U.S. patent application number 10/610748 was filed with the patent office on 2004-05-20 for methods of obtaining 2-methoxyestradiol of high purity.
Invention is credited to Agoston, Gregory E., Shah, Jamshed H., Treston, Anthony M..
Application Number | 20040097479 10/610748 |
Document ID | / |
Family ID | 32302125 |
Filed Date | 2004-05-20 |
United States Patent
Application |
20040097479 |
Kind Code |
A1 |
Agoston, Gregory E. ; et
al. |
May 20, 2004 |
Methods of obtaining 2-methoxyestradiol of high purity
Abstract
2-methoxyestradiol having greater than 98% purity is obtained by
synthetic or purification methods. This highly pure 2-methoxy
estradiol, lacking estrogenic components, is particularly suitable
for clinical use in humans. The purification methods of the
invention involve the use of liquid-solid chromatography (LSC) to
separate 2-ME2 from other compounds. The chromatographic media is
preferably silica. The solvent system comprises a non-polar
solvent, such as chloroform, and a polar solvent, such as
methanol.
Inventors: |
Agoston, Gregory E.;
(Germantown, MD) ; Shah, Jamshed H.; (Brookeville,
MD) ; Treston, Anthony M.; (Rockville, MD) |
Correspondence
Address: |
JOHN S. PRATT, ESQ
KILPATRICK STOCKTON, LLP
1100 PEACHTREE STREET
SUITE 2800
ATLANTA
GA
30309
US
|
Family ID: |
32302125 |
Appl. No.: |
10/610748 |
Filed: |
July 1, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10610748 |
Jul 1, 2003 |
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09644387 |
Aug 23, 2000 |
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60150293 |
Aug 23, 1999 |
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Current U.S.
Class: |
514/182 |
Current CPC
Class: |
C07J 1/007 20130101 |
Class at
Publication: |
514/182 |
International
Class: |
A61K 031/56 |
Claims
What is claimed is:
1. A composition comprising 2-methoxyestradiol having a purity
greater than 99.5%.
2. The composition of claim 1, containing less than 0.03% estradiol
and less than 0.02% estrone.
3. The composition of claim 2, containing less than 0.01% estradiol
and less than 0.01% estrone.
4. The composition of claim 2, further containing less than 0.02%
2-hydroxyestradiol.
5. The composition of claim 2, further containing less than 0.02%
4-hydroxyestradiol.
6. The composition of claim 2, further containing less than 0.02%
4-methoxyestradiol.
7. The composition of claim 1, containing 0.01% or less estradiol,
0.02% or less 2-hydroxyestradiol, 0.01% or less 4-hydroxyestradiol,
0.01% or less 4-methoxyestradiol, and 0.01% or less estrone.
8. A composition comprising 2-methoxyestradiol having a purity
greater than 98.0% and containing less than 0.03% estradiol and
less than 0.02% estrone.
9. The composition of claim 8, containing less than 0.01% estradiol
and less than 0.01% estrone.
10. The composition of claim 8, containing 0.01% or less estradiol,
0.02% or less 2-hydroxyestradiol, 0.01% or less 4-hydroxyestradiol,
0.01% or less 4-methoxyestradiol, and 0.01% or less estrone.
11. The composition of claim 8, wherein the 2-methoxyestradiol has
a purity greater than 99.0%.
12. The composition of claim 11, containing less than 0.01%
estradiol and less than 0.01% estrone.
13. The composition of claim 11, containing 0.01% or less
estradiol, 0.02% or less 2-hydroxyestradiol, 0.01% or less
4-hydroxyestradiol, 0.01% or less 4-methoxyestradiol, and 0.01% or
less estrone.
14. A method for purifying 2-methoxyestradiol to a produce a
2-methoxyestradiol having a purity greater than 98% and containing
les's than 0.03% estradiol and less than 0.02% estrone comprising:
adding a solution comprising 2-methoxyestradiol to a chromatography
medium; and eluting the 2-methoxyestradiol off of the medium with a
solvent system comprising a polar solvent and a non-polar
solvent.
