant gave 1.20 g (55%) of
compound 17: mp 195 °C; IR (KBr) 2980-2810, 1765, 1730,
1715, 1665, 1600, 1500, 918, 880 cm-1; 1H NMR (CDCl3) δ 0.89
(s, 3 H), 1.30-1.85 (m, 9 H), 1.90-2.45 (m, 11 H), 2.47-2.85
(m, 2 H), 4.15 (q, J ) 6.4 Hz, 2 H), 4.78 (t, J ) 8.5 Hz, 1 H),
6.95 (s, 1 H), 7.77 (s, 1 H). Anal. (C24H30O6) C, H.
2-(2′,2′,2′-Tr iflu or oeth oxy)-3,17â-bis(ben zyloxy)estr a -
1,3,5(10)-tr ien e (13). Powdered K2CO3 (1.5 g, 11 mmol) was
added to a solution of compound 12 (4.0 g, 8.5 mmol) in
anhydrous DMF (50 mL), followed by dropwise addition of CF3-
CH2I (5.0 mL, 51 mmol) at room temperature. The resulting
reaction mixture was heated at 110 °C for 3 h. Additional K2-
CO3 (2.5 g) and CF3CH2I (6 mL) were added, and the mixture
was heated again at 130 °C for 2 h. The mixture was cooled
in an ice bath and poured onto ice cold 3 N HCl (125 mL). The
aqueous layer was extracted with ether (2 × 200 mL). The
combined ether layer was washed with 3 N HCl (100 mL),
water (100 mL), and brine (100 mL) and dried over anhydrous
MgSO4. The ether layer was evaporated under reduced
pressure, and the resulting crude product on purification on
a silica gel column gave product 13 (4.5 g, 95%) as a colorless
oil: IR (neat) 2910, 2850, 1600, 1500, 730, 690 cm-1; 1H NMR

Synthesis of Analogs of 2-Methoxyestradiol
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 15 2331
3,17â-Bis(a cetyloxy)-2-(2′,2′,2′-tr iflu or oeth oxy)-6-estr a -
1,3,5(10)-tr ien e (18). A solution of chromium trioxide (2.0
g, 20 mmol) in 90% glacial acetic acid (10 mL) was added
dropwise at 9-10 °C to a solution of 2-(trifluoroethoxy)-3,17â-
O-diacetyl derivative 16 (2.1 g, 4.6 mmol) in glacial acetic acid
(14 mL). The resulting mixture was stirred at 10 °C for 60
min. The mixture was poured onto an ice-water mixture (150
mL), and the compound was extracted into ethyl acetate (3 ×
200 mL). The combined organic layer was washed with water
(100 mL), NaHCO3 solution (100 mL), water (100 mL), and
brine (100 mL) and dried over anhydrous sodium sulfate. The
organic layer, on evaporation under reduced pressure, provided
a residue, which on purification over a silica gel column using
hexane-ethyl acetate mixture as eluant gave the desired
compound 18 (1.35 g, 61%): mp 131-133 °C; Rf 0.59 [hexane-
ethyl acetate (7:3), silica gel]; IR (KBr) 2960-2840, 1770, 1730,
1675, 1610, 1500, 890, 850 cm-1; 1H NMR (CDCl3) δ 0.83 (s, 3
H), 2.83-1.27 (m, 19 H), 4.45 (q, J ) 8.5 Hz, 2 H), 4.75 (t, J )
8.5 Hz, 1 H), 6.93 (s, 1 H), 7.80 (s, 1 H). Anal. (C24H27 F3O6)
C, H.
6-(Oxim in o)-2-(2′,2′,2′-t r iflu or oet h oxy)est r a -1,3,5(10)-
tr ien e-3,17â-d iol (22). Sodium acetate (2.86 g, 35 mmol) was
added to a stirred solution of 6-oxo derivative 20 (0.45 g, 1.2
mmol) in methanol (55 mL). Hydroxylamine hydrochloride
salt (2.2 g, 32 mmol) in water (3 mL) was added at room
temperature. The mixture was stirred at 90 °C for 6 h.
Solvent was removed under reduced pressure, the resulting
residue was diluted with water (60 mL), and the product was
extracted into ethyl acetate (3 × 120 mL). The combined
organic layer was washed with water (70 mL) and brine (70
mL) and dried over anhydrous sodium sulfate. The organic
layer on evaporation under reduced pressure provided the
crude product, which on crystallization from ethyl acetate-
hexane mixture gave oxime 22 (0.3 g, 65%): mp 187-191 °C;
IR (KBr) 3330, 2960-2860, 1500, 880, 750 cm-1
;
