Synthesis and in vivo antitumor evaluation of 2-methoxyestradiol 3-phosphate, 17-phosphate, and 3,17-diphosphate

Article abstract of DOI:10.1021/jm070639e

A prodrug strategy was investigated to address the problem of limited aqueous solubility and the resulting limited bioavailability of the antitumor agent 2-methoxyestradiol. The 3-phosphate, 17-phosphate, and 3,17-diphosphate of 2-methoxyestradiol were sy

Full text of DOI:10.1021/jm070639e

6700
J. Med. Chem. 2007, 50, 6700–6705
Synthesis and in Vivo Antitumor Evaluation of 2-Methoxyestradiol 3-Phosphate, 17-Phosphate,
and 3,17-Diphosphate
Allison B. Edsall,† Gregory E. Agoston,‡ Anthony M. Treston,‡ Stacy M. Plum,‡ Robert H. McClanahan,§ Tian-Sheng Lu,§
Wei Song,§ and Mark Cushman*,†
Department of Medicinal Chemistry and Molecular Pharmacology, School of Pharmacy and Pharmaceutical Sciences, and the Purdue Cancer
Center, Purdue UniVersity, West Lafayette, Indiana 47907, EntreMed, Inc., RockVille, Maryland 20850, and Department of Toxicology and
Pharmacology, Ricera Biosciences, LLC, 7528 Auburn Road, Concord, Ohio 44077
ReceiVed June 4, 2007
A prodrug strategy was investigated to address the problem of limited aqueous solubility and the resulting
limited bioavailability of the antitumor agent 2-methoxyestradiol. The 3-phosphate, 17-phosphate, and 3,17-
diphosphate of 2-methoxyestradiol were synthesized. 2-Methoxyestradiol 3-phosphate was metabolized more
efficiently to the parent compound in vivo than 2-methoxyestradiol 17-phosphate, and it was also more
cytotoxic in cancer cell cultures than either the 17-phosphate or the 3,17-diphosphate. These results agree
with the in vivo anticancer activity of 2-methoxyestradiol 3-phosphate in a mouse Lewis lung carcinoma
experimental metastasis model as opposed to the 17-phosphate and 3,17-diphosphate, both of which were
inactive. The in vivo antitumor activity of 2-methoxyestradiol 3-phosphate at a dose of 200 mg/kg per day
was comparable to that of a maximally tolerated dose of cyclophosphamide.
Scheme 1a
The endogenous human estrogen metabolite 2-methoxyestra-
diol (1), formed by hepatic cytochrome P450 2-hydroxylation
of ꢀ-estradiol followed by 2-O-methylation by catechol
O-methyltranseferase,1–3 has attracted interest because of its
potent inhibition of tumor vasculature and tumor cell growth.4
Because solid tumor growth is dependent on angiogenesis, the
potent antiangiogenic activity and tubulin polymerization inhibi-
tion of 2ME2 in vivo are of potential therapeutic value and have
warranted further investigation in clinical trials.4–10
The cytotoxicity of 2-methoxyestradiol has been associated
with uneven chromosome distribution, inhibition of mitosis, and
an increase in the number of abnormal metaphases.5,6 Competi-
tive binding studies with [3H]colchicine have demonstrated that
a Reagents and conditions: (a) (1) DMAP, CH3CN, CCl4, DIEA, 0 °C
the inhibitory effect of 2-methoxyestradiol on tubulin poly-
(30 min); (2) dibenzylphosphite, 0 °C to room temperature (16 h); (b) (1)
Pd/C, H2, MeOH, 40 psi, room temperature (24 h); (2) 60 psi, room
temperature (24 h).
merization is mediated through the colchicine binding site on
tubulin.11,12 The inhibition of tubulin polymerization by 2-meth-
oxyestradiol results in the inhibition of hypoxia inducible
factor-1 (HIF) at the post-transcriptional level.13 This dysregu-
lation of HIF downstream from the 2ME2/tubulin interaction
inhibits the transcriptional activation of HIF response genes,
including vascular endothelial growth factor (VEGF), a major
participant in the process of angiogenesis.13
There are many ways to improve the bioavailability of clinical
agents. 2-Methoxyestradiol in its original capsule formulation
displayed low plasma concentrations relative to the oral doses
administered. This indicates low bioavailability of 2-methoxy-
estradiol, possibly as a function of its low aqueous solubility.10,14
To address this issue from a formulation standpoint, EntreMed
has developed an alternate formulation that is currently under
clinical evaluation in oncology. An alternative approach is to
prepare water-soluble prodrugs that would be efficiently me-
tabolized to 2-methoxyestradiol as presented in this paper.
