Synthesis of 2- and 17-substituted estrone analogs and their antiproliferative structure-activity relationships compared to 2-methoxyestradiol

Article abstract of DOI:10.1016/j.bmc.2009.08.038

A novel series of 17-modified and 2,17-modified analogs of 2-methoxyestradiol (2ME2) were synthesized and characterized. These analogs were designed to retain or potentiate the biological activities of 2ME2 and have diminished metabolic liability. The ana

Full text of DOI:10.1016/j.bmc.2009.08.038

Bioorganic & Medicinal Chemistry 17 (2009) 7344–7352
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of 2- and 17-substituted estrone analogs and their antiproliferative
structure–activity relationships compared to 2-methoxyestradiol
Jamshed H. Shah, Gregory E. Agoston, Lita Suwandi, Kimberly Hunsucker, Victor Pribluda,
*
Xiaoguo H. Zhan, Glenn M. Swartz, Theresa M. LaVallee, Anthony M. Treston
EntreMed, Inc., 9640 Medical Center Drive, Rockville, MD 20850, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 17-modified and 2,17-modified analogs of 2-methoxyestradiol (2ME2) were synthesized
and characterized. These analogs were designed to retain or potentiate the biological activities of 2ME2
and have diminished metabolic liability. The analogs were evaluated for antiproliferative activity against
MDA-MB-231 breast tumor cells, antiangiogenic activity in HUVEC, and estrogenic activity on MCF-7 cell
proliferation. Several analogs were evaluated for metabolic stability in human liver microsomes and
in vivo in a rat cassette dosing model. This study lead to several 17-modified analogs of 2ME2 that have
similar or improved antiproliferative and antiangiogenic activity, lack estrogenic properties and have
improved metabolic stability compared to 2ME2.
Received 24 June 2009
Accepted 17 August 2009
Available online 21 August 2009
Keywords:
2
-Methoxyestradiol analogs
Antiproliferative
Antiangiogenic
Structure–activity relationships
Ó 2009 Elsevier Ltd. All rights reserved.
1
. Introduction
estradiol, 2ME2 is metabolized principally by oxidative and conju-
gation processes. These transformations lead to a rapid decrease in
Endogenous 2ME2 is produced by hydroxylation of estradiol
E2) at the 2-position and subsequent 2-O-methylation by cate-
the concentration of the active drug.
Several previous studies have been conducted to determine
structure–activity relationships (SAR) of 2ME2 analogs. However,
(
1,2
10
chol-O-methyltransferase (COMT).
The apoptotic activity of
2
ME2 requires proliferating cells, as it is not cytotoxic to quiescent
the rationale for the exploration of those analogs was to increase
the interaction with tubulin, a known target of 2ME2, or to
enhance in vitro cytotoxicity, and not to diminish metabolism.
The approach described in this paper, based on data from animal
models and human clinical PK and metabolism information, was
to generate in vitro-active analogs with modifications expected
to reduce or prevent metabolism at the 17 position.
In order to block the oxidative and conjugation metabolism dis-
cussed above, a series of 2ME2 analogs with polar, ionizable, alkyl,
endocyclic, or exocyclic olefins at position 17 were prepared and
tested for activity in vitro and for metabolic stability in a variety
of models. Additionally, several 17-deoxyestrone analogs were also
modified at position 2 as a pilot study to explore the possibility of
blocking demethylation of the methoxy group in the A ring. For all
these analogs, data are presented on the synthesis and on prelimin-
ary biological in vitro screening. To assess the antiangiogenic and
antitumor activity of these analogs, inhibition of proliferation
was assessed in non-transformed human cells (human umbilical
vein endothelial cells, HUVEC) and in three human tumor cell lines
(breast carcinoma MDA-MB-231, glioblastoma U87-MG, and pros-
tate carcinoma PC3). Estrogenic activity was assessed by ability to
stimulate proliferation of the estrogen-dependent human breast
cancer cell line MCF-7. A study was also conducted to determine
the metabolic stability of selected analogs toward glucuronidation
1
cell cultures. In numerous tumor models in mice, oral administra-
tion of 2ME2 resulted in strong inhibition of tumor growth and
tumor neovascularization at doses that produced no apparent signs
3
of toxicity. 2ME2 inhibits several stages of the angiogenic cascade
including endothelial cell proliferation, migration, and tubule for-
mation.1 2ME2 (Panzem ) has undergone evaluation in several
Ò
4
Phase I and Phase II oncology clinical trials and may have a role
in other angiogenic diseases such as rheumatoid arthritis.
Orally-delivered steroids such as estradiol and 17-ethynyl-
estradiol are extensively metabolized during uptake from the gas-
trointestinal tract and by first-pass metabolism in the liver. Two
major metabolic pathways that lead to rapid deactivation and
excretion of E2 are oxidation by 17-b-hydroxysteroid dehydroge-
nase of the D-ring 17-hydroxy group to form estrone (E1), and con-
jugation with sulfate and/or glucuronide of the hydroxyl group at
position 3 on the A-ring and at position 17 on the D-ring. Pharma-
cokinetic (PK) and metabolism data from Phase I studies of 2ME2
in humans and adsorption distribution metabolism and excretion
5
6
7
(
ADME) studies in rats demonstrated similar metabolic transfor-
4
,8,9
mations for 2ME2.
As reported for estradiol and 17-ethynyl
*
Corresponding author. Tel.: +1 240 864 2600; fax: +1 240 864 2601.
E-mail address: tonyt@entremed.com (A.M. Treston).
0
968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.038

J. H. Shah et al. / Bioorg. Med. Chem. 17 (2009) 7344–7352
7345
by human liver microsomes, in the presence of the cofactor uridine
H C
3
CH3
O
0
5
-diphosphoglucuronic acid (UDPGA). Several lead substituents
Hydrazine/
were also evaluated in a cassette dosing PK study in rats.
KOH/DiEt-glycol
n-Bu-OH
HO
HO
1
0
2
. Chemistry
Estrone
HOAc
Treatment of 2-methoxyestrone (2ME1) with anhydrous hydra-
HNO3
zine in refluxing diethyleneglycol and 1-butanol mixture for 1 h
followed by the addition of 3 M equiv of KOH pellets gave 2-meth-
oxy-17-deoxyestrone (1) in 90% yield1 as shown in Scheme 1. 17-
Alkyl and alkenyl 2-methoxyestrone analogs were synthesized by
treating alkyl triphenylphosphonium bromide with potassium
tert-amylate in refluxing anhydrous benzene, followed by the addi-
CH3 Pd-C (10 %)
EtOH/Dioxane/
HCHO
CH3
1
Me N
2
O N
2
HO
H2 gas, 40 psi
HO
1
2
11
Pd-C 10%/
1
2,13
tion of 2ME1.
After work-up, the resulting intermediate was
purified on a silica column to give compounds 2–5 (54–84% yield).
Hydrogenation of compounds 2–5 in a Parr hydrogenator using
Pd/C 10% in ethyl acetate gave 17-b-alkyl analogs 6, 7, 8, and 9
as shown in Scheme 1 (85% yield or greater).
The syntheses of compounds 11–16 are shown in Scheme 2. Es-
trone was reduced to 17-deoxyestrone (10) using a similar proce-
MeOH/Dioxane
CH3
CH3
O
H
HCOOH/
Tol.
H N
2
NH
HO
HO
dure as shown above for 1. Reaction of 10 with 1 equiv of HNO
3
in
13
1
5
glacial acetic acid for 18 h at room temperature gave compound 11
1
0a
in 60% yield.
Reduction of 11 in the presence of formaldehyde
AlH /THF
1) NaNO2/ AcOH
3
(
37% in H O) and Pd/C (10%) in a Parr hydrogenator at 40 psi of
2
2
) NaN / H O
3 2
hydrogen gas in 1:3 mixture of dioxane/MeOH gave the 2-dimeth-
ylamine-17-deoxyestrone 12 in 75% yield.14 Reduction of 11 with-
CH3
CH3
out formaldehyde under similar conditions as for 12 gave
H CHN
3
compound 13 in 90% yield.10a Heating 13 in refluxing toluene in
N3
HO
the presence of formic acid resulted in the formation of formamide
16
HO
1
5
14
1
3
5 in 75% yield. Reduction of 15 with AlH in THF gave the sec-
16
ondary amine 16 in 70% yield. Reaction of 13 in acetic acid gla-
cial, with sodium nitrite at 0 °C followed by treating with sodium
azide in water gave compound 14 in 80% yield.
Scheme 2.
17
As shown in Scheme 3, compound 17 was synthesized with 90%
yield by heating 2ME1 and p-toluenesulfonylhydrazide in refluxing
methanol. Similarly the reaction of 2ME1 with hydroxylamine in
refluxing ethanol gave compound 21 in 90% yield. Reaction of 17
with n-BuLi in THF gave compound 20 in 80% yield.19 Compounds
sized following the procedure of McGuire,21 and was then con-
verted to 23 by treating it with SOCl at 70 °C followed by NH
2 3
gas in THF.