1H NMR
(CDCl3 + DMSO-d6 + D2O) δ 0.77 (s, 3 H), 1.10-2.38 (m, 11
H), 2.67 (s, 2 H), 3.05-3.27 (m, 2 H), 3.72 (t, J ) 8.5 Hz, 1 H),
4.50 (q, J ) 8.5 Hz, 2 H), 6.88 (s, 1 H), 7.57 (s, 1 H), 10.20 (s,
1 H). Anal. (C20H24NF3O4) C, H.
2-E t h oxy-6-oxoest r a -1,3,5(10)-t r ien e-3,17â-d iol 6-H y-
d r a zon e (23). Glacial acetic acid (0.5 mL) and anhydrous
hydrazine (0.2 mL) were added under Ar to a suspension of
6-oxo derivative 19 (0.4 g, 1.2 mmol) in anhydrous ethanol (25
mL). The mixture was stirred at 95 ( 5 °C. After 2 h,
anhydrous hydrazine (0.1 mL) was added to the reaction
mixture, and the reaction was continued for 1 h. The solvent
was removed under reduced pressure, and the residue was
diluted with water (25 mL). The aqueous solution was
extracted with dichloromethane (3 × 100 mL). The combined
organic layer was washed with water (100 mL) and dried over
anhydrous sodium sulfate. The organic layer on evaporation
under reduced pressure gave the desired hydrazone 23 (364
mg, 84%): mp 132-138 °C; Rf 0.24 [ethyl acetate-hexane (4:
1), silica gel]; IR (KBr) 3370, 2960, 2860, 1610, 1570, 1500
2-Eth oxy-6-oxoestr a -1,3,5(10)-tr ien e-3,17â-d iol (19). A
20% solution of KOH in methanol (5 mL) was added dropwise
to a suspension of compound 17 (0.95 g, 2.3 mmol) in
anhydrous methanol (12 mL). The reaction mixture was
stirred at room temperature for 4 h. The solvent was removed
under reduced pressure at 42-45 °C. The residue was diluted
with an ice-water mixture (30 mL) and extracted with ethyl
acetate (3 × 125 mL). The combined organic layer was washed
with ice cold water (30 mL) and brine (30 mL) and dried over
anhydrous sodium sulfate. The organic layer, on evaporation
under vacuum, gave 19 (0.7 g, 92%). An analytical sample
was prepared by a short column chromatography on silica gel
using EtOAc, followed by EtOAc-MeOH (18:1), and finally
chloroform: mp 194-196 °C; IR (KBr) 3400-3200, 2960-2842,
1
1
1650, 1590, 1500, 880, 852 cm-1; H NMR (DMSO-d6 + D2O)
cm-1; H NMR (CDCl3) δ 7.55 (s, 1 H), 6.78 (s, 1 H), 5.70 (bs,
δ 0.85 (s, 3 H), 1.20-1.80 (m, 10 H), 1.82-2.82 (m, 8 H), 3.72
(t, J ) 8.5 Hz, 1 H), 4.20 (q, J ) 6.4 Hz, 2 H), 6.85 (s, 1 H),
7.58 (s, 1 H). Anal. (C20H26O4‚0.25H2O) C, H.
1 H, exchangeable with D2O), 5.20 (bs, 3 H, exchangeable with
D2O), 4.15 (q, J ) 6.4 Hz, 2 H), 3.77 (dd, J ) 8.5 Hz, 1 H),
2.45-1.15 (m, 17 H), 0.8 (s, 3 H); MS m/ z (relative intensity)
345 (MH+, 100), 330 (43), 263 (30), 243 (100), 202 (27), 189
(25), 165 (52); exact mass calcd (MH)+ 345.2178, found
345.2187. Anal. (C20H28N2O3‚0.4CH2Cl2) C, H, N.
6-Oxo-2-(2′,2′,2′-tr iflu or oeth oxy)estr a -1,3,5(10)-tr ien e-
3,17â-d iol (20). A 20% solution of KOH in methanol (7 mL)
was added dropwise to a suspension of compound 18 (1.1 g,
2.4 mmol) in anhydrous methanol (20 mL). The resulting
reaction mixture was stirred at room temperature for 5 h. The
solvent was removed under reduced pressure at 42-45 °C. The
residue was neutralized with ice cold 3 N HCl, and the
resulting precipitate was removed by filtration. The filtrate
was suspended in ethyl acetate, and residual insoluble mate-
rial was removed by filtration. The organic layer, on dilution
with hexane, deposited a solid which was collected by filtration
and dried to afford 20. The filtrate, on purification over a silica
gel column, gave an additional amount of 2-(trifluoroethoxy)-
6-oxoestra-1,3,5(10)-triene-3,17â-diol (20) (total yield 0.75 g,
83%): mp 186-188 °C; IR (KBr) 3400, 2940, 1660, 1600, 1505,
2-Eth oxy-6-a m in oestr a -1,3,5(10)-tr ien e-3,17â-d iol (24).