Ample precedent exists for the successful use of phosphate
prodrugs to increase aqueous solubility, including applications
with estramustine, epinine, anandamide, buparvaquone, camp-
tothecin, paclitaxel, etoposide, cortisone, resveratrol, and com-
bretastatin A-4.15–24 Phosphate prodrugs are typically converted
to the parent molecule via nonspecific alkaline or acidic
phosphatases.15,17,20,22 This approach has allowed intravenous
administration of phosphate prodrugs, including some steroid
hormones, to overcome otherwise bleak pharmacokinetic pro-
files. In particular, combretastatin A-4, another microtubule-
targeting and antiangiogenic agent, has recently benefited from
a prodrug approach.22,25
Chemistry
Three potential phosphate prodrugs of 2-methoxyestradiol
were synthesized as described in Schemes 1–3. These were the
3-phosphate 3 (Scheme 1), the 17-phosphate 7 (Scheme 2), and
the 3,17-diphosphate 9 (Scheme 3) derivatives.
The regioselective phosphorylation of 2-methoxyestradiol
at the 3-hydroxyl group as opposed to the 17-hydroxyl group
relied on the selective deprotonation of the phenol under basic
conditions, resulting in the more nucleophilic phenoxide
* Author to whom correspondence should be addressed [telephone (765)
494-1465; fax (765) 494-6790; e-mail cushman@pharmacy.purdue.edu].
†
Purdue University.
EntreMed.
Ricera Biosciences, LLC.
‡
§
10.1021/jm070639e CCC: $37.00
2007 American Chemical Society
Published on Web 12/06/2007

Prodrug Strategy for 2-Methoxyestradiol
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6701
Scheme 2a
a Reagents and conditions: (a) Ti(t-BuO)4, Et3N, (EtO)2P(O)Cl, CH2Cl2,
room temperature (24 h); (b) Pd/C, H2, MeOH, room temperature (17 h);
(c) (1) TMSBr, CHCl3, room temperature, (30 h); (2) MeOH, reflux (3 h).
Scheme 3a
a Reagents and conditions: Ti(t-BuO)4, Et3N, (EtO)2P(O)Cl, CH2Cl2,
room temperature (11 h); (b) (1) TMSBr, CHCl3, room temperature (9 h);
(2) MeOH.
anion. Treatment of 2-methoxyestradiol (1) with dimethyl-
aminopyridine, carbon tetrachloride, diisopropylethylamine,
and dibenzyl phosphite successfully produced the phosphoric
acid dibenzyl ester 2 in high yield (Scheme 1). Hydrogenoly-
sis of the two benzyl moieties of 2 provided the 3-phosphate
analogue 3.
To allow phosphorylation of the 17-hydroxyl group and not
the 3-hydroxyl group, 2-methoxyestradiol (1) was first benzy-
lated regioselectively on the 3-hydroxy group to afford 2-meth-
oxyestradiol-3-benzyl ether (4),26 which was then phosphory-
lated at the 17-position using titanium tert-butoxide, triethylamine,
and diethylchlorophosphate to afford the diethylphosphate ester
5 (Scheme 2).27 The titanium tert-butoxide was prepared by
heating tert-butyl acetate and titanium isopropoxide at reflux.28
Hydrogenolysis of the benzyl ether 5 yielded intermediate 6.
Reaction of the diethyl phosphate 6 with trimethylsilyl bromide
afforded the desired 17-phosphate 7.
The diphosphate intermediate 8 (Scheme 3) was synthe-
sized utilizing methods similar to those employed for the
preparation of intermediate 5. Phosphorylation of the 3- and
17-hydroxyl groups of 2-methoxyestradiol (1) using titanium
tert-butoxide, triethylamine, and diethylchlorophosphate pro-
vided intermediate 8. Cleavage of the ethyl moieties with
trimethylsilyl bromide in refluxing MeOH provided the
desired 3,17-diphosphate 9.
Biological Results and Discussion
The three phosphates 3, 7, and 9, as well as their synthetic
precursors 6 and 8, were examined for antiproliferative activity

6702 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Edsall et al.