15. The method of claim 14, wherein the medium is silica and
wherein the 2-methoxyestradiol is eluted using a step gradient of
99:1 CHCl.sub.3:MeOH to 98:2 CHCl:MeOH.
16. A method for producing 2-methoxyestradiol having a purity
greater than 98% and containing less than 0.03% estradiol and less
than 0.02% estrone comprising: protecting the 3- and 17-hydroxyl
groups of estradiol; reacting the protected estradiol with bromine
and acetic acid to produce a 2-brominated derivative of estradiol;
reacting the 2-brominated derivative of estradiol with sodium
methoxide in the presence of a copper catalyst; removing the
protecting groups on the 3- and 17-hydroxyl groups to produce
2-methoxyestradiol; and purifying the 2-methoxyestradiol using
liquid chromatography on an adsorption/partition medium with a
solvent system comprising a polar and a nonpolar solvent.
17. A method for producing 2-methoxyestradiol having a purity
greater than 98% and containing less than 0.03% estradiol and less
than 0.02% estrone comprising: ring-brominating estradiol by
reacting estradiol with bromine in the presence of acetic add to
produce a ring-brominated intermediate; reacting the
ring-brominated intermediate with sodium methoxide in the present
of a copper catalyst to produce 2-methoxyestradiol; and purifying
the 2-methoxyestradiol using liquid chromatography on an
adsorption/partition medium with a solvent system comprising a
polar and a nonpolar solvent.
18. A method for producing 2-methoxyestradiol having a purity
greater than 98% and containing less than 0.03% estradiol and less
than 0.02% estrone comprising: protecting the 3- and 17-hydroxyl
groups of estradiol; reacting the protected estradiol with nitric
acid and acetic acid to produce a 2-nitro derivative of estradiol;
reducing the 2-nitro derivative of estradiol to produce the
corresponding 2-amino derivative of estradiol; reacting the 2-amino
derivative of estradiol under Sandmeyer conditions to produce a
3-,17-hydroxyl protected 2-methoxyestradiol; and removing the
protecting groups on the 3- and 17-hydroxyl groups to produce
2-methoxyestradiol.
19. A method for producing 2-methoxyestradiol having a purity
greater than 98% and containing less than 0.03% estradiol and less
than 0.02% estrone comprising: protecting the 3-hydroxyl group of
estrone; reacting the protected estrone with nitric acid and acetic
acid to produce a 2-nitro derivative of estrone; reducing the
2-nitro derivative of estrone to produce the corresponding 2-amino
derivative of estrone; reacting the 2-amino derivative of estrone
under Sandmeyer conditions to produce a 3-hydroxyl protected
2-methoxyestrone; removing the protecting group on the 3-hydroxyl
group to produce 2-methoxyestrone; and reducing the 17-keto group
of 2-methoxyestrone to produce 2-methoxyestradiol.
20. A method for producing 2-methoxyestradiol having a purity
greater than 98% and containing less than 0.03% estradiol and less
than 0.02% estrone comprising: brominating estradiol in the
presence of acetic acid to produce a mixture of ring-brominated
estradiols; isolating 2-bromoestradiol from the mixture of
estradiols; and reacting the 2-bromoestradiol with sodium methoxide
in the presence of a copper catalyst to produce
2-methoxyestradiol.
21. 2-methoxyestradiol having a purity greater than 98% and
containing less than 0.03% estradiol and less than 0.02% estrone
produced by the process comprising: protecting the 3- and
17-hydroxyl groups of estradiol; reacting the protected estradiol
with bromine and acetic add to produce a 2-brominated derivative of
estradiol; reacting the 2-brominated derivative of estradiol with
sodium methoxide in the presence of a copper catalyst; removing the
protecting groups on the 3- and 17-hydroxyl groups to produce
2-methoxyestradiol; and purifying the 2-methoxyestradiol using
liquid chromatography on an adsorption/partition medium with a
solvent system comprising a polar and a nonpolar solvent.