TiC14 (1 mL) was introduced via a syringe under Ar to an ice
cold suspension of sodium borohydride (0.8 g, 21 mmol) in
anhydrous DME (30 mL). A solution of the oxime 21 (0.7 g,
2.03 mmol) in anhydrous DME (15 mL) was added dropwise.
The resulting mixture was stirred at 0 °C for 1.5 h and allowed
to warm to room temperature with stirring. After 2 days, an
additional amount of sodium borohydride (0.24 g) was added,
and stirring was continued for 1.5 days at room temperature.
The mixture was cooled to 0 °C, and water (10 mL) was added
very cautiously, followed by neutralization with saturated
sodium bicarbonate solution. The aqueous layer was extracted
with ethyl acetate (2 × 200 mL) and dichloromethane (2 ×
200 mL). Both organic layers were washed separately with
water (100 mL), saturated sodium bicarbonate (100 mL), and
brine (100 mL) and dried over anhydrous sodium sulfate. The
organic layers on evaporation under reduced pressure gave
0.4 g of crude product. This crude compound was dissolved
in anhydrous THF (25 mL), and 5% Pd-C (0.6 g) was added
under Ar. The resulting reaction mixture was hydrogenated
at 50-55 psi. After 5 h, an additional amount of 5% Pd-C
(0.16 g) was added, and the reaction mixture was hydrogenated
at 50-55 psi for an additional 15 h. The catalyst was removed
by filtration through a Celite pad, and the Celite pad was
washed with dichloromethane (100 mL). The organic layer
on evaporation under reduced pressure provided a crude
mixture, which on purification on a silica gel column using
hexane-ethyl acetate and ethyl acetate-methanol mixtures
as eluants provided compound 24 (160 mg, 22%): mp 208-
1
1440, 870 cm-1; H NMR (DMSO-d6 + D2O) δ 0.83 (s, 3 H),
1.20-2.85 (m, 13 H), 3.05 (bs, 1 H), 3.75 (t, J ) 8.5 Hz, 1 H),
4.58 (q, J ) 8.5 Hz, 2 H), 6.92 (s, 1 H), 7.63 (s, 1 H), 8.6 (s, 1
H). Anal. (C20H23F3O4) C, H.
2-E t h oxy-6-(oxim in o)est r a -1,3,5(10)-t r ien e-3,17â-d iol
(21). Sodium acetate (2.0 g, 30 mmol) was added to a stirred
solution of 6-oxo derivative 19 (0.34 g, 1.03 mmol) in methanol
(35 mL). Hydroxylamine hydrochloride salt (2.0 g, 29 mmol)
in water (3 mL) was added at room temperature. The reaction
mixture was stirred at 90 °C for 4 h. The solvent was removed
under reduced pressure, the resulting residue was diluted with
water (50 mL), and the product was extracted with ethyl
acetate (3 × 150 mL). The combined organic layer was washed
with water (100 mL) and brine (100 mL) and dried over
anhydrous sodium sulfate. The organic layer, on evaporation
under reduced pressure, provided the crude product, which
was purified on a silica gel column using hexane-ethyl acetate
mixture as eluant. Appropriate fractions were combined and
evaporated under reduced pressure to afford 21 (60-68%): mp
228-230 °C; IR (KBr) 3530, 3360-3140, 2930, 1605, 1505, 870,
798 cm-1; 1H NMR (CDCl3 + DMSO-d6 + D2O) δ 0.85 (s, 3 H),
1.10-2.45 (m, 18 H), 3.20 (dd, J ) 4.3 and 12.8 Hz, 1 H), 3.75
(t, J ) 8.5 Hz, 1 H), 4.18 (q, J ) 8.5 Hz, 2 H), 6.80 (s, 1 H),
7.55 (s, 1 H). Anal. (C20H27NO4) C, H.
1
212 °C; H NMR (DMSO-d6) δ 0.7 (s, 3 H), 1.05-2.40 (m, 14
H), 3.40 (s, 2 H), 3.55 (t, J ) 8.5 Hz, 1 H), 4.05 (q, J ) 8.5 Hz,
2 H), 4.40 (bs, 1 H), 4.60 (s, 1 H), 6.85 (s, 1 H), 7.05 (s, 1 H),
8.38 (bs, 2 H), 8.80 (s, 1 H). Anal. (C20H29NO3‚HCl‚0.5H2O)
C, H, N.
2,3,17â-O-Tr ia cetylestr a -1,3,5(10)-tr ien e (25). This mi-
nor product resulted from acylation of 2,3,17-trihydroxyestra-

2332 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 15
Cushman et al.