Table 2. Concentrations of 2-Methoxyestradiol 3-Phosphate (3),
2-Methoxyestradiol (1), and 2-Methoxyestrone in Rat Plasma after
Intravenous Administration of 1 mg/kg of 2-Methoxyestradiol
3-Phosphate
Table 4. Concentrations of 2-Methoxyestradiol and 2-Methoxyestrone
in Rat Plasma after Intravenous Administration of 1 mg/kg of
2-Methoxyestradiol
concentration (ng/mL)
concentration (ng/mL)
time (min)
2ME2a
BLQc
763.5 ( 256.1
168.0 ( 10.1
44.7 ( 3.2
11.3 ( 3.3
BLQ
2ME1b
time (min)
2ME2-3Pa
2ME2b
2ME1c
BLQ
16.3 ( 2.7
12.7 ( 1.4
8.8 ( 1.5
2.7 ( 3.8
BLQ
0
5
15
30
60
120
BLQ
6.3 ( 8.9
BLQ
5.4 ( 0.2
BLQ
BLQ
0
5
15
30
60
120
BLQd
BLQ
366.4 ( 14.8e
31.3 ( 5.5
7.1 ( 2.0
1.2 ( 1.6
BLQ
153.4 ( 13.8
57.3 ( 13.0
22.3 ( 0.1
5.3 ( 4.0
BLQ
a 2-Methoxyestradiol. b 2-methoxyestrone. c Below the limit of quanti-
tation (1 ng/mL for 2ME2-3P and 5 ng/mL for 2ME2 and 2ME1).
a 2-Methoxyestradiol 3-phosphate. b 2-methoxyestradiol. c 2-methoxyestrone.
d Below the limit of quantitation (1 ng/mL for 2ME2-3P and 5 ng/mL for 2ME2
and 2ME1). e Each value is the average of three separate determinations.
Table 5. Antitumor Activity of 2-Methoxy-ꢀ-estradiol 3-Phosphate in
the LLC Experimental Metastasis Assay Following Intraperitoneal
Administration
Table 3. Concentrations of 2-Methoxyestradiol 17-Phosphate (7),
2-Methoxyestradiol (1), and 2-Methoxyestrone in Rat Plasma after
Intravenous Administration of 1 mg/kg of 2-Methoxyestradiol
17-Phosphate
treatment group
dosing regimen
mean lung wt (g (SD) T/Ca
vehicle control
2ME2
0.1 mL qd × 12
0.60 ( 0.18
200 mg/kg qdc × 12
200 mg/kg qd × 12
100 mg/kg qd × 12
0.44 ( 0.17
0.23 ( 0.06
0.44 + 0.21
0.19 ( 0.03
0.55
0.10
0.55
0.02
concentration (ng/mL)
2ME2-3Pb
2ME2-3P
time (min)
2ME2-17Pa
2ME2b
2ME1c
cyclophosphamide 150 mg/kg qodd × 3
0
5
15
30
60
120
BLQd
BLQ
BLQ
BLQ
BLQ
BLQ
BLQ
BLQ
a T/C was determined following correction for normal (non-tumor-
bearing) lung weight as described in the Experimental Section. b 2-Methoxy-ꢀ-
estradiol 3-phosphate. c qd, daily dosing. d qod, dosing every other day.
1316.1 ( 73.1e
351.4 ( 120.0
137.7 ( 59.3
21.2 ( 12.8
6.9 ( 5.8
34.1 ( 5.0
15.0 ( 3.9
9.2 ( 2.0
BLQ
2). The level of 2-methoxyestradiol (1) surpassed that of the
prodrug 3 15 min after administration and remained at higher
concentration thereafter. The concentrations of all three com-
pounds (1, 3, and 2-methoxyestrone) approached the lower limits
of detection 30 min after administration, and they were
undetectable after 1 h.
In contrast, the 17-phosphate 7 was metabolized to 2-meth-
oxyestradiol much less efficiently (Table 3). The concentrations
of 2-methoxyestradiol (1) achieved after iv administration of
the 17-phosphate were significantly lower than those realized
after administration of the 3-phosphate 3, and the concentrations
of 2-methoxyestradiol (1) never surpassed those of the prodrug
7. The rate of disappearance of the 17-phosphate was slower
than that of the 3-phosphate.
At the doses administered, the concentrations of 2-methox-
yestradiol were higher after iv administration of 2-methox-
yestradiol itself than after administration of either of the prodrugs
(Table 4). The main advantage offered by the prodrugs is that
they are more water soluble and can therefore be administered
intravenously in higher doses.
The efficacies of 2ME2 and the two monophosphates 3 and
7, as well as the diphosphate 9, were examined in a mouse Lewis
lung carcinoma (LLC) experimental metastasis model following
intraperitoneal (ip) administration. In this experimental model,
lung weight is used to indicate tumor burden. 2ME2 at a dose
of 200 mg/kg resulted in lung weights corresponding to a
decrease in tumor burden without morbidity being observed.