22. 2-methoxyestradiol having a purity greater than 98% and
containing less than 0.03% estradiol and less than 0.02% estrone
produced by the process comprising: ring-brominating estradiol by
reacting estradiol with bromine in the presence of acetic add to
produce a ring-brominated intermediate; reacting the
ring-brominated intermediate with sodium methoxide in the present
of a copper catalyst to produce 2-methoxyestradiol; and purifying
the 2-methoxyestradiol using liquid chromatography on an
adsorption/partition medium with a solvent system comprising a
polar and a nonpolar solvent.
23. 2-methoxyestradiol having a purity greater than 98% and
containing less than 0.03% estradiol and less than 0.02% estrone
produced by the process comprising: protecting the 3- and
17-hydroxyl groups of estradiol; reacting the protected estradiol
with nitric add and acetic add to produce a 2-nitro derivative of
estradiol; reducing the 2-nitro derivative of estradiol to produce
the corresponding 2-amino derivative of estradiol; reacting the
2-amino derivative of estradiol under Sandmeyer conditions to
produce a 3-,17-hydroxyl protected 2-methoxyestradiol; and removing
the protecting groups on the 3- and 17-hydroxyl groups to produce
2-methoxyestradiol.
24. 2-methoxyestradiol having a purity greater than 98% and
containing less than 0.03% estradiol and less than 0.02% estrone
produced by the process comprising: protecting the 3-hydroxyl group
of estrone; reacting the protected estrone with nitric acid and
acetic acid to produce a 2-nitro derivative of estrone; reducing
the 2-nitro derivative of estrone to produce the corresponding
2-amino derivative of estrone; reacting the 2-amino derivative of
estrone under Sandmeyer conditions to produce a 3-hydroxyl
protected 2-methoxyestrone; removing the protecting group on the
3-hydroxyl group to produce 2-methoxyestrone; and reducing the
17-keto group of 2-methoxyestrone to produce
2-methoxyestradiol.
25. 2-methoxyestradiol having a purity greater than 98% and
containing less than 0.03% estradiol and less than 0.02% estrone
produced by the process comprising: brominating estradiol in the
presence of acetic acid to produce a mixture of ring-brominated
estradiols; isolating 2-bromoestradiol from the mixture of
estradiols; and reacting the 2-bromoestradiol with sodium methoxide
in the presence of a copper catalyst to produce 2-methoxyestradiol.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application Serial No. 60/150,293 filed Aug. 23, 1999.
FIELD OF THE INVENTION
[0002] The invention relates to the estradiol metabolite
2-methoxyestradiol and to methods of obtaining purified
2-methoxyestradiol.
BACKGROUND OF THE INVENTION
[0003] 2-Methoxyestradiol,
1,3,5(10)-estratrien-2,3,17.beta.-triol-2-methy- l-ether (2-ME2) is
an endogenous metabolite of estradiol, the major ovarian estrogen.
The chemical formula of 2-ME2 is C.sub.19H.sub.26O.sub.3, and the
compound has a molecular weight of 302.4. 2-ME2 has low of
estrogenic activity but has been found to have other biological
effects.
[0004] U.S. Pat. Nos. 5,504,074, 5,661,143, and 5,892,069 to
D'Amato et al. disdose methods of treating mammalian diseases
characterized by abnormal cell mitosis using 2-ME2. Undesirable
cell mitosis is characteristic of many diseases, including, but not
limited to, cancer, atherosclerosis, proliferation of solid tumors,
vascular malfunctions, endometriosis, retinopathies, arthropathies,
and abnormal wound healing. In addition, cell mitosis is important
in a wide variety of biological functions, including but not
limited to the normal development of the embryo, formation of the
corpus luteum, cyclic proliferation of uterine endometrium, wound
healing, and inflammatory and immune responses.
[0005] U.S. Pat. No. 5,521,168 to Clark discloses using 2-ME2 for
lowering intraocular pressure. 2-ME2 also inhibits estrogen-induced
pituitary tumor angiogenesis and suppresses tumor growth in Fisher
344 rats as reported by Banerjee, S. K. et al., Proc. Amer. Assoc.