1
8
3
. Results and discussion
Before addressing the metabolic stability of the 2ME2 analogs
1
8 and 19 were synthesized by reductive amination of 2ME1 with
ammonium acetate and propylamine in the presence of NaBH CN
in absolute methanol (67–70% yield).20 Compound 22 was synthe-
3
modified at positions 2 and/or 17, an SAR study was conducted
in order to select compounds that retain a similar in vitro activity
profile to 2ME2. Antiproliferative activity was assessed in endothe-
lial cells (HUVEC) as an in vitro surrogate for antiangiogenic activ-
ity, and in three tumor cell lines (MDA-MB-231, U87-MG, and PC3)
as a measure of antitumor activity. From the compounds listed in
Table 1 only the 17-methylene (2), 17-ethylene (3), and 16,17-ole-
fin (20) exhibited better antiproliferative activities than 2ME2.
Increasing carbon chain length at position 17 resulted in a moder-
ate to significant reduction in antiproliferative activity (see com-
pound sub-groups 2 to 5 and 6 to 9) suggesting that bulkier
groups are not well tolerated at that position. We have previously
shown a similar limitation on substituent size at the neighboring
H C
3
R
H C
Benzene
K-t-Amyl
3
O
MeO
HO
MeO
HO
R-PPh Br
3
reflux, 3h
2
3
4
5
: R = H
: R = Me
: R = Et
: R = Pr
2
-Methoxyestrone (2ME1)
Hydrazine/
KOH/DEG/
n-But-OH
H / Pd-C (10%)
2
1
0j
EtOAc
C-16 position. Introduction of non-acidic polar groups at posi-
tion 17 such as amine (18), hydroxyl amine (21), and carboxyam-
ide (23) had little effect on antiproliferative activity compared to
CH3
H C
3
R
MeO
HO
2
ME2. However, 17-carboxylic acid (22) showed complete loss of
MeO
HO
activity suggesting that an acidic group is not tolerated at this po-
sition. It is also possible that the carboxylic acid prevents the com-
pound from entering the cell due to poor cell permeability.
Although slightly less active than 2ME2, the 17-deoxy analog 1
has significantly reduced likelihood of metabolism compared to
1
6
7
8
9
: R = Me
: R = Et
: R = Pr
: R= Bu
2
ME2 or several other analogs reported here as it lacks a moiety
which could easily be conjugated (e.g., OH, NH ) or an exocyclic
or endocyclic olefin which may be subject to oxidation. D-ring
2
Scheme 1.

7
346
J. H. Shah et al. / Bioorg. Med. Chem. 17 (2009) 7344–7352
H C
3
N-OH
H C
3
NH-R
MeO
HO
MeO
HO
21
1
1
8: R = H
9: R = i-Pr
NH OH
MeOH
2
R-NH / NaCNBH
2
3/
MeOH/Reflux
H C
3
O
1
. MsCl,TEA, CH Cl
2 2
MeO
2. TMSCn, ZnI2, CH2Cl2
. POCl , Pyr
3
3
HO
H N-NHSO Tol
6
4. H2, 10% Pd/C, CH2Cl2
5. NaOH, ethyleneglycol, 160 C
H C COOH
2
2
o
MeOH/
Reflux
3
H C
3
N-NHSO Tol
2
MeO
HO
MeO
HO
2
2
17
o
1
. SOCl , 70 C
2
THF/ n-BuLi
H C
2. NH (g), THF
3
H C CONH2
3
3
MeO
HO
MeO
HO
20
2
3
Scheme 3.
Table 1
Antiproliferative activities of 17-modified analogs of 2ME2
Compound #
R
2
R
17
MDA-MB-231 (IC50
l
M)
U87-MG (IC50
lM)
PC3 (IC50
lM)
HUVEC (IC50
l
M)
MC7 SI Relative to 2ME2
2
1
2
3
4
5
6
7
8
9
1
1
1
2
2
2
2
ME2
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
CH
3
O–
O–
O–
O–
O–
O–
O–
O–
O–
O–
O–
O–
O–
O–
O–
O–
O–
OH–
H
@CH
@CHCH
@CHCH
@CHCH
1.00 ± 0.05
2.20 ± 0.52
0.24 ± 0.00
0.58 ± 0.31
7.89 ± 2.87
14.00 ± 6.74
3.33 ± 0.23
48.06 ± 0.91
95.31 ± 3.09
33.47 ± 3.67
72.54 ± 13.3
9.7 7 ± 1.64
17.80 ± 4.06
0.73 ± 0.13
8.68 ± 0.75
>100
8.54 ± 2.29
7.59 ± 0.21
N/A
N/A
N/A
2.65 ± 0.52
8.49 ± 2.49
N/A
N/A
N/A
N/A
6.46 ± 2.31
8.70 ± 0.76
85.44 ± 6.05
28.82 ± 10.2
27.19 ± 8.91
13.82 ± 7.91
23.40 ± 8.33
0.62 ± 0.02
8.11 ± 0.94
>100
0.84 ± 0.02
1.32 ± 0.56
0.19 ± 0.19
0.26 ± 0.23
3.23 ± 0.63
3.47 ± 0.68
2.40 ± 0.45
3.63 ± 0.12
40.79 ± 1.01
12.64 ± 6.45
7.95 ± 2.18
13.37 ± 2.30
18.81 ± 0.48
0.71 ± 0.08
4.27 ± 2.11
35.9 ± 2.27
4.55 ± 1.72
1.00 ± 0.05
0.24 ± 0.06
0.89 ± 0.01
0.89 ± 0.01
0.47 ± 0.03
0.51 ± 0.06
0.89 ± 0.02
0.49 ± 0.31
0.69 ± 0.04
0.59 ± 0.14
0.43 ± 0.29
0.19 ± 0.09
0.11 ± 0.04
0.30 ± .02
1.22 ± 0.11
N/A
2
3
2
2
CH
CH
3
2
CH
3
N/A
CH
3
9.39 ± 0.43
12.77 ± 0.90
87.88 ± 2.53
21.11 ± 1.23
27.32 ± 0.51
19.01 ± 2.07
49.18 ± 4.01
0.88 ± 0.07
9.40 ± 0.64
>100
–CH
–CH
–CH
2
2
2
CH
3
CH
CH
2
CH
CH
Ph-CH
2 3
3
2
2
CH
3
7
8
9
0
1
2
3
@NNHSO
NH
NH-Pr
2
16,17-Olefin
@N–OH
COOH
CONH
2
2.51 ± 0.02
11.18 ± 0.04
21.5 ± 3.85
N/A
N/A = Not available.
SI = Stimulation index.
olefin compound 20 exhibited slightly better activity than 2ME2
even though it lacks any substituent at the 17 position. This effect
may be related to that reported by Rao,1 where activity of a small
series of 2ME2 analogs modified in the D ring was correlated with
the angle of the D-ring to the plane of the ABC ring system.
Previous in vitro metabolism studies indicated that the meth-
oxy moiety of 2ME2 could be oxidatively removed by the action
of cytochromes P450 (data not shown). The resultant 2-hydroxy-
estradiol, if formed in vivo, could be estrogenic. Table 2 shows data
from the sub-group of analogs in which we attempted to replace
the methoxy group at position 2 by substituents which would
not be subject to cytochrome-P450-mediated demethylation. In
this subset of molecules with a 17-deoxy moiety, all 2-position
substituents tested failed to produce analogs which were equipo-
tent to 2ME2.
0i
2ME2 has low binding affinity for both estrogen receptors
a and
b, does not engage the estrogen receptor for its antiproliferative
activity, nor do estrogen receptor agonists or antagonists influence
2
2
the activity of 2ME2. Therefore, one goal of this program was to
identify analogs that exhibit similar selectivity between estrogenic

J. H. Shah et al. / Bioorg. Med. Chem. 17 (2009) 7344–7352
7347
Table 2
Antiproliferative activities of 2,17-modified 2ME2 analogs
Compound #
R
2
MDA-MB-231 (IC50
lM)
U87-MG (IC50
l
M)
PC3 (IC50
l
M)
HUVEC (IC50
l
M)
MC7 SI Relative to 2ME2
1
CH
H–
3
O–
2.20 ± 0.52
41.26 ± 1.99
39.33 ± 12.6
>100
7.12 ± 0.24
24.59 ± 1.32
26.76 ± 5.31
7.59 ± 0.21
22.06 ± 1.64
34.16 ± 6.39
>100
8.49 ± 2.49
1.32 ± 0.56
8.22 ± 1.78
7.43 ± 0.96
60.05 ± 0.58
2.83 ± 0.30
6.80 ± 1.74
4.11 ± 1.51
0.24 ± 0.06
1.91 ± 0.31
2.17 ± 0.05
1.49 ± 0.23
2.04 ± 1.43
1.71 ± 0.34
1.22 ± 0.74
1
1
1
1
1
1
0
2HCl
3
4
5
6HCl
21.99 ± 2.92
26.00 ± 4.18
>80
HClꢀ(CH
3 2
) N–
2
H N
N3–
HCOHN–
HClꢀCH HN–
NT
NT
42.86 ± 3.89
22.31 ± 1.11
25.99 ± 0.66
22.82 ± 2.1
3
SI = Stimulation index.