2-Methoxyestradiol 17-monophosphate (7) and 3,17-diphosphate
(9) did not exhibit any effect on lung weight compared to the
vehicle control-treated animals, indicating that neither compound
demonstrates antitumor activity at the doses used (data not
shown). In contrast, administration of 2-methoxyestradiol
3-phosphate (3) at doses of 100 and 200 mg/kg per day resulted
in lung weights corresponding to a dose-related decrease in
tumor burden (Table 5). At the higher dose of 200 mg/kg per
day, the inhibition of tumor growth was comparable to that
observed after administration of a maximally tolerated dosing
regimen of cyclophosphamide (positive control treatment), and
it is significantly better than that observed with 2-methoxyestra-
diol itself at the same dose. However, morbidity requiring
BLQ
a 2-Methoxyestradiol 17-phosphate. b 2-methoxyestradiol. c 2-methoxyestrone.
d Below the limit of quantitation (1 ng/mL for 2ME2-17P and 5 ng/mL for
2ME2 and 2ME1). e Each value is the average of three separate determinations.
against the human cancer cell lines in the National Cancer
Institute screen, in which the activity of each compound was
evaluated with approximately 55 different cancer cell lines of
diverse tumor origins.29,30 The GI50 values obtained with
selected cell lines, along with the mean graph midpoint (MGM)
values, are summarized in Table 1. The MGM is based on a
calculation of the average GI50 for all of the cell lines tested
(approximately 55) in which GI50 values below and above the
test range (10-8-10-4 molar) are taken as the minimum (10-8
molar) and maximum (10-4 molar) drug concentrations used
in the screening test. For comparison purposes, the activity of
2-methoxyestradiol (1) is also included in Table 1.
Comparison of the MGM values of the three phosphates
reveals that the 3-phosphate 3 (MGM ) 2.7 µM) is the most
cytotoxic, followed by the 17-phosphate 7 (MGM ) 31.6 µM)
and the 3,17-diphosphate (MGM ) 93.3 µM). The overall
cytotoxicity of the 3-phosphate 3 is comparable to that of the
parent compound 2-methoxyestradiol (1), although it was
slightly less potent overall. Of the 52 cell lines that both
compounds 1 and 3 were tested in successfully, 2-methox-
yestradiol was more potent in 38 cell lines, its 3-phosphate was
more potent in 12 cell lines, and in 2 cell lines the potencies
were equal. Both the diethylphosphate synthetic precursor 6 and
the bis(diethylphosphate) 8 were more potent than the corre-
sponding phosphates 7 and 9.
The pharmacokinetics of 2-methoxyestradiol (1), its 3-phos-
phate 3, and its 17-phosphate 7 were investigated in rats. The
3-phosphate 3 and 17-phosphate 7 were not effective as prodrugs
in improving the oral bioavailability of 2-methoxyestradiol in
rats (data not shown). The pharmacokinetics were therefore
studied after intravenous (iv) administration, and the results are
listed in Tables 2–4. 2-Methoxyestradiol 3-phosphate (3), its
metabolite 2-methoxyestradiol (1), and 2-methoxyestrone (me-
tabolite of 2ME2) were apparent at the earliest time point
evaluated following administration of the 3-phosphate 3 (Table

Prodrug Strategy for 2-Methoxyestradiol
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6703
2-Methoxy-ꢀ-estradiol 3-Phosphate Monosodium Salt (3).
Palladium hydroxide, 20 wt % [10% palladium (dry basis) on
carbon, wet, Degussa type, water ∼50%, 0.011 g, 0.021 mmol]
was added to a high-pressure vessel and covered with MeOH (5
mL). A solution of 2-methoxy-ꢀ-estradiol-3-dibenzylphosphate (2,
120 mg, 0.213 mmol) in MeOH (5 mL) was added to the vessel,
and MeOH (5 mL) was used to rinse the residual palladium catalyst
from the sides. The reaction mixture was shaken in a Parr
hydrogenator under a 40 psi atmosphere of hydrogen for 24 h. Thin
layer chromatography (TLC) indicated that no reaction had taken
place. More palladium catalyst (20 mg, 0.038 mmol) was added,
and the reaction mixture was shaken under a 60 psi atmosphere of
hydrogen for 24 h. TLC indicated that the starting material had
been completely converted to a product that could not be moved
above the baseline on silica gel TLC in 5% MeOH/CHCl3. The
reaction mixture was filtered through a Celite pad, which was
washed with MeOH. The organic solution was condensed in vacuo
to provide a colorless oil. Water (20 mL) and diethyl ether (20
mL) were added, and the mixture was separated. The ether layer
was washed with water (5 mL), and the combined water layers
were frozen in a round-bottom flask. After freeze-drying, crude
product was recovered as a fluffy white solid. A cellulose column
was washed repeatedly (H2O × 2, 0.1 M HCl × 2, H2O × 2, 0.1
M NaOH × 2, H2O × 2, MeOH × 2) to remove impurities from
the column. (Note: a yellow impurity was removed during the base
washing.) The crude product was purified via gravity chromato-
graphy (cellulose, MeOH), and the desired product was recovered
from in vacuo condensation of the combined fractions to afford 3
(80 mg, 98%): mp (decomposed above 65 °C); 1H NMR (300 MHz,
DMSO) δ 7.01 (s, 1 H), 6.84 (s, 1 H), 3.70 (s, 3 H), 3.5 (t, J )
8.06 Hz, 1 H), 2.64 (m, 2 H), 2.27–1.01 (m, 16 H), 0.66 (s, 3 H);
31P NMR (121.5 MHz, DMSO, 85% phosphoric acid standard) δ
4.70 (m, 1 P); IR (film) 3369, 2927, 2867, 2340, 1648, 1508, 1447,
1264, 1208, 1117, 962, 883 cm-1; low-resolution ESIMS m/z (rel
intensity) 383 (100, MH+). Anal. C19H27O6Na·1.6H2O: C, H.