1,3,5(10)-triene, which was present as an impurity in the
starting material 14 used for the preparation of 16. Compound
reduced pressure. The resulting residue was suspended in
THF (15 mL), and 3 N HCl (15 mL) was added to the
suspension. The reaction mixture was stirred at room tem-
perature for 45 min at 0 °C. The solvent was evaporated under
reduced pressure, and the residue was diluted with dichlo-
romethane (2 × 150 mL). The combined organic layer was
washed with water (25 mL) and brine (25 mL) and dried over
MgSO4. The solvent was evaporated under reduced pressure,
and the resulting crude compound on purification on a silica
gel column gave pure 6,7-dehydro compound 29 (0.63 g, 30%):
Rf 0.66 [dichloromethane-methanol (95:5), silica gel]; IR (KBr)
25: mp 162-164 °C; Rf 0.33 [hexane-ethyl acetate (4:1)]; IR
1
(KBr) 2910, 2820, 1765, 1715, 1490, 910 cm-1
;
H NMR
(CDCl3) δ 0.87 (s, 3 H), 1.15-2.40 (m, 22 H), 2.75-2.95 (m, 2
H), 4.72 (t, J ) 8.5 Hz, 1 H), 6.87 (s, 1 H), 7.05 (s, 1 H). Anal.
(C24H30O6) C, H.
2-E t h oxy-1,3,5(10)-t r ien e-3,6r,17â-t r iol (26). Sodium
borohydride (0.5 g, 13.2 mmol) was added under Ar at room
temperature to a solution of 6-oxo derivative 19 (0.85 g, 2.57
mmol) in anhydrous methanol (30 mL). After 6 h, the solvent
was evaporated under reduced pressure. The residue was
diluted with water (40 mL), and dichloromethane (170 mL)
and ice cold 3 N HCl (5 mL) were added. The dichloromethane
layer was removed, and the aqueous layer was extracted again
with dichloromethane (150 mL). The combined organic layer
was washed with water (50 mL), sodium bicarbonate (50 mL),
and brine (50 mL) and dried over anhydrous sodium sulfate.
The organic layer upon evaporation under reduced pressure
followed by purification on a silica gel column provided a
diastereomeric mixture of alcohols (0.5 g, 59%): mp 97-106
°C; Rf 0.27 [dichloromethane-methanol (95:5), silica gel].
Recrystallization from ethyl acetate gave a mixture enriched
in 6R-diastereomer (>95% 6R-isomer): IR (KBr) 3380, 2910,
2850, 1500, 1270, 1105, 1050, 870, 720 cm-1; 1H NMR (CDCl3)
δ 7.12 (s, 1 H), 6.78 (s, 1 H), 5.65 (s, 1 H), 4.60 (bm, 1 H), 4.10
(q, J ) 6.4 Hz, 2 H), 3.75 (t, J ) 6.4 Hz, 1 H), 2.45-1.10 (m,
18 H), 0.8 (s, 3 H). Anal. (C20H28O4‚0.7H2O): C, H.
3400, 2919, 2841, 1600, 1569, 1502, 1279 cm-1 1H NMR
;
(CDCl3, 300 MHz), 6.85 (s, 1 H), 6.72 (s, 1 H), 6.40 (d, J ) 8.5
and 2.1 Hz, 1 H), 5.88 (d, J ) 8.5 and 2.1 Hz, 1 H), 5.50 (s, 1
H), 4.15 (q, J ) 8.5 Hz, 2 H), 3.75 (t, J ) 8.5 Hz, 1 H), 2.45-
1.15 (m, 15 H), 0.8 (s, 3 H). Anal. (C20H26O3‚0.25H2O) C, H.
2,4-Dibr om o-3-h ydr oxyestr a-1,3,5(10)-tr ien -17-on e (31).
Estrone (1.5 g, 5.6 mmol) was dissolved in acetic acid (240 mL)
at 75-80 °C on a steam bath and the mixture cooled to 17 °C.