and antiproliferative activities as seen for 2ME2 compared to estra-
diol. The estrogenic activity of 2ME2 and its analogs on estrogen
dependent MCF-7 breast cancer cell line was investigated, and
activities relative to 2ME2 are presented in the Tables. As shown
in Table 1, the only analog exhibiting more estrogenic activity than
several compounds for pharmacokinetic properties simultaneously
in a single animal. Plasma values for 2ME2 were below the limit of
quantification in this experiment, which can be explained by the
low dose used in a cassette dosing experiment. In contrast, all ana-
logs tested showed significant oral bioavailability, with acceptable
2
ME2 is compound 21 which contains a highly polar hydroxyl-
C
max, AUC and half life (Table 4). 17-Ethylene analog 3 had the
amine. All other analogs are further reduced in undesired estro-
genic effect compared to 2ME2, and several compounds 1, 18, 19,
and 20 exhibited significant reduction in estrogenicity while show-
ing improved antiproliferative properties. The 2- and 17-doubly-
modified compounds shown in Table 2 had greater estrogenic
and weaker antiproliferative activity compared to 2ME2.
greatest bioavailability, AUC and Cmax of the compounds tested.
This analog also had a long t1/2 (16.4 h). The 17-deoxy analog 1
had the longest t1/2 of any of the analogs tested in this experiment
(198.9 h). Despite showing acceptable stability in the in vitro
microsomal stability study described above, 17-methyl analog 6
had a short t1/2 (0.5 h) and had poor bioavailability (9.4%). Based
on these cassette dosing data, the leading substituents for position
17 with respect to metabolic stability are 17-deoxy, 17-methylene,
and 17-ethylene.
The substituents introduced at the 17 position were chosen to
reduce or prevent the facile oxidation or conjugation identified
for the 17-hydroxyl moiety of 2ME2. We chose several of the anti-
proliferative and non-estrogenic analogs for testing in an in vitro
metabolism analysis. This study was conducted to determine the
metabolic stability of the selected 2ME2 analogs toward glucuron-
idation in human liver microsomes, in the presence of the cofactor
4. Conclusions
0
uridine 5 -diphosphoglucuronic acid (UDPGA). Under these condi-
A novel series of compounds has been synthesized that retain
tions it is expected that steroid analogs could be conjugated at
either or both positions 3 or 17, as long as there is an availability
of appropriate functionality. 2ME2, 17-methylene analog 2, 17-
ethylene analog (3), and 17-amino derivative (18) were metabo-
lized extensively after 60 min of incubation with human liver
microsomes as shown in Table 3 (<10% remaining). The 17-methyl
derivative 6 under the same conditions was 47% metabolized in
.
many of the in vitro biological activities of 2ME2 Analogs lacking
the hydroxyl moiety at position 17 cannot be metabolized to 2-
methoxyestrone nor conjugated at that position and, in general,
they retain antiproliferative activity on both endothelial and tumor
cells (Table 1). In vitro metabolic stability studies confirm that sev-
eral of these analogs are metabolically more stable than 2ME2 (Ta-
ble 3). Further replacement of the 2-methoxy group of 1 by other
moieties (Table 2) such as 2-N-formamide (15), 2-N-methylamino
(16), or 2-N,N-dimethylamino (12), was intended to prevent meta-
bolic demethylation to potentially estrogenic 2-hydroxyl deriva-
tives. However these substitutions resulted in analogs that
exhibited weaker activity profiles and increased estrogenicity,
and were therefore not pursued further. The most significant
improvement in overall properties came from replacement of the
17-hydroxy group of 2ME2 with a hydrogen or an exocyclic or
endocyclic olefin (i.e., 1 (17-deoxy), 2 (17-methylene), and 3 (17-
ethylene). These substituents resulted in analogs which showed
diminished estrogenic activity, improved metabolic stability in
the in vivo rat cassette dosing model, and increased or similar anti-
proliferative and antiangiogenic activity compared to 2ME2.
6
0 min, whereas analogs 1 (17-deoxy), 7 (17-ethyl), and 15 (17-
deoxy, 2-N-formamide derivative) were significantly more stable
than 2ME2 under these conditions (117%, 72%, and 68% remaining
after 60 min, respectively). All analogs tested retain the 3-hydroxyl
moiety present in E2 and 2ME2, which is a known target for met-
abolic conjugation. The variation in stability between analogs such
as 1, 6, and 7 compared to 2 and 3 suggests that certain D-ring
modifications not only prevent conjugation at position 17 but
may also exert an effect at the 3 position that can result in de-
creased conjugation at that site.
Several analogs (1, 2, 3, 6, and 2ME2) were tested using rat cas-
sette dosing as a screen for in vivo pharmacokinetic properties. The
cassette dosing technique has the advantage of being able to screen
Table 3
Metabolic stability analysis of position 17 and 2-substituted 2ME2 analogs
Time (min)
Control (UDPGA)
Control (+BSA,ꢁHLM)
Compound #
2ME2 %
1 %
2 %
3 %
6 %
7 %
15 %
18 %
0
100
—
—
100
—
—
100
58
29
100
94
101
117
100
76
67
100
75
75
100
67
57
100
91
76
100
101
86
100
32
30
9
1
3
6
5
0
0
113
93
9.4
5.8
0.8
47
72
68
0
Incubations with human liver microsomes (HLM) were performed in 50 mM tris, pH 7.5 containing 10 mM magnesium chloride, 2 mM uridine-5 -diphosphoglucuronic acid
UDPGA), 25 g alamethicin/mg liver microsomal protein and 5 M of each test compound. Control incubations containing the specific concentration of substrate and bovine
(
l
l
serum albumin (BSA) but lacking human liver microsomes were conducted.
Amount of substrate analog remaining at each timepoint (shown as % remaining relative to 0 min) was determined by LCMS analysis.

7
348
J. H. Shah et al. / Bioorg. Med. Chem. 17 (2009) 7344–7352
Table 4
of Dr. Dorraya El Ashry, of Georgetown University. For MCF-7
Rat cassette dosing summary of selected analogs
estrogen-dependent proliferation assays the cells were seeded in
complete media at 20–30,000 cells/well in 24-well plates. After
allowing the cells to adhere overnight the seeding density was
determined by cell count. Cells were then washed with PBS
(37 °C) and starved by placing them in IMEM–phenol red free med-
ia containing 2% charcoal–dextran fetal bovine–stripped serum
Compound #
PO Cmax
nM)
PO t1/2
(h)
PO AUC
(nM h)
Oral bioavailability
(%F)
(
2
1
2
3
6
ME2
BLOQ
40
40
80
50
BLOQ
198.9
24.9
16.4
0.5
—
—
670
572
1163
165
44.5
32.8
84.2
9.4
(
3
Georgetown University) and 1X antibiotic–antimitotic. After
days of starvation, cells were treated with or without increasing
concentrations of each compound, replacing the media every 2–
days, and counted after 8 or 10 days of treatment. Proliferation
was measured by cell counting using a Coulter Z1 cell counter
Coulter Corporation, Hialeah, FL). Each condition was done in trip-
3
The in vitro metabolism data indicate that the 3-position in this
series of analogs remains a target for conjugation. The lead substit-
uents at position 17 identified here have been combined with
modifications at the 3-position to further improve the pharmacoki-
netic properties, metabolism, and antitumor activity of this series
of molecules. The data from these studies are being reported
(
licate in at least 2 independent experiments. Results are presented
as the stimulation index (SI) relative to 2ME2 (defined as 1.00).
Ex vivo liver microsome studies were performed at BD Gentest
in Woburn MA. The metabolic study with human liver microsomes
were performed in 50 mM tris, pH 7.5 containing 10 mM magne-
2
3
separately.
0
sium chloride, 2 mM uridine-5 -diphosphoglucuronic acid (UDP-
GA), 25 lg alamethicin/mg liver microsomal protein, and 5 lM of
5
. Experimental
each test compound. Control incubations containing the specific
concentration of substrate and bovine serum albumin (BSA) but
lacking human liver microsomes were conducted. For control sam-
ples lacking cofactor UDPGA, incubation were performed in 50 mM
Chemicals and reagents of high purity were obtained from Al-
drich, Fluka, or Acros chemical companies and were used without
further purification. Chemical reactions were monitored by thin
layer chromatography using Merck precoated Silica Gel 60F254
tris pH 7.5 containing 10 mM magnesium chloride, 25
lg alame-
thicin/mg liver microsomal protein and 5 M of each test com-
l
TM
TM
plates. Biotag FLASH-12 or FLASH-40 were used for flash chro-
pound. After incubation times of 0, 15, 30 and 60 min, an aliquot
was removed and added to 1.5 ꢂ vol/vol acetonitrile containing
internal standard. The samples were frozen at ꢁ20 °C for subse-
quent analysis. LCMS analysis was performed using Waters C18
Symmetry 2.1 mm ꢂ 50 mm column eluted with water (A)/metha-
nol (B) using gradient from 50% B to 100% B over 5 min, 100% B to
1
13
matography. H and C Nuclear Magnetic Resonance (NMR) spec-
tra were recorded on a Bruker DPX 300 MHz spectrometer.