3-Benzyloxy-2-methoxy-ꢀ-estradiol-17-phosphoric Acid
Diethyl Ester (5). Compound 426 (500 mg, 1.27 mmol) was
dissolved in anhydrous dichloromethane (10 mL) in a flame-dried
round-bottom flask under an argon atmosphere. Titanium tert-
butoxide28 (0.05 mL, 0.13 mmol) was added, followed by the
addition of triethylamine (0.27 mL, 1.91 mmol) and diethylchlo-
rophosphate (0.18 mL, 1.27 mmol). The reaction mixture was
allowed to stir at room temperature for 24 h before water (10 mL)
was added. The aqueous mixture was extracted with ethyl acetate
(3 × 25 mL), dried over anhydrous magnesium sulfate, and
condensed in vacuo. Purification via flash chromatography (silica
gel, hexane/ethyl acetate 3:1 by volume until the starting material
eluted, followed by flushing the product from the column with
acetone/hexane 1:1) gave the desired product 5 (468 mg, 89%) as
euthanasia was observed with both the 100 and 200 mg/kg doses
of 3 (2/10 and 1/10, respectively) as opposed to 0% mortality
with cyclophosphamide or with 2ME2. The fact that 2ME2-3P
is more active than 2ME2 at the same dose suggests that 2ME2-
3P has greater intrinsic activity than 2ME2 and/or that it is
transported to the site of action more effectively than 2ME2
and is then hydrolyzed to 2ME2. The observed toxicity of
2ME2-3P will be addressed in any future development of this
molecule.
The fact that 2-methoxyestradiol 3-phosphate (3) is active in
vivo, whereas 2-methoxyestradiol 17-phosphate is inactive,
agrees with the more efficient conversion of the 3-phosphate to
the parent drug 2-methoxyestradiol (Tables 2 and 3). It also
reflects the more potent activity of the 3-phosphate 3 versus the
17-phosphate 7 in the in vitro proliferation assays (Table 1).
Conclusion
The significance of the results of this study can be sum-
marized in the following points: (1) Methods were developed
for the efficient syntheses of 2-methylestradiol 3-phosphate,
2-methoxyestradiol 17-phosphate, and 2-methoxyestradiol 3,17-
diphosphate. In principle, the routes to these three compounds
are general and could be extended to the regioselective syntheses
of the three possible phosphates of estradiol and a variety of its
derivatives as well. (2) The three phosphates of 2-methoxyestra-
diol are not effective as prodrugs by the oral route of
administration because they did not lead to increased levels of
2-methoxyestraiol when compared with administration of 2-meth-
oxyestradiol. However, the prodrugs are more water soluble and
can therefore be administered in higher doses than 2-methox-
yestradiol by the intravenous route. This could increase the
efficacy and versatility of 2-methoxyestradiol as an antitumor
agent. (3) 2-Methoxyestradiol 3-phosphate (3) exhibits a
significant, dose-related reduction in tumor weight in the Lewis
lung carcinoma metastasis model following intraperitoneal
administration to mice. The activity underscores the importance
of continued development of 2-methoxyestradiol for treatment
of cancer in humans. (4) 2-Methoxyestradiol 3-phosphate was
metabolized more efficiently to the parent drug than the 17-
phosphate after intravenous administration. This fact should be
taken into consideration in the future design of phosphate
prodrugs of estradiol and 2-methoxyestradiol analogues.