Some of the estrone precipitated during cooling. Iron (34 mg)
was added. A solution of bromine (2 mL, 40 mmol) in glacial
acetic acid (40 mL) was added dropwise to the cold stirred
mixture, and stirring was continued for 30 min at 17 °C. The
reaction mixture was poured onto an ice-water mixture (800-
900 mL), and a yellow solid separated, which was collected by
filtration and washed with water. The yellow solid was
dissolved in ethyl acetate (300 mL). The organic layer was
washed with water (100 mL), saturated sodium bicarbonate
(2 × 100 mL), water (100 mL), and brine (100 mL) and dried
over anhydrous sodium sulfate. The organic layer, on evapo-
ration under vacuum, gave 2.5 g of a mixture of products,
which was separated by silica gel column chromatography
using ethyl acetate-hexane as eluant. The desired compound
31 (1.5 g, 62%) was a major product. An analytical sample
was prepared by crystallization from ethyl acetate: mp 220-
226 °C (lit.43 mp 225-226 °C); IR (KBr) 3260, 2930, 2860, 1715,
1535, 1458, 750 cm-1; 1H NMR (CDCl3) δ 7.45 (s, 1 H), 5.87 (s,
1 H, exchangeable with D2O), 3.10-2.90 (dd, J ) 8.5 and 21.4
2-Eth oxy-6-oxoestr a -1,3,5(10)-tr ien -3,17â-d iol 6-Tosyl-
h yd r a zon e (27). Acetic acid (0.5 mL) was added to a stirred
solution of 6-oxo derivative 19 (0.8 g, 2.42 mmol) in ethanol
(30 mL). Tosylhydrazine (0.484 g, 2.6 mmol) was added at
room temperature. The resulting mixture was stirred at 90-
95 °C for 5.5 h. More tosylhydrazine (0.2 g) was added, and
heating continued for 3.5 h. The solvent was removed under
reduced pressure, the resulting residue was diluted with water
(50 mL), and the product was extracted with ethyl acetate (3
× 150 mL). The combined organic layer was washed with
water (100 mL) and brine (100 mL) and dried over anhydrous
sodium sulfate. The organic layer on evaporation under
reduced pressure provided an impure compound 27 which, on
crystallization from an ethyl acetate-hexane mixture, gave
pure compound 27 (0.6 g, 50%): mp 240 °C; Rf 0.24 [dichlo-
romethane-methanol (95:1), silica gel]; IR (KBr) 3497, 3223,
Hz, 1 H), 2.80-1.35 (m, 14 H), 0.91 (s, 3 H). Anal. (C18H20
Br2O2) C, H.
-
2,4-Dieth oxy-3-h ydr oxyestr a-1,3,5(10)-tr ien -17-on e (32).
Sodium ethoxide (2.1 g, 31 mmol) was dissolved in anhydrous
ethanol (20 mL) and stirred for 15-20 min at room temper-
ature. Anhydrous DMF (20 mL) was added to the solution
under Ar, and stirring was continued for 20 min at room
temperature. CuI (2.3 g, 24 mmol) and 2,4-dibromo compound
31 (0.7 g, 2.0 mmol) were added successively, and the resulting
mixture was stirred at 105-110 °C for 2 h. The reaction
mixture was stirred overnight at room temperature. The
reaction mixture was poured into ice cold water (200 mL) and
neutralized with 3 N HCl. Insoluble material was removed
by filtration through a Celite pad, and the aqueous layer was
extracted with ethyl acetate (2 × 250 mL). The combined
organic layer was washed with water (150 mL), saturated
aqueous sodium bicarbonate (150 mL), and brine (150 mL) and
dried over anhydrous sodium sulfate. The resulting com-
pound, after removal of solvent, was purified by silica gel
column chromatography using a hexane-dichloromethane
system to afford the product in 37% yield: mp 57-62 °C; IR
(KBr) 3540, 2980-2860, 1730, 1495 cm-1; 1H NMR (CDCl3) δ
6.65 (s, 1 H), 5.45 (s, 1 H), 4.10 (q, J ) 6.4 Hz, 4 H), 2.95 (dd,
J ) 8.5 and 21.4 Hz, 1 H), 2.80-1.10 (m, 20 H), 0.95 (s, 3 H).
Anal. (C22H30O4‚H2O) C, H.
1
2974-2939, 1621, 1592, 1577, 1499 cm-1; H NMR (CDCl3) δ
0.75 (s, 3 H), 1.15-2.38 (m, 16 H), 2.45 (s, 3 H), 2.65 (dd, J )
4.3 and 17.2 Hz, 1 H), 3.72 (t, J ) 8.5 Hz, 1 H), 4.15 (q, J )
6.4 Hz, 2 H), 5.60 (s, 1 H), 6.73 (s, 1 H), 7.37 (d, J ) 8.5 Hz, 2
H), 7.57 (s, 1 H), 7.67 (s, 1 H), 7.95 (d, J ) 8.5 Hz, 2 H).
2-E t h oxy-6-(O-m et h yloxim in o)est r a -1,3,5(10)-t r ien e-
3,17â-d iol (28). A solution of methoxylamine (1.84 g) in
distilled water (3.5 mL) was added to a suspension of 6-oxo
derivative 19 (0.33 g, 1 mmol) and anhydrous sodium acetate
(1.94 g) in anhydrous MeOH (100 mL), and the mixture was
stirred at 95 ( 5 °C for 4.5 h. The solvent was evaporated
under reduced pressure. The residue was dissolved in water
(50 mL) and the solution extracted with ethyl acetate (2 × 200
mL). The organic layer was washed with water (50 mL) and
brine (50 mL) and dried over anhydrous Na2SO4. The organic
layer on evaporation gave an amorphous powder (0.32 g). The
crude mixture was chromatographed over silica gel and eluted
with hexane-ethyl acetate (1:1) to afford the desired product.