Chemical shifts are reported in parts per million (d) relative to tet-
ramethylsilane as internal standard (0.0 d). IR spectra were re-
corded on a Perkin–Elmer 783 spectrometer and wave numbers
are reported in cm . Melting points were determined on a Kofler
apparatus and are uncorrected. Elemental analysis was performed
by Atlantic Micro Lab., Norcross, GA, USA.
ꢁ
1
7
min, 50% B to 8.5 min and 50% B to 10 min at flow rate of 0.2 mL/
min. Mass spectral data was recorded in the negative ion mode.
The cassette dosing experiment was conducted by Ricerca Bio-
sciences (Concord OH) using a standard protocol. Six Sprague-
Dawley rats were implanted with jugular vein canulae. The com-
pounds (3–5/cassette) were administered to three rats intrave-
nously and three rats orally. Blood was taken at 10 timepoints
for intravenous and eight time points for the oral route. Com-
pounds were extracted by plasma protein precipitation and ana-
lyzed by LCMSMS. Quantification was carried out against a
standard curve prepared by spiking known amounts of the same
mixture of compounds into blank rat plasma. PK parameters were
calculated using WinNonlin (Pharsight Inc.).
5
5
.1. Biology
.1.1. Proliferation assays
MDA-MB-231 human breast carcinoma cells, U87-MG human
glioblastoma cells and PC3 human prostate carcinoma cells were
grown in DMEM containing 10% fetal bovine serum (FBS) (Hyclone
Laboratories, Logan UT) supplemented with 2 mM
L-glutamine,
1
00 units/mL penicillin, and 100 g/mL streptomycin (Irvine Scien-
l
tific, Santa Anna, CA). Proliferation was assessed by detection of
DNA synthesis by use of the 5-bromo-2 -deoxyuridine (BrdU) cell
0
proliferation colorimetric ELISA kit from Roche (Indianapolis, IN)
according to the manufacturer’s instructions. For BrdU assays, the
cells were seeded at 2000 cells/well in a 96-well plate, allowed
to attach overnight and then exposed to increasing concentrations
of the individual compounds for 48 h. Each condition was assessed
in triplicate and the experiments were carried out a minimum of
two times. Results in Table 1 are means of the data from each inde-
pendent experiment ± SD.
5.2. Chemistry
5.2.1. Synthesis of 2-methoxyestra-1,3,5(10)-triene-3-ol (1)
Into a stirring suspension of 2-methoxyestrone (9.0 g, 30 mmol)
in 60 mL diethyleneglycol, 20 mL 1-butanol and 2 mL anhydrous
hydrazine (60 mmol) was added. The reaction mixture was heated
under reflux for 1 h until the solution cleared. After the mixture
cooled to 50 °C, KOH pellets (5.04 g, 90 mmol) was added and the
butanol was distilled. The reaction mixture was heated at 50 °C
for 2 h and then cooled to room temperature. After pouring into
ice (50 g), 20 mL 6 N HCl was added and the reaction mixture stir-
red to give a white solid product. The product was separated by fil-
tration, washed with cold water and dried under vacuum to give
7.5 g (80%) of product. The product was purified on a silica gel col-
HUVEC were grown in EGM (Clonetics). HUVEC were seeded at
5
000 cells/well in 96-well plates. After being allowed to attach
overnight, the cells were washed with PBS and incubated in the ab-
sence of growth factor for 24 h (EBM, 2% FBS, Clonetics). Cells were
then treated with increasing concentrations of drug in EBM con-
taining 2% FBS and 10 ng/mL bFGF for 48 h at 37 °C. The prepara-
tion of the drugs and BrdU proliferation assay were performed as
indicated above.
1
umn eluted with CHCl . Mp 111–112 °C; H NMR (CDCl ) d, 6.93
3
3
MCF-7, an estrogen dependent breast carcinoma cell line, was
maintained in DMEM/F12 (1:1) containing 10% (v/v) FBS (Hyclone
Laboratories, Logan, UT) and 1X antibiotic–antimytotic. MCF-7
cells, used between passage 60 and passage 90, were the kind gift
(1H, s, aromatic), 6.75 (1H, s, aromatic), 5.35 (1H, s, phenol), 3.95
(3H, s, methoxy), 2.95 (2H, dd, J = 5.0, 3.0 Hz), 2.25 (2H, m), 1.95
(2H, m), 1.95–1.05 (11H, m), 0.8 (3H, s). Anal. Calcd for C19H26O2:
C, 79.68; H, 9.15. Found: C, 79.79; H, 9.03.

J. H. Shah et al. / Bioorg. Med. Chem. 17 (2009) 7344–7352
7349
5
(
.2.2. Synthesis of 17(20)-methyleneestra-1,3,5(10)-triene-3-ol
2). (General procedure A)
Potassium-tert-amylate in 4.35 mL toluene (1.54 M, 6.69 mmol,
5.2.5. Preparation of 2-methoxy-17(20)-(E/Z)-butylideneestra-
1,3,5(10)-triene-3-ol (5)
Reaction conditions as above in general procedure A except
reaction scale was doubled and butyl triphenylphosphonium bro-
mide was used, from 2-methoxyestrone (593.6 mg, 1.97 mmol) ob-
13
prepared as reported by Schow et al. ) was added to a suspension
of methyl triphenylphosphonium bromide (2.39 g, 6.69 mmol) in
anhydrous benzene (50 mL) and refluxed for 30 min. 2-Methoxye-
strone (300 mg, 1 mmol) in warm benzene (5 mL) was then added
and the mixture was refluxed for 3 h. The reaction was cooled to
room temperature, poured into 100 mL water and washed with
ether (2 ꢂ 100 mL). The combined organics were washed with
tain 532.1 mg (1.56 mmol, 79% yield) of final product. Selected
1
spectral data: H NMR (300 MHz, CDCl
3
) d 6.82 (s, 1H), 6.66 (s,
1H), 5.43 (s, 1H), 5.08 (t, J = 7.4 Hz, 1H), 3.88 (s, 3H), 2.81–2.77
(m, 2H), 2.54–2.01 (m, 7H), 1.99–1.87 (m, 1H), 1.76 (app t, J = 9.6,
12.6 Hz, 2H), 1.49–1.26 (m, 6H), 0.97–0.89 (m, Z isomer, terminal
butyl methyl) and 0.80 (s, E isomer, total 6H, Z/E ratio 9:1, respec-
6
M HCl (1 ꢂ 100 mL), saturated NaHCO
3
(1 ꢂ 100 mL), water
1
3
(
1 ꢂ 100 mL), and brine (1 ꢂ 100 mL), then dried with sodium sul-
3
tively by difference in integration). C NMR (75 MHz, CDCl ) d
fate, filtered, and concentrated via rotary evaporation to give a
semi-solid yellowish oil. The crude product was purified by silica
gel column chromatography using 95:5 hexane/ethyl acetate as
152.2 (E isomer) and 149.8 (Z isomer), 145.0, 143.8, 132.4, 130.0,
120.5 (Z isomer) and 116.7 (E isomer), 115.0, 108.4, 56.5, 55.7 (Z
isomer) and 54.1 (E isomer), 44.9, 44.5 (Z isomer) and 44.3 (E iso-
mer), 39.1 (E isomer) and 38.8 (Z isomer), 37.8 (Z isomer) and 36.6
(E isomer), 32.0, 31.0 (E isomer) and 30.1 (Z isomer), 29.5, 28.2 (E
isomer) and 28.0 (Z isomer), 27.7 (Z isomer) and 27.4 (E isomer),
24.5, 24.3 (Z isomer) and 23.3 (E isomer), 19.6 (E isomer) and
an eluent to yield 220 mg (73% yield) of white solid as 17(20)-
1
methyleneestra-1,3,5(10)-triene-3-ol (2).
CDCl
J = 2.26 Hz, 2H), 3.89 (s, 3H), 2.86–2.74 (m, 2H), 2.64–2.49 (m,
H NMR (300 MHz,
3
) d 6.83 (s, 1H), 6.67 (s, 1H), 5.44 (br s, 1H), 4.70 (t,
1
0
1
2
8
H), 2.39–2.17 (m, 3H), 2.02–1.78 (m, 3H), 1.65–1.19 (m, 4H),
32 2
17.9 (Z isomer), 14.4. Anal. Calcd for C23H O : C, 81.13; H, 9.47.
Found: C, 81.32; H, 9.55.