Experimental Section
1
a clear oil: H NMR (300 MHz, CDCl3) δ 7.46–7–29 (m, 5 H),
2-Methoxy-ꢀ-estradiol-3-phosphoric Acid Dibenzyl Ester
(2). 2-Methoxyestradiol (100 mg, 0.33 mmol) and dimethylami-
nopyridine (8 mg, 0.066 mmol) were dissolved in anhydrous
acetonitrile (10 mL) in a flame-dried round-bottom flask under
argon. The reaction mixture was cooled to 0 °C, and carbon
tetrachloride (0.32 mL, 3.3 mmol) and diisopropylethylamine (0.29
mL, 1.65 mmol) were added via syringe. After 30 min, dibenzyl
phosphite (0.22 mL, 1.00 mmol) was added, and the reaction
mixture was allowed to warm to room temperature and stirred for
16 h. The reaction mixture was poured into a 0.5 M monobasic
sodium phosphate solution (20 mL), and the aqueous mixture was
extracted with ethyl acetate (3 × 50 mL). The combined organic
layers were condensed in vacuo and purified via flash chromato-
graphy (silica gel, hexane/ethyl acetate 3:1–1:1 gradient) to provide
6.84 (s, 1 H), 6.62 (s, 1 H), 5.10 (s, 2 H), 4.30 (m, 1 H), 4.12 (m,
4 H), 3.86 (s, 3 H), 2.72 (m, 2 H), 2.26–1.22 (m, 13 H), 1.35 (m,
overlapped, 6 H), 0.84 (s, 3 H); IR (film) 2929, 1607, 1515, 1455,
1262, 1025, 978, 740, 698 cm-1; low-resolution ESIMS m/z (rel
intensity) 529 (MH+, 100). Anal. C30H41O6P: C, H.
2-Methoxy-ꢀ-estradiol-17-phosphoric Acid Diethyl Ester
(6). Palladium hydroxide, 20 wt % [20% palladium (dry basis) on
carbon, wet, Degussa type, water ∼50%, 0.015 g, 0.03 mmol] and
5 (150 mg, 0.28 mmol) were added to a round-bottom flask that
had been evacuated and filled with argon three times. MeOH (5
mL) was added, and the reaction mixture was evacuated and filled
with argon two times and then with hydrogen. After 17 h of stirring
at room temperature, the reaction mixture was filtered through a
Celite pad, which was washed with MeOH. The organic solution
was condensed in vacuo to provide a clear oil. Purification via flash
chromatography (silica gel, 1% MeOH in chloroform) gave the
desired product 6 (118 mg, 96%) as a colorless solid: mp 106–108
1
2 (165 mg, 89%) as a yellow oil: H NMR (300 MHz, CDCl3) δ
7.27 (m, 10 H), 6.80 (s, 1 H), 6.77 (s, 1 H), 5.12 (d, J ) 7.79 Hz,
4 H), 3.70 (s, 3 H), 3.65 (t, J ) 8.36 Hz, 1 H), 2.63 (m, 2 H),
2.22–1.09 (m, 14 H), 0.71 (s, 3 H); 31P NMR (121.5 MHz, CDCl3,
85% phosphoric acid standard) δ –5.26 (m, 1 P); IR (film) 3537,
1
°C; H NMR (300 MHz, CDCl3) δ 6.78 (s, 1 H), 6.64 (s, 1 H),
2931, 1508, 1456, 1273, 1207, 1015, 934, 884, 738, 697 cm-1
;
5.46 (bs, 1 H), 4.30 (m, 1 H), 4.11 (m, 4 H), 3.86 (s, 3 H), 2.76
(m, 2 H), 2.28–1.16 (m, 13 H), 1.35 (m, overlapped, 6 H), 0.84 (s,
3 H); IR (film) 3271, 2928, 1509, 1262, 1028, 978 cm-1; low-
low-resolution ESIMS m/z (rel intensity) 563 (100, MH+). Anal.
C33H39O6P: C, H.

6704 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26
Edsall et al.
resolution ESIMS m/z (rel intensity) 461 (MNa+, 100). Anal.
C23H35O6P·0.05CHCl3: C, H.
eight-week-old mice were injected with 0.2 mL of the cell
suspension (1 × 105 tumor cells/mouse) via the lateral tail vein
with a 27-gauge needle. Beginning 3 days after tumor cell injection,
cohorts of mice (10 mice/treatment group) were treated intraperi-
toneally daily × 12 days with (a) 0.1 mL of vehicle control, (b)
100 or 200 mg/kg 2-methoxy-ꢀ-estradiol 3-phosphate monosodium
salt, (c) 100 or 200 mg/kg 2-methoxyestradiol 17-O-phosphate, or
(d) 100 or 200 mg/kg 2-methoxyestradiol 3,17-O,O-diphosphate.
Following 12 days of treatment (15 days after tumor cell injection),
mean lung weight was determined from each treatment group and
corrected for non-tumor-bearing lung weight. Antitumor activity
was then determined by calculating a treatment/control (T/C) value
using the formula T/C ) (A – C)/(B – C), where A ) mean lung
weight of treated tumor-bearing mice, B ) mean lung weight of
vehicle control treated tumor-bearing mice, and C ) mean lung
weight of non-tumor-bearing mice.