This was further purified by crystallization from hexane-ethyl
acetate to afford 28 (0.3 g, 83%): mp 92-97 °C; Rf 0.56
(hexane-ethyl acetate (1:1), silica gel]; IR (KBr) 3460, 2960-
2840, 1590, 1560, 1490, 1435, 890-872 cm-1; 1H NMR (CDCl3,
300 MHz) δ 7.55 (s, 1 H), 6.78 (s, 1 H), 5.50 (s, 1 H), 4.15 (q,
2 H), 3.97 (s, 3 H), 3.85-3.65 (m, 1 H), 3.05 (dd, J ) 8.5 and
21.4 Hz, 1 H), 2.45-1.15 (m, 15 H), 0.95 (t, J ) 8.5 Hz, 1 H),
0.8 (s, 3 H). Anal. (C21H29NO4‚0.1n-C6H12) C, H, N.
2,4-Dieth oxy-3-h yd r oxyestr a -1,3,5(10)-tr ien -17â-ol (33).
Sodium borohydride (0.12 g, 3.2 mmol) was added slowly under
Ar to a solution of 2,4-diethoxy-17-keto derivative 32 (0.22 g,
0.61 mmol) in anhydrous methanol (8 mL). After 5.5 h, the
solvent was removed under reduced pressure, and the residue
was acidified with 3 N HCl. The compound was extracted with
ethyl acetate (2 × 100 mL). The organic layer was washed
with water (80 mL), saturated sodium bicarbonate (2 × 80
mL), water (100 mL), and brine (100 mL) and dried over
anhydrous sodium sulfate. Evaporation of the solvent under
reduced pressure, followed by purification on a silica gel
column, gave 33 (55 mg, 25%): mp 67-72 °C; IR (KBr) 3400,
2-E t h oxy-6,7-d eh yd r oest r a -1,3,5(10)-t r ien e-3,17â-d iol
(29). A solution of 2-ethoxy-6-ketoestradiol (19) (1.1 g, 3.3
mmol) in anhydrous methanol (30 mL) was prepared by
dissolving the solid in warm methanol and then cooling to room
temperature. Sodium borohydride (0.8 g, 21 mmol) was added
in portions to the solution, and the mixture was stirred at room
temperature for 3.5 h. The solvent was evaporated under
1
2960-2845, 1600, 1590 cm-1; H NMR (CDCl3 + D2O) δ 6.65
(s, 1 H), 4.82 (bs, 2 H), 4.07 (q, 4 H), 3.72 (t, J ) 8.5 Hz, 1 H),

Synthesis of Analogs of 2-Methoxyestradiol
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 15 2333
2.92 (dd, J ) 8.5 and 17.1 Hz, 1 H), 2.75-2.50 (m, 1 H), 2.40-
1.10 (m, 19 H), 0.82 (s, 3 H). Anal. (C22H32O4‚0.25H2O) C, H.
3-(Ben zyloxy)-2-eth oxyestr a -1,3,5(10)-tr ien -17â-ol (34).
Benzyl bromide (4.5 g, 38 mmol) was added to a suspension of
2-ethoxy-â-estradiol 2 (4.5 g, 13 mmol) and potassium carbon-
ate (8.6 g, 62 mmol) in anhydrous ethanol (100 mL) under Ar
at 0 °C. The resulting mixture was stirred at 100 ( 5 °C for
7-8 h. The solvent was evaporated under reduced pressure,
the residue was diluted with water, and the mixture was
extracted with ethyl acetate (3 × 200 mL). The combined
organic solution was washed with water (100 mL), sodium
bicarbonate (120 mL), and brine (100 mL) and dried over
sodium sulfate. The ethyl acetate layer on evaporation under
reduced pressure gave a viscous oil (5.8 g), which was used in
the next step without any further purification: 1H NMR
(CDCl3) δ 7.35 (s, 5 H), 6.75 (s, 1 H), 6.65 (s, 1 H), 5.5 (s, 1 H),
4.55 (s, 2 H), 4.04 (q, J ) 6.4 Hz, 2 H), 3.5 (t, J ) 8.5 Hz, 1 H),
2.75 (m, 3 H), 2.20-1.95 (m, 6 H), 1.90-1.80 (m, 2 H), 1.75-
1.5 (m, 2 H), 1.45-1.1 (m, 7 H), 0.85 (s, 1 H).
3.60-3.20 (m, 4 H), 3.00 (t, J ) 8.5 Hz, 1 H), 2.82-2.42 (m, 4
H), 2.40-1.90 (m, 4 H), 1.88-1.55 (m, 3 H) 1.27 (t, J ) 8.5, 3
H), 0.78 (s, 3 H). Anal. (C20H29ClNO2‚HCl‚0.5C2H5OH) C, H,
N.