1
3
3
.85 (s, 3H). C NMR (75 MHz, CDCl ) d 162.2, 144.9, 143.8,
32.3, 130.0, 115.0, 108.5, 101.2, 77.6, 56.5, 53.9, 44.7, 39.2, 36.2,
9.9, 29.5, 28.1, 27.3, 24.3, 19.0. Anal. Calcd for C20
0.48; H, 8.79. Found: C, 80.60; H, 8.77.
26 2
H O : C,
5
.2.6. Synthesis of 2-methoxy-17-b-methylestra-1,3,5(10)-
triene-3-ol (6). (General procedure B)
Compound 2 (471.9 mg, 1.58 mmol) was dissolved in ethyl
acetate (20 mL), flushed with argon, Pd/C 10% (47.5 mg) was
added and the reaction mixture was then subjected to hydrogena-
tion in a Parr hydrogenator for 1 h under 30 psi of hydrogen. The
reaction mixture was then filtered through a Celite pad and sol-
5
.2.3. Synthesis of 2-methoxy-19(E/Z)-norpregna-1,3,5(10)17(20)-
tetraene-3-ol (3)
Reaction conditions as above except reaction scale was doubled
and ethyl triphenylphosphonium bromide was used, from 2-meth-
oxyestrone (613 mg, 2.04 mmol) obtain 540 mg (1.73 mmol, 84%
yield) of final product. Selected spectral data: 1H NMR (300 MHz,
vent was removed to yield 472.5 mg white crystals (97% yield)
1
of the final product. Mp 133–134 °C; H NMR (CDCl
3
) d, 6.82 (s,
3
CDCl ) d 6.82 (s, 1H), 6.67 (s, 1H), 5.44 (s, 1H), 5.23–5.07 (m, 1H),
1
H), 6.66 (s, 1H), 5.43 (s, 1H), 3.88 (s, 3H), 2.85–2.70 (m, 2H),
3.88 (s, 3H), 2.86–2.72 (m, 2H), 2.51–2.38 (m, 2H), 2.38–2.17 (m,
3H), 1.99–1.88 (m, 1H), 1.83–1.68 (m, 4H), 1.49–1.20 (m, 5H),
0.94 (s, Z isomer) and 0.80 (s, E isomer, total 3H, ratio 5:1, respec-
2
.32–2.15 (m, 2H), 1.94–1.68 (m, 4H), 1.52–1.12 (m, 8H), 0.90
13
(
d, J = 6.9 Hz, 3H), 0.61 (s, 3H).
3
C NMR (CDCl ) d, 144.90,
1
4
1
43.75, 132.65, 130.11, 114.99, 108.51, 56.46, 55.21, 45.58,
4.85, 42.74, 39.39, 37.97, 30.65, 29.52, 28.38, 27.21, 24.83,
4.34, 12.44. Anal. Calcd for C20H O : C, 79.96; H, 9.39. Found:
28 2
1
3
3
tively). C NMR (75 MHz, CDCl ) d 153.0 (E isomer) and 150.7 (Z
isomer), 145.0, 143.8, 132.4, 130.0, 115.0, 113.8, 110.6 (Z isomer)
and 108.4 (E isomer), 56.5, 55.6, 54.1, 45.0 (Z isomer) and 44.5 (E
isomer), 39.0 (E isomer) and 38.7 (Z isomer), 37.7 (Z isomer) and
C, 79.98; H, 9.49.
3
2
6.6 (E isomer), 31.9, 29.5, 28.1 (E isomer) and 28.0 (Z isomer),
7.7 (Z isomer) and 27.4 (E isomer), 24.5 (Z isomer) and 24.4 (E iso-
5
3
.2.7. Synthesis of2-methoxy-17-b-ethyl-estra-1,3,5(10)-triene-
-ol (7)
Compound 7 was synthesized according to the general proce-
mer), 19.5 (E isomer) and 17.4 (Z isomer), 14.0 (E isomer) and 13.6
Z isomer). Anal. Calcd for C21 : C, 80.73; H, 8.79. Found: C,
(
8
28 2
H O
dure B above for 6 except compound 3 was used for hydrogenation
to give 91% reaction yield. Mp 118–119 °C; H NMR (300 MHz,
0.60; H, 8.77.
1
CDCl
3
) d 6.82 (s, 1H), 6.66 (s, 1H), 5.43 (s, 1H), 3.88 (s, 1H), 2.78
5
1
.2.4. Synthesis of 2-methoxy-17(20)-E/Z-propylideneestra-
,3,5(10)-triene-3-ol (4)
Reaction conditions as general procedure A except reaction
(
app t, J = 10.2, 4.5 Hz, 3H), 2.30–2.15 (m, 2H), 2.00–1.82 (m, 3H),
1
3
1
3
.79–1.69 (m, 1H), 1.62–1.08 (m, 11H), 0.92 (app t, J = 7.5, 6.6 Hz,
H), 0.63 (s, 3H). C NMR (75 MHz, CDCl ) d 144.90, 143.74,
3
32.66, 130.10, 114.98, 108.49, 56.45, 55.36, 53.58, 44.91, 42.85,
9.19, 38.47, 29.51, 28.65, 28.35, 27.24, 24.67, 23.57, 13.78,
1
3
scale was doubled and propyl triphenylphosphonium bromide
was used, from 2-methoxyestrone (614.2 mg, 2.04 mmol) obtain
3
58.9 mg (1.10 mmol, 54% yield) of final product. Selected spectral
1
12.95. Anal. Calcd for C21
9.95l; H, 9.71.
30 2
H O : C, 80.21; H, 9.62. Found: C,
data: H NMR (300 MHz, CDCl
3
) d 6.81 (s, 1H), 6.66 (s, 1H), 5.44 (s,
7
1
2
1
H), 5.07 (t, J = 7.4 Hz, 1H), 3.88 (s, 3H), 2.88–2.71 (m, 2H), 2.53–
.04 (m, 6H), 2.00–1.87 (m, 1H), 1.76 (t, J = 9.9, 12 Hz, 2H), 1.49–
.18 (m, 6H), 0.99 (t, J = 7.5 Hz, 3H), 0.94 (s, Z isomer) and 0.81
5.2.8. Synthesis of2-methoxy-17-b-propylestra-1,3,5(10)-triene-
3-ol (8)
Compound 8 was synthesized according to the general proce-
dure B above for compound 6 except compound 4 was used to
1
3
(
(
1
1
s, E isomer, total 3H, Z/E ratio 5:1, respectively).
75 MHz, CDCl 151.5 (E isomer) and 149.2 (Z isomer),
45.0,143.8, 132.4, 130.0, 122.3 (Z isomer) and 118.6 (E isomer),
15.0, 108.4, 56.5, 55.7 (Z isomer) and 54.0 (E isomer), 45.0 (Z iso-
C NMR
3
)
d
1
give 95% yield. Mp 124–126 °C; H NMR (CDCl
3
) d, 6.82 (s, 1H),
mer) and 44.9 (E isomer), 44.5 (Z isomer) and 44.2 (E isomer), 39.1
E isomer) and 38.8 (Z isomer), 37.7 (Z isomer) and 36.6 (E isomer),
6.66 (s, 1H), 5.43 (s, 1H), 3.88 (s, 3H), 2.84–2.70 (m, 2H), 2.31–
(
2.16 (m, 2H), 1.97–1.67 (m, 4H), 1.52–1.06 (m, 12H), 0.93 (t,
1
3
3
2
1.9, 29.5, 28.2 (E isomer) and 28.0 (Z isomer), 27.7 (Z isomer) and
7.4 (E isomer), 26.7 (E isomer) and 24.5 (Z isomer), 22.2 (E isomer)
3
J = 6.7 Hz, 3H), 0.63 (s, 3H). C NMR (75 MHz, CDCl ) d 144.91,
143.75, 132.66, 130.10, 115.00, 108.50, 56.46, 55.31, 51.34,
44.92, 42.83, 39.21, 38.41, 33.15, 29.52, 28.98, 28.37, 27.24,
and 21.3 (Z isomer), 19.5 (E isomer) and 17.9 (Z isomer), 15.8 (Z
isomer) and 14.7 (E isomer). Anal. Calcd for C22
H, 9.26. Found: C, 80.71; H, 9.30.