Pharmacokinetics. Sprague–Dawley rats used in this study were
obtained from Charles River Laboratories (Portage, MI) and were
surgically implanted with jugular cannula to facilitate blood
sampling. The animals were laboratory bred and were experimen-
tally naive at the outset of the study.
The test articles, received as solids, were separately weighed into
appropriate containers and formulated in 45% (w/v) hydroxypropyl-
ꢀ-cyclodextrin (HPꢀCD) in water at concentrations of 0.5 mg/mL
for intravenous injection, 1.25 mg/mL for oral administration, and
2.5 mg/mL for intraperitoneal administration. Formulations were
prepared as close as possible to the time of dose administration.
Animals were observed once each morning and afternoon
throughout the study for viability. Daily observations were recorded.
Body weights were measured prior to dosing. The animals each
received a single dose of the test compound. The dose volume for
each compound was 2 mL/kg and was based on the most recent
body weight obtained prior to dosing. Intravenous administration
was via the tail vein. Animals were fasted overnight prior to dose
administration. Predose samples were collected within 1 h of dosing.
Following dosing, blood samples (ca. 200 µL) were collected into
separate tubes containing anticoagulant (K-EDTA) via the jugular
cannula at 2, 4, 6, 8, 12, and 24 h. For samples at 1 h and later,
replacement saline was added via the jugular cannula. Blood
samples were transferred to the study director or designate for
preparation of plasma samples. Plasma samples were prepared as
soon as possible after collection by centrifugation for 10 min using
a tabletop centrifuge, immediately harvested, and stored at freezer
temperatures (<-20 °C) pending analysis by LC-MS/MS. After
the blood sampling, all animals were anesthetized with either carbon
dioxide or carbon dioxide and oxygen, killed by opening the
thoracic cavity, or killed by cervical dislocation and discarded.
For the preparation of standard curves, each analyte was spiked
separately into 100 µL of rat plasma at various concentrations
(5–2000 ng/mL for 2ME1, 5–2500 ng/mL for 2ME2, 1–1000 ng/
mL for 2ME2-3P and 2ME2-17P), and vortex-mixed well. For
standard curve samples and study samples, an aliquot of 10 µL of
formic acid was added to each sample tube and mixed well. The
samples were then extracted by protein precipitation using aceto-
nitrile. The supernatant was then transferred and dried over a
nitrogen flow under vacuum. The residue was reconstituted in 50:
50 MeOH/H2O and transferred to LC vial with low volume insert
for LC-MS/MS analysis.
2-Methoxyestradiol 17-O-Phosphate (7). Trimethylsilyl bro-
mide (1.20 mL, 9.13 mmol) was added via syringe to a solution of
compound 6 (400 mg, 0.91 mmol) in anhydrous chloroform (5 mL).
The reaction mixture was allowed to stir at room temperature for
30 h before it was condensed in vacuo. MeOH (5 mL) was added
to the reaction flask, and the reaction mixture was heated at reflux
for 3 h. After the reaction mixture had cooled, the solvent was
evaporated in vacuo, and water (5 mL) was added to the solid
residue. The turbid aqueous mixture was extracted with ethyl acetate
(2 × 15 mL). The organic extracts were combined and evaporated
in vacuo to give a pink solid. A cellulose column was washed
repeatedly (H2O × 2, 0.1 M HCl × 2, H2O × 2, 0.1 M NaOH ×
2, H2O × 2, MeOH × 2) to remove impurities from the column.
(Note: a yellow impurity was removed during the base washing.)
The crude product was purified via gravity chromatography
(cellulose, MeOH), and the desired product was recovered from in
vacuo condensation of the combined fractions to afford 7 (320 mg,
1
92%): mp 82–85 °C; H NMR [300 MHz, (CD3)2CO] δ 8.52 (bs,
3 H), 6.97 (s, 1 H), 6.65 (s, 1 H), 4.46 (m, 1 H), 3.86 (s, 3 H), 2.85
(m, 2 H), 2.91–1.32 (m, 13 H), 1.01 (s, 3 H); IR (KBr pellet) 3414,
2728, 1595, 1509, 1448, 1264, 1208, 1117, 1016, 873, 761, 511
cm-1; low-resolution negative ESIMS m/z (rel intensity) 381 [(M
- H+)-, 100]. Anal. C19H27O6P: C, H.