Bis[2-e t h oxy-3-h yd r oxye st r a -1,3,5(10)-t r ie n -17â-yl]
Su lfite (38). SOCl2 (2 mL) was added under Ar at 0 °C to a
stirred suspension of 2-ethoxy-â-estradiol (2) (0.5 g, 1.6 mmol)
in anhydrous benzene (2 mL). The reaction mixture was
allowed to warm to room temperature, and stirring was
continued for 14 h. The solvent was evaporated under reduced
pressure, and the residue was diluted with dichloromethane
(200 mL). The organic layer was washed with water (50 mL)
and dried over sodium sulfate. The organic layer, on evapora-
tion under reduced pressure, gave a mixture of compounds,
which on purification over a silica gel column using hexane-
ethyl acetate mixture as eluant gave compound 38 (0.18 g,
35%): Rf ) 0.46 [hexane-ethyl acetate (4:1)]; IR (KBr) 3550,
3428, 2974-2867, 1738, 1611, 1518, 1474, 756 cm-1; 1H NMR
(CDCl3) δ 6.78 (s, 2 H), 6.68 (s, 2 H), 5.55 (s, 2 H), 4.55 (t, 1
H), 4.3 (t, 1 H), 4.08 (q, 4 H), 2.78 (bs, 4 H), 2.5-1.05 (m, 32
H), 0.85 (d, 3 H); MS (FAB) m/ z 678 (M+), (negative ion FAB)
m/ z 677 [(M - H)-]. Anal. (C40H54SO7‚0.2CH3CO2Et) C, H,
S.
2,4-Dibr om oestr a -1,3,5(10)-tr ien e-3,17â-d iol (39). Es-
tradiol (2.0 g, 7.4 mmol) was dissolved in glacial acetic acid
(300 mL) at 80 °C and cooled to 15-17 °C, resulting in partial
precipitation of solid. A solution of bromine (0.75 mL, 14.6
mmol) in glacial acetic acid was added dropwise, and the
mixture was stirred at 15-17 °C for 32 min. The reaction
mixture was poured into an ice-water mixture (1 L). A yellow
solid precipitated. It was removed by filtration and dissolved
in ethyl acetate (400 mL). The ethyl acetate solution was
washed with water (100 mL) and brine (100 mL) and dried
over sodium sulfate. The organic layer on evaporation under
reduced pressure gave a crude mixture, which on crystalliza-
tion from ethyl acetate gave compound 39 (1.85 g, 72%): mp
220-222°C (lit.44 mp 214-215 °C); Rf (dichloromethane, silica
gel) 0.26; 1H NMR (CDCl3) δ 7.45 (s, 1 H), 5.88 (s, 1 H,
exchangeable with D2O), 3.75 (t, 1 H), 3.05-2.80 (dd, 1 H),
2.78-2.45 (m, 1 H), 2.45-1.10 (m, 13 H), 0.82 (s, 3 H).
Tu bu lin Assa ys. Electrophoretically homogeneous tubulin
was purified from bovine brain as described previously.45
Determination of IC50 values for the polymerization of purified
tubulin was performed as described in detail elsewhere.5 In
brief, tubulin was preincubated at 26 °C with varying com-
pound concentrations, reaction mixtures were chilled on ice,
GTP (required for the polymerization reaction) was added, and
polymerization was followed at 26 °C by turbidimetry at 350
nm in Gilford recording spectrophotometers equipped with
electronic temperature controllers. The extent of polymeri-
zation after 20 min was determined. IC50 values were deter-
mined graphically. All compounds were examined in at least
two independent assays. Inhibition of colchicine binding to
tubulin was performed as described previously.5 Reaction
mixtures contained 1.0 µM tubulin (0.1 mg/mL), 5.0 µM [3H]-
colchicine, and 50 µM inhibitor. Incubation was for 10 min at
37 °C.
2-Eth oxy-3-(ben zyloxy)estr a-1,3,5(10)-tr ien -17-on e (35).
Activated MnO2 (3 g) was added under Ar to a solution of crude
alcohol 34 (1.6 g, 3.9 mmol) in anhydrous dichloromethane (50
mL). After 7 h, an additional amount of MnO2 (5 g) was added,
and the reaction mixture was left overnight at room temper-
ature. MnO2 (4 g) was added again, and stirring was contin-
ued for 7 h. The mixture was filtered through a Celite pad,
and the filtrate was evaporated under reduced pressure to give
crude product, which was purified by column chromatography
on silica gel using hexane, followed by hexane-CH2Cl2 (2:8)
to afford pure 35 (0.42 g, 8%): IR (neat) 3065, 2929, 2869,
1
2825, 1727, 1607, 1504, 1450 cm-1; H NMR (CDCl3) δ 0.92
(s, 3 H), 1.30-2.60 (m, 16 H), 2.83 (m, 2 H), 4.07 (q, J ) 8.5
Hz, 2 H), 5.10 (s, 2 H), 6.68 (s, 1 H), 6.88 (s, 1 H), 7.20-7.60
(m, 5 H).