H
30
O
2
: C, 80.94;
24.77, 22.39, 15.06, 12.97. Anal. Calcd for C22
9.82. Found: C, 80.20; H, 9.86.
32 2
H O : C, 80.44; H,

7
350
J. H. Shah et al. / Bioorg. Med. Chem. 17 (2009) 7344–7352
5
3
.2.9. Synthesis of 2-methoxy-17-b-butylestra-1,3,5(10)-triene-
-ol (9)
Compound 9 was synthesized according to the general proce-
dure B above for compound 6 except compound 5 was used to give
4 h. After filtering the reaction mixture through Celite filtering
agent, the solvents were evaporated under vacuum to give white
1
crystals (140 mg, 95%). Mp 222–223 °C; H NMR (CDCl
3
,) d, 6.75
(1H, s, aromatic), 6.44 (1H, s, aromatic), 3.80 (2H, br, amine) 2.75
(2H, dd, J = 5.0, 3.0 Hz), 2.20 (2H, m), 1.95 (2H, m), 1.75–1.05
1
8
1
2
1
5% yield. Mp 119–120 °C; H NMR (CDCl
3
) d, 6.82 (s, 1H), 6.66 (s,
H), 5.43 (s, 1H), 3.89 (s, 3H), 2.78 (app t, J = 10.8, 4.5 Hz, 2H), 2.30–
.16 (m, 2H), 1.96–1.83 (m, 3H), 1.79–1.69 (m, 1H), 1.51–1.07 (m,
5H), 0.92 (t, J = 9, 6.9 Hz, 4H), 0.63 (s, 3H). 13C NMR (75 MHz,
(11H, m), 0.80 (3H, s). Anal. Calcd for C18
H25NO: C, 79.66; H,
9.28; N, 5.16. Found: C, 79.96; H, 8.78; N, 5.14.
CDCl
3
) d 144.90, 143.74, 132.66, 130.09, 114.99, 108.48, 56.46,
5.2.14. Synthesis of 2-azido-estra-1,3,5(10)-triene-3-ol (14)
Into a solution of 2-amino-estradiol (144 mg, 0.5 mmol) in 3 mL
acetic acid glacial, a solution of sodium nitrite (48 mg, 0.7 mmol) in
1 mL water was added at 0 °C. The color of the reaction mixture
changed to orange-yellow. After stirring at 0 °C for 30 min a solu-
tion of sodium azide (45 mg, 0.7 mmol) in water was added. The
color of the reaction mixture changed to orange-red. Temperature
was maintained at 0 °C for 30 min and then raised to room temper-
ature. After stirring for 1 h, the solvents were evaporated under
vacuum and the remaining solid was dissolved in chloroform.
The chloroform layer was washed with water and brine and dried
5
2
C
5.31, 51.52, 44.92, 42.84, 39.20, 38.42, 31.56, 30.49, 29.52,
9.02, 28.36, 27.23, 24.75, 23.63, 14.60, 12.96. Anal. Calcd for
23
34 2
H O : C, 80.65; H, 10.01. Found: C, 80.90; H, 10.10.
5
.2.10. Synthesis of estra-1,3,5(10)-triene-3-ol (10)
Into a stirring suspension of estrone (8.1 g, 30 mmol) in 60 mL
diethylene glycol, 20 mL 1-butanol and 2 mL hydrazine (anhy-
drous) (60 mmol) were added. The reaction mixture was heated
under reflux for 1 h until the solution cleared. After cooling the
reaction mixture to 50 °C, KOH pellets (5.04 g, 90 mmol) was
added and butanol was distilled. The reaction mixture was heated
at 50 °C for 2 h and then cooled to room temperature. The reaction
mixture was poured onto ice (50 g), and then 20 mL 6 N HCl was
added with stirring to give a white solid product. The product
was isolated by filtration, washed with cold water and dried under
vacuum to give 7.5 g (90%) product. The product was purified on a
over CaSO
brown foamy solid. The product was purified by flash silica gel col-
umn eluted with CHCl /MeOH 99:1 mixture to give 100 mg light
yellow foamy solid (70%). IR and H NMR in CDCl
4
anhydrous, filtered and evaporated to give a light
3
1
3
confirmed the
product as 2-azido-estra-1,3,5(10)-triene-3-ol. H NMR (CDCl ) d,
1
3
7.03 (1H, s, aromatic), 6.65 (1H, s, aromatic), 5.25 (1H, s, phenol),
silica gel column eluted with CHCl
3
/MeOH 99:1. Mp 136–137 °C;
,) d, 7.18 (1H, d, J = 8.0 Hz, aromatic), 6.64 (1H,
d, J = 8.0 Hz, aromatic), 6.58 (1H, s, aromatic), 4.58 (1H, s, phenol),
.85 (2H, dd, J = 5.0, 3.0 Hz), 2.25 (2H, m), 1.95 (2H, m), 1.80–1.05
11H, m), 0.85 (3H, s). Anal. Calcd for C18 24O: C, 84.32; H, 9.44.
2.85 (2H, dd, J = 5.0, 3.0 Hz), 2.25 (2H, m), 1.95 (2H, m), 1.95–
1
H NMR (6) (CDCl
3
23 3
1.05 (11H, m), 0.78 (3H, s). Anal. Calcd for C18H N O: C, 72.70;
H, 7.80; N, 14.13. Found: C, 72.87; H, 7.91; N, 14.33.
2
(
H
5.2.15. Synthesis of 2-N-formamidoestra-1,3,5(10)-triene-3-ol
(15)
Found: C, 84.01; H, 9.67.
Formic acid (0.2 mL) was added into a hot solution (60 °C) of 9
(135 mg, 0.5 mmol) in 5 mL toluene. The toluene and water mix-
ture was azeotroped for 1 h. When 1/3 of the toluene was distilled
the reaction mixture was cooled to room temperature to give
5
.2.11. Synthesis of 2-nitroestra-1,3,5(10)-triene-3-ol (11)
Into a solution of 17-deoxyestrone (6), (760 mg, 3 mmol) in
0 mL acetic acid glacial, 0.19 mL (3 mmol) of concd HNO was
2
3
added at 2 °C. The reaction mixture was brought to room temper-
ature and stirred for 18 h. The reaction mixture was diluted with
water and products were extracted with ethyl ether. The ether
white crystals of 2-N-formamido-17-deoxyestrone (100 mg, 75%).
1
Mp 190–191 °C; H NMR (CDCl
3
,) d, 8.2 (1H, s), 7.6 (1H, br), 7.05
(1H, s, aromatic), 6.84 (1H, s, aromatic), 2.90 (2H, dd, J = 5.0,
layer was washed with water and brine and dried over CaSO
anhydrous), filtered and evaporated to give a yellow solid. The
product was purified on flash silica gel column eluted with hex-
ane/CH Cl 4:1 mixture to give a yellow crystal product (400 mg,
5%). Mp = 118–119 °C; H NMR (7) (CDCl
4
3.0 Hz), 2.20 (2H, m), 1.85 (2H, m), 1.8–1.15 (11H, m), 0.75 (3H,
(
s). Anal. Calcd for C19
2
H25NO : C, 76.22; H, 8.42; N, 4.68. Found: C,
76.28; H, 8.72; N, 4.52.
2
2
1
5
3
,) d, 10.42 (1H, s, phe-
5.2.16. Synthesis of 2-N-methylaminoestra-1,3,5(10)-triene-3-
nol), 8.05 (1H, s, aromatic), 6.84 (1H, s, aromatic), 2.95 (2H, dd,
ol (16)
J = 5.0, 3.0 Hz), 2.30 (2H, m), 1.95 (2H, m), 1.80–1.15 (11H, m),
Into solution of AlH
(anhydrous), amide 10 (150 mg, 0.5 mmol) in 5 mL THF was added
at room temperature and stirred for 2 h. THF/H O (0.4 mL, 1:1)
3
(5 mmol) (formed in situ) in 15 mL THF
0
.80 (3H, s). Anal. Calcd for C18
3
H23NO : C, 71.73; H, 7.69; N, 4.65.
Found: C, 71.69; H, 7.64; N, 4.39.
2
was added dropwise, followed by 1.5 mL NaOH (15% solution). This
suspension was stirred for 20 min after which, ether was added
(20 mL). The Al(OH) precipitates were removed by filtration. Sol-
3
5
3
.2.12. Synthesis of2-N,N-dimethylaminoestra-1,3,5(10)-triene-
-ol (12)
Compound 7 (301 mg, 1.0 mmol) was dissolved in 30 mL diox-
vents were evaporated and the product was purified on a flash silica
gel column eluted with a hexane/ether 1:1 mixture to give a white
solid product (120 mg, 70%). The HCl salt of the product was formed
by dissolving 11 in IPA/HCl (gas) solution. The salt was precipitated
by adding ethyl ether, which was isolated by filtration, washed by
ane/methanol 1:3 mixture and was hydrogenated in a Parr hydrog-
enator at 40 psi of hydrogen gas in the presence of 0.4 mL
formaldehyde (37%) and Pd/C 10% (200 mg) for 4 h. After filtering
the reaction mixture through a Celite filter pad the solvents were
evaporated under vacuum to give a white powder (240 mg, 95%).
ether and dried under vacuum. Mp 266–267 °C (HCl salt, decom-
1
1
H NMR (CDCl
.85 (2H, dd, J = 5.0, 3.0 Hz), 2.65 (6H, s, dimethylamino) 2.25
2H, m), 1.95 (2H, m), 1.75–1.05 (11H, m), 0.80 (3H, s). Anal. Calcd
for C20 29NO: C, 80.22; H, 9.76; N, 4.68. Found: C, 80.07; H, 10.13;
N, 4.25.