Cancer Res. 39, March 1998.
[0006] Presently, commercially available preparations of 2-ME2 are
either less than 98% pure or contain undesirable steroid
contaminants that are of concern for pharmaceutical uses. Important
contaminants of these preparations are estradiol,
4-hydroxyestradiol, 4-methoxyestradiol, 2-hydroxyestradiol,
estrone, and 2-methoxyestrone. The amounts of these contaminants
that are found in presently available 2-ME2 preparations are
unacceptable for pharmaceutical applications.
[0007] Any therapeutic use of 2-ME2 in humans requires 2-ME2 having
a high level of purity. In general, therapeutic agents are required
to be substantially pure to avoid negative side effects of
contaminants. In particular, since 2-ME2 has effects that are
counteracted by estradiol and other estrogenic metabolites, it is
crucial to have a 2-ME2 preparation substantially free of such
contaminants. Effects that may be seen from contaminating
estradiol, estrone, and 2-hydroxyestradiol include estrogenic
effects such as feminization, endometrial proliferation, increased
risk of uterine and breast cancer, developmental effects on sexual
organs, inhibition of leukopoesis, and effects on hematopoetic
cells. 4-hydroxyestradiol, 4-methoxyestradiol, and estradiol are
known mutagens and carcinogens.
[0008] Accordingly, what is needed is a composition of 2-ME2 which
is greater than 98% pure and which contains substantially no
estradiol or other steroids having estrogenic or carcinogenic
effects.
[0009] What is also needed is a composition containing 2-ME2 that
is greater than 99.5% pure.
[0010] What is also needed are methods for making 2-ME2 of greater
than 98% purity and containing substantially no estradiol or other
steroids having estrogenic or carcinogenic effects.
[0011] Also needed are methods of substantially separating 2-ME2
from estradiol, related molecules, and other contaminants,
resulting in 2-ME2 having a purity of greater than 99.5%.
SUMMARY OF THE INVENTION
[0012] The present invention provides 2-ME2 having greater than 98%
purity, more preferably greater than 99% purity, most preferably
greater than 99.5% purity. The 2-ME2 preparations preferably
contain less than 0.03% estradiol, 0.02% or less
2-hydroxyestradiol, 0.02% or less 4-hydroxyestradiol, 0.02% or less
4-methoxyestradiol, and less than 0.02% estrone. More preferably,
the 2-ME2 preparations contain 0.01% or less estradiol, 0.02% or
less 2-hydroxyestradiol, 0.01% or less 4-hydroxyestradiol, 0.01% or
less 4-methoxyestradiol, and 0.01% or less estrone.
[0013] The present invention also provides methods of obtaining
2-ME2 of greater than 98% purity, more preferably greater than 99%
purity, most preferably greater than 99.5% purity. In some
embodiments, the methods involve synthetic techniques. In other
embodiments, the methods involve purification techniques to
separate the 2-ME2 from other compounds. In yet other embodiments,
the methods involve both synthetic techniques and purification
techniques described herein.
[0014] The purification methods involve the use of liquid-solid
chromatography (LSC) to separate 2-ME2 from other compounds. The
chromatographic media is preferably silica. The solvent system
comprises a non-polar solvent, such as chloroform, and a polar
solvent, such as methanol.
[0015] Accordingly, an object of the present invention is to
provide 2-ME2 having a purity greater than 98%.
[0016] Another object of the present invention is to provide 2-ME2
substantially free of estradiol, related compounds, and other
unwanted impurities.
[0017] Still another object of the invention is to provide methods
of obtaining substantially pure 2-ME2 by synthetic techniques.
[0018] Another object of the invention is to provide methods of
obtaining substantially pure 2-ME2 by purification techniques.
[0019] Other features and advantages of the invention will be
apparent from the following description of preferred embodiments
thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 is a chromatogram from the reversed phase BPLC
analysis of 2-methoxyestradiol available from Sigma Chemical
Company (45H4033). This graph shows that the Sigma product contains
about 0.034% estradiol.
[0021] FIG. 2 is an expanded view