2-Eth oxy-3-(ben zyloxy)-17â-(ben zyla m in o)estr a -1,3,5-
(10)-tr ien e (36). Glacial acetic acid (0.8 mL), benzylamine
(1.6 mL, 15 mmol), and sodium cyanoborohydride (0.9 g, 14
mmol) were added under Ar to a solution of 17-keto derivative
35 (2 g, 5 mmol) in anhydrous 1,2-dichloroethane (30 mL). The
mixture was stirred at room temperature for 72 h. Additional
amounts of anhydrous benzylamine (0.4 mL), acetic acid (0.2
mL), and sodium cyanoborohydride (0.25 g) were added to the
mixture, and stirring was continued for 24 h. The solvent was
removed under reduced pressure, and the residue was diluted
with water (50 mL) and extracted with ethyl acetate (3 × 150
mL). The combined organic layer was washed with water (100
mL), sodium bicarbonate solution (100 mL), and brine (100
mL) and dried over anhydrous sodium sulfate. The organic
layer on evaporation under reduced pressure gave crude
product (3.1 g), which was subjected to column chromatogra-
phy on silica gel using hexane, followed by hexane-CH2Cl2
(1:1), and finally CH2Cl2. Appropriate fractions were combined
and evaporated under reduced pressure to give pure 36 (2.2
g, 89%) as a sticky solid which was used without further purifi-
cation: IR (KBr) 2921, 1608, 1508, 1453, 1264, 1209, 1119,
1019, 729 cm-1; 1H NMR (CDCl3) δ 7.52-7.25 (m, 11 H), 6.85
(s, 1 H), 6.65 (s, 1 H), 4.20 (q, J ) 8.5 Hz, 2 H), 3.80 (s, 2 H)
2.75 (m, 1 H) 2.65 (t, J ) 8.5 Hz, 1 H), 2.35-2.12 (m, 4 H),
2.10 (s, 2 H), 1.85 (m, 4 H), 1.75 -1.52 (m, 6 H) 1.40 (t, J )
8.5, 3 H), 0.78 (s, 3 H).
Mea su r em en t of Estr ogen Recep tor Rela tive Bin d in g
Affin ities (RBA). Estrogen receptor RBA’s were determined
using competitive radiometric binding assays using a tritium-
labeled estrogen as the tracer and immature rat uterine cytosol
as the source of receptor, according to the methods described
previously.46 By definition, estradiol is given an RBA value
of 100. The assays of 21, 22, and 28 were conducted at 0 °C
for 20 h, while those for 1 and 2 were performed at 0 °C for 18
h. The coefficient of variation in replicate experiments is
typically less than 30%.
2-Eth oxy-3-h ydr oxy-17â-am in oestr a-1,3,5(10)-tr ien e Hy-
d r och lor id e (37). 20% Pd(OH)2-C (1.7 g) was added care-
fully under Ar to a solution of 17-benzylamino derivative 36
(1.52 g, 3.0 mmol) in anhydrous THF (20-30 mL). The
resulting mixture was hydrogenated at 45-50 psi on a Parr
apparatus for 27 h. After completion, the catalyst was
removed via filtration using a Celite pad under Ar, and the
pad was washed with THF (150 mL), followed by a 1:1 mixture
of THF-CH2Cl2 (100 mL). The filtrate obtained on evapora-
tion under reduced pressure gave impure compound (1.08 g),
which was purified by column chromatography. The resulting
material was then converted to the hydrochloride salt (0.16 g,
15%) by dissolving it in ether (100 mL) and bubbling HCl gas
through the solution: mp 270 °C; IR (KBr) 3137, 2931-2261,
Ack n ow led gm en t. This research was made possible
by contracts NO1-CM-17512 and NO1-CM-27764,
awarded to M.C. and Pharm-Eco, respectively, by the
National Cancer Institute, DHHS. We are indebted to
Kathryn E. Carlson and Karen Avenatti for performing
estrogen receptor binding affinity measurements. The
authors (Y.P.S. and S.R.) are thankful to Mr. Paul J .
Lydon and Ms. Danguole Senuta Falguni Kher for
1612, 1499, 1396 cm-1 1H NMR (CDCl3) δ 8.60 (bs, 1 H,
;
exchangeable with D2O), 8.20 (bs, 2 H, exchangeable with
D2O), 6.75 (s, 1 H), 6.52 (s, 1 H), 3.95 (q, J ) 8.5 Hz, 2 H),

2334 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 15
Cushman et al.
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Institute, is gratefully acknowledged.
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