3
,) d 7.15 (1H, s, aromatic), 6.74 (1H, s, aromatic),
pose); H NMR (HCl salt) (DMSO-d
6
,) d, 10.25 (1H, s), 10.16 (1H,
2
(
br), 7.15 (1H, s, aromatic), 6.84 (1H, s, aromatic), 2.85 (3H, s, methyl-
amine), 2.75 (2H, dd, J = 5.0, 3.0 Hz), 2.20 (2H, m), 1.95 (2H, m), 1.75–
H
1.00 (11H, m), 0.70 (3H, s). Anal. Calcd for C19
9.53; N, 4.91. Found: C, 80.06; H, 9.78; N, 5.13.
H27NO: C, 79.95; H,
5
.2.13. Synthesis of 2-aminoestra-1,3,5(10)-triene-3-ol (13)
Compound 7 (170 mg, 0.5 mmol) was dissolved in 30 mL diox-
5.2.17. Synthesis of 2-methoxyestra-1,3,5(10)-triene-17-p-toluene-
sulfonhydrazone-3-ol (17)
ane/methanol 1:5 mixture and hydrogenated in a Parr hydrogena-
tor at 40 psi of hydrogen in the presence of Pd/C 10% (200 mg) for
To a solution of 2-methoxyestrone (0.60 g, 2 mmol) in 2 mL
methanol, p-toluene-sulfonylhydrazide (0.46 g, 2.5 mmol) was

J. H. Shah et al. / Bioorg. Med. Chem. 17 (2009) 7344–7352
7351
added. The reaction mixture was heated under reflux for 1 h. The
methanol was evaporated and product was crystallized from ethyl
ether to give 720 mg (80%) white solid. 1H NMR (CDCl
,) d, 7.90
2H, d, J = 7.3, aromatic), 7.37 (2H, d, J = 7.35, aromatic), 6.95 (1H,
s), 6.70 (1H, s, aromatic), 6.63 (1H, s, aromatic), 5.50 (1H, br),
(s, 3H). Anal. Calcd for C19
80.12; H, 8.60.
H
24
O
2
: C, 80.24; H, 8.50. Found: C,
3
(
5.2.21. Synthesis of 2-methoxyestra-1,3,5(10)-triene-17-one-oxime-
3-ol (21)
3
2
.95 (3H, s, methoxy), 2.85 (3H, s), 2.89 (2H, dd, J = 5.0, 3.0 Hz),
.55 (2H, m), 2.30 (2H, m), 2.10–1.35 (11H, m), 0.95 (3H, s). Anal.
To a solution of 2-methoxyestrone (0.90 g, 3 mmol) in 10 mL
methanol, sodium acetate (anhydrous) (3 g, 30 mmol), and hydrox-
ylamine hydrochloride (2.1 g in 3 mL water) were added. The reac-
tion mixture was heated under reflux for 3 h to give a clear
solution. The solvents were evaporated and the product was ex-
tracted with chloroform. The chloroform layer was washed with
Calcd for C26
H
32
N
2
O
4
S: C, 66.64; H, 6.88; N, 5.98. Found: C,
6
6.61; H, 6.80; N, 6.01.
5
3
.2.18. Synthesis of 2-methoxyestra-1,3,5(10)-triene-17-amino-
-ol (18)
4
water (20 mL), brine and dried over CaSO (anhydrous), filtered
A solution of 2-methoxyestrone (300 mg, 1 mmol), ammonium
and evaporated to give 820 mg (90%) of white solid. Mp 208–
209 °C; H NMR (CDCl ,) d, 6.90 (1H, s, aromatic), 6.75 (1H, s, aro-
3
matic), 5.55 (1H, br), 3.90 (3H, s, methoxy), 2.85 (2H, dd, J = 5.0,
3.0 Hz), 2.55 (2H, m), 2.30 (2H, m), 2.10–1.35 (11H, m), 1.03 (3H,
1
acetate (0.77 g, 10 mmol) and NaBH
absolute methanol was stirred for 48 h at 25 °C. After adding
.5 mL concentrated HCl the methanol was evaporated. After dilut-
3
CN (33 mg, 7 mmol) in 10 mL
0
ing the reaction mixture with 10 mL water, it was basified with 1 N
NaOH until pH >10 and the product was extracted with 2 ꢂ 20 mL
portions of ethyl acetate. The combined extracts were dried over
s). Anal. Calcd for C19
72.39; H, 7.91; N, 4.43.
3
H25NO : C, 72.35; H, 7.99; N, 4.44. Found: C,
MgSO
white solid (67%). Product was recrystallized from the isopropa-
nol/hexane mixture. 1H NMR (CDCl
) d, 6.93 (1H, s, aromatic),
.69 (1H, s, aromatic), 5.50 (1H, br), 3.91 (3H, s, methoxy), 2.85
2H, dd, J = 5.0, 3.0 Hz), 2.55 (2H, m), 2.30 (2H, m), 2.10–1.35
11H, m), 1.03 (3H, s). Anal. Calcd for C19 : C, 75.71; H,
4
(anhydrous), filtered and evaporated to give 200 mg of
5.2.22. Synthesis of 3-hydroxy-2-methoxy-estra-1,3,5(10)-
triene-17 /b-carboxylic acid (22)
a
3
A slurry of steroid (3-hydroxy-2-methoxy-estra-1,3,5(10)-tri-
ene-17 cyano) (590 mg, 1.5 mmol) in ethylene glycol (22 mL)
was added to a solution of NaOH (1.9 g) in ethylene glycol
(13 mL) and refluxed for 4 h. The reaction was cooled to room tem-
perature, water was added, and solution was acidified to pH 1 with
HCl. A precipitate started to form, and the mixture was placed in
fridge overnight. Crystals were isolated and purified by chromatog-
6
(
(
H27NO
2
9
.03; N, 4.65. Found: C, 75.74; H, 8.83; N, 4.35.
5
.2.19. Synthesis of2-methoxyestra-1,3,5(10)-triene-17-propyl-
amino-3-ol (19)
raphy with 9:1:0.1 CHCl
3
/MeOH/NH
4
OH. Yield: 50%, 250 mg,
1
To a solution of 2-methoxyestrone (300 mg, 1 mmol) in 10 mL
absolute methanol was added propylamine (0.49 mL, 6 mmol),
0.76 mmol. H NMR (CDCl ) d, 6.72 (s, beta isomer) and 6.69 (s, al-
3
pha isomer, total 1H, ratio 2:1, respectively), 6.57 (s, 1H), 5.38 (s,
br, 1H), 3.79 (s, beta isomer) and 3.77 (s, alpha isomer, total 3H, ra-
tio 2:1, respectively), 2.75–2.66 (m, 2H), 2.58 (dd, J = 7.9, 3.4 Hz, al-
pha isomer) and 2.42 (app t, J = 9.1 Hz, beta isomer, total 1H, ratio
1:2, respectively), 2.28–1.10 (m, 13H), 0.86 (s, alpha isomer) and
0.71 (s, beta isomer, total 3H, ratio: 1:2, respectively). High-resolu-
0
3
.4 mL 5 N HCl/MeOH and LiBH CN (33 mg, 7 mmol) and stirred
for 60 h at 25 °C. The methanol was evaporated and after diluting
the reaction mixture with 10 mL water, it was basified with 1 N
NaOH until pH >10 and product was extracted with 2 ꢂ 20 mL por-
tions of ethyl acetate. The combined extracts were dried over
MgSO
4
(anhydrous), filtered and evaporated to give 220 mg of
26 4
tion mass spectrometric analysis (C20H O as the sodium adduct)
Calculated MNa : 353.1729, observed MNa : 353.1727.
+
+
white solid (70%). Product was recrystallized from an isopropa-
1
nol/hexane mixture, mp 233.5–234.5 °C. H NMR (CDCl
1H, s, aromatic), 6.79 (1H, s, aromatic), 5.70 (1H, br), 3.93 (3H, s,
methoxy), 3.10–2.91 (3H, m), 2.85 (2H, dd, J = 5.0, 3.0 Hz), 2.55
2H, m), 2.30 (2H, m), 2.10–1.35 (14H, m), 1.03 (3H, s), 0.93 (3H,
t, J = 5.5 Hz). Anal. Calcd for C22 : C, 76.92; H, 9.68; N, 4.08.
Found: C, 77.12; H, 9.60; N, 3.99.
3
) d, 7.03
(
5.2.23. Synthesis of 3-hydroxy-2-methoxy-estra-1,3,5(10)-triene-
17
a
/b-carboxamide (23)
Steroid 22 (0.4 mmol) in freshly distilled SOCl
red for 2 h at 70 °C, than evaporated. THF (2 mL) was added and
NH (g) was bubbled through for 5 min at room temperature. The
(
2
(2 mL) was stir-
H33NO
2
3
mixture was diluted with THF (2 mL) and solid byproduct was fil-
5
.2.20. Synthesis of 2-methoxy-1,3,5(10)16-estratetraene-3-ol
tered off. The filtrate was evaporated and purified by chromatogra-
1
(
20)
phy (95:5:0.1 CHCl
(CDCl
3
4
/MeOH/NH OH) to give compound 23. H NMR
2
-Methoxyestrone-p-tosylhydrazone (17) (1.5757 g, 3.3 mmol)
3
) d, 6.80 (s, 1H), 6.66 (s, 1H), 5.54–5.27 (m, 3H), 3.88 (s, beta
was dissolved in anhydrous tetrahydrofuran (50 mL) in a flame
dried 250 mL round bottomed flask and cooled to ꢁ10 °C. The mix-
ture was stirred and n-butyl lithium (5.3 mL, 2.5 M in hexanes, Al-
drich) was added dropwise. The mixture was warmed to room
temperature over 6 h. Ice (ꢃ5 g) was added, followed by saturated
ammonium chloride (ꢃ50 mL). The mixture was transferred to
isomer) and 3.87 (s, alpha isomer, total 3H, ratio 3:1, respectively),
2.85–2.74 (m, 2H), 2.43–1.22 (m, 14H), 0.92 (s, alpha isomer) and
0.79 (s, beta isomer, total 3H, ratio 1:3, respectively. High-resolu-
+
tion mass spectrometric analysis (C20
330.2069, observed MH : 330.2068.
H
26NO
3
) Calculated MH :
+
2
50 mL separatory funnel, shaken, and separated. The aqueous
References and notes
layer was washed with ethyl acetate (50 mL) and the organics were
combined. The organics were washed with 1 N HCl (20 mL) and
brine (50 mL), then dried with magnesium sulfate, filtered, and sol-
vent was removed under reduced pressure. The resulting solid was
dissolved in acetone (ꢃ10 mL) and solvent was removed under re-
duced pressure two times. Product was purified by silica gel col-
1.
Pribluda, V. S.; Gubish, E. R.; LaVallee, T. M.; Treston, A. M.; Swartz, G. M.;
Green, S. J. Cancer Met. Rev. 2000, 19, 173.
2
3
.
.
Zhu, B. T.; Conney, A. H. Cancer Res. 1998, 58, 2269.
Fotsis, T.; Zhang, Y.; Pepper, M. S.; Adlercrcutz, H.; Montesano, R.; Nawreth, P.
P.; Schweigerer, L. Nature 1994, 368, 237.
4. Wilding, G.; Sweeney, C.; King, D. M.; Horvath, D.; Staab, M. J.; Fife, K.;
Armstrong, V.; Soriano, J. V.; Pribluda, V.; Gubish, E. R.; Sidor, C. Proc. Am. Soc.
Clin. Oncol. 2002, 21, #338.
5. Fogler, W. E. Drug Disc. World 2008, Fall, 15.
6. Martucci, C. P. Metabolic Fate of Catecolestrogens. In Catecol Estrogens;
umn eluted with hexanes/CHCl
3
1:1 mixture to obtain crystalline
) d 6.81 (s,
solid (745 mg, 80% yield). 1H NMR (300 MHz, CDCl
3
1
H), 6.68 (s, 1H), 5.97–5.91 (m, 1H), 5.80–5.74 (m, 1H), 5.43
(
s, 1H), 3.88 (s, 3H), 2.90–2.71 (m, 2H), 2.38–1.34 (m, 11H), 0.82
Merriam, G. R., Lipsett, M. B., Eds.; Raven Press: NY, 1983; pp 115–122.

7
352
J. H. Shah et al. / Bioorg. Med. Chem. 17 (2009) 7344–7352
7
.
.
Adlercreutz, H.; Martin, F. J. Steroid Biochem. 1980, 13, 231; Adlercreutz, H. N.
Engl. J. Med. 1974, 290, 1081.
2002, 67, 1079; (j) Agoston, G. E.; Shah, J. H.; LaVallee, T. M.; Zhan, X.;
Pribluda, V. S.; Treston, A. M. Bioorg. Med. Chem. 2007, 15, 7524.
11. Shapiro, R. J.; Djerassi, C. J. Org. Chem. 1964, 86, 2825.
12. Peters, R. H.; Crowe, F. C.; Avery, M. A.; Chong, W. K. M.; Tanabe, M. J. Med.
Chem. 1989, 32, 1642.
13. Schow, S. R.; McMorris, T. C. J. Org. Chem. 1979, 44, 3760.
14. Romanelli, M. G.; Becker, E. I. Org. Syn. Coll. Vol. 5; John Wiley and Sons, 1973.
pp 552–554.
15. Fieser, L. F.; Jones, J. E. Org. Syn. Coll. Vol. 3; John Wiley and Sons, 1955. pp 590–
591.
16. Shah, J. H.; Izenwasser, S.; Geter-Douglass, B.; Witkin, J. M.; Newman, A. H. J.
Med. Chem. 1995, 38, 4284.
8
(a) For data in humans: Sledge, G. W.; Miller, K. D.; Haney, L. G.; Nguyen, D. D.;
Storniole, A. M.; Philips, E. N.; Pribluda, V. S.; Gubish, E. R. 38th Annual Meeting,
American Society of Clinical Oncology, May 2002, Orlando, FL.; (b) Miller, K. D.;
Murray, D. J.; Curry, E.; Haney, L. G.; McClaskey, G. W; Storniolo, A. M.; Philips,
E.; Pribluda, V. S.; Gubish, E. R. 38th Annual Meeting, American Society of
Clinical Oncology, May 2002, Orlando, FL.; (c) Fpr data in rats, Unpublished
data, EntreMed, Inc.
9
.
Lakhani, N. J.; Sparreboom, A.; Xu, X.; Veenstra, T. D.; Venitz, J.; Dahut, W. L.;
Figg, W. D. J. Pharm. Sci. 2007, 96, 1821.
1
0. (a) Cushman, M.; He, M.-H.; Katzenellenbogen, J. A.; Lin, C. M.; Hamel, E.
J. Med. Chem. 1995, 38, 2041; (b) Cushman, M.; He, M.-H.;
Katzenellenbogen, J. A.; Varma, R. K.; Hamel, E.; Lin, C. M.; Ram, S.;
Sachdeva, Y. P. J. Med. Chem. 1997, 40, 2323; (c) Miller, T. A.; Bulman, A.
L.; Thompson, C. D.; Garst, M. E.; Macdonald, T. L. J. Med. Chem. 1997, 40,
17. Carroll, F. I.; Gao, Y.; Abraham, P.; Lewin, A. H.; Lew, R.; Patel, A.; Boja, J. W.;
Kuhar, M. J. J. Med. Chem. 1992, 35, 1813.
18. Kabalka, G. W.; Hutchins, R.; Natale, N. R.; Yang, D. T. C.; Broach, V. Org. Syn.
Coll. Vol. 6; John Wiley and Sons, 1973. pp 293–298.
3
836; (d) Miller, T. A.; Bulman, A. L.; Thompson, C. D.; Garst, M. E.;
19. Berliner, D. L., Adams, N. W.; White, J. U.S. Patent 5783571, 1998.
20. Borch, R. F.; Bernstein, K. D.; Durst, D. H. J. Am. Chem. Soc. 1971, 93, 2897.
21. McGuire, M. A.; Sorenson, E.; Owing, F. W.; Resnick, M. F.; Baine, N. H. J. Org.
Chem. 1994, 59, 6683.
22. LaVallee, T. M.; Zhan, X. H.; Herbstritt, C. J.; Kough, E. C.; Green, S. J.; Pribluda,
V. S. Cancer Res. 2002, 62, 3691.
23. (a) Suwandi, L. S.; Agoston, G. E.; Shah, J. S.; Hanson, A. H.; Zhan, X. H.; LaVallee,
T. M.; Pribluda, V.; Treston A. M., Bioorg. Med. Chem. Lett. in press.; (b)
Agoston, G. E.; Shah, J. H.; Suwandi, L. S.; Hanson, A. D.; Zhan, X. H.; LaVallee, T.
M.; Pribluda, V. S.; Treston, A. M., Bioorg. Med. Chem. Lett. in press.
Macdonald, T. L. Biooorg. Med. Chem. Lett. 1997, 7, 1851; (e) Wang, Z.;
Yang, D.; Mohanakrishnan, A. K.; Fanwick, P. E.; Nampoothiri, P.; Hamel,
E.; Cushman, M. J. Med. Chem. 2000, 43, 2419; (f) Hughes, R. A.; Harris, T.;
Altman, E.; McCallister, D.; Vlahos, R.; Robertson, A.; Cushman, M.; Wang,
Z.; Stewart, A. G. Mol. Pharmacol. 2002, 61, 1053; (g) Cushman, M.;
Mohanakrishnan, A. K.; Hollingshead, M.; Hamel, E. J. Med. Chem. 2002, 45,
4
748; (h) Edsall, A. B.; Mohanakrishhnan, A. K.; Yang, D.; Fanwick, P. E.;
Hamel, E.; Hanson, A. D.; Agoston, G. E.; Cushman, M. J. Med. Chem. 2004,
7, 5126; (i) Rao, P. N.; Cessac, J. W.; Tinley, T. L.; Mooberry, S. L. Steroids
4