2-Methoxy-ꢀ-estradiol-3,17-diphosphoric Acid Diethyl Ester
(8). Compound 1 (5.0 g, 16.5 mmol) was dissolved in anhydrous
dichloromethane (50 mL) in a flame-dried round-bottom flask under
an argon atmosphere. Titanium tert-butoxide (1.28 mL, 3.31 mmol)
was added, followed by triethylamine (8.05 mL, 57.75 mmol) and
diethylchlorophosphate (5.96 mL, 41.25 mmol). The reaction
mixture was allowed to stir at room temperature for 11 h before
water (50 mL) was added. After the chlorinated organic layer was
removed, the aqueous mixture was extracted with ethyl acetate (4
× 100 mL). The combined organic extracts were dried over
anhydrous magnesium sulfate and condensed in vacuo. Purification
via flash chromatography (silica gel, 500 g, ethyl acetate) yielded
the desired product 8 (6.8 g, 72%) as a white solid: mp 65–68 °C;
1H NMR (300 MHz, CDCl3) δ 6.96 (s, 1 H), 6.84 (s, 1 H),
4.31–3.82 (m, 9 H), 3.82 (d, J ) 1.27 Hz, 3 H), 2.77–2.75 (m, 2
H), 2.28–1.18 (m, 25 H), 0.83 (s, 3 H); IR (film) 2981, 2931, 1509,
1273, 1033, 980, 920, 799 cm-1; low-resolution ESI m/z (rel
intensity) 575 (MH+, 100). Anal. C27H44O9P2: C, H.
2-Methoxyestradiol 3,17-O,O-Diphosphate (9). Trimethylsilyl
bromide (13.8 mL, 104.4 mmol) was added via syringe to a solution
of compound 8 (4.0 g, 6.96 mmol) in anhydrous chloroform (50
mL). The reaction mixture was allowed to stir at room temperature
for 9 h, and then it was condensed in vacuo. MeOH (50 mL) was
added to the reaction flask, and the solvent was evaporated in vacuo
using heat. Additional MeOH (50 mL) was added, and the solvent
was evaporated again in vacuo using a heated water bath. The pink-
brown mixture was taken up in ethyl acetate (20 mL) and extracted
into deionized water (4 × 100 mL). Lyophilization of the water
gave the pure product (2.8 g, 89%) as a pink solid: mp 165–169
°C; 1H NMR [300 MHz, (CD3)2CO] δ 6.96 (s, 1 H), 6.95 (s, 1 H),
6.17 (bs, 7 H), 4.12 (dt, J ) 8.8 and 8.07 Hz, 1 H), 3.83 (s, 3 H),
2.74 (m, 2 H), 1.87–0.91 (m, 13 H), 0.61 (s, 3 H); 31P NMR [121.5
MHz, CD3OD (85% phosphoric acid internal standard)] δ 0.87 (s,
1 H), 3.53 (m, 1 H); IR (KBr pellet) 3550, 2930, 1506, 1457, 1206,
1018, 873, 492 cm-1; low-resolution ESIMS m/z (rel intensity) 463
(MH+, 100). Anal. C19H28O9P2 ·1.52H2O: C, H.
LC-MS/MS analysis was conducted using a PE Sciex API 3000
LC-MS/MS system. Electrospray ionization in negative ion MRM
mode was conducted for quantitation of analytes. A Luna Phenyl-
Hexyl HPLC column (150 × 2 mm, 5 µm particle size) was used.
Mobile phase A was 5 mM NH4OAc in H2O, and mobile phase B
was MeOH, and a linear step gradient (hold at 50% B for 0.3 min,
ramp to 95% B in 3.2 min, hold at 95% B for 5.5 min, return to
50% B in 0.1 min) was used. The following precursor product ion
pairs (MRM pairs) were used for quantitation: 2ME1 (299.4, 284.1),
2ME2 (301.3, 286.3), 2ME2 3-phosphate (381.2, 79.0), and 2ME2
17-phosphate (381.2, 79.0).
Lewis Lung Carcinoma (LLC) Experimental Metastasis
Model. Male, pathogen-free mice of the inbred mouse strain
C57BL/6J were obtained from The Jackson Laboratory (Bar Harbor,
ME). The mice were housed in a barrier environment under specific
pathogen-free conditions and provided sterilized mouse chow
(LM485, Harlan-Sprague–Dawley, Indianapolis, IN) and water ad
libitum. LLC cells in exponential growth phase were harvested by
a brief trypsinization (0.25% Difco trypsin and 0.02% EDTA for 1
min at 37 °C), washed, and resuspended to 5 × 105 cells/mL in
Ca2+, Mg2+ free PBS (BioWhittaker, Walkersville, MD). Six- to

Prodrug Strategy for 2-Methoxyestradiol
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 26 6705
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Supporting Information Available: Elemental analyses of
compounds 2, 3, and 5-9. This materials ia available free of charge
via the Internet at http://pubs.acs.org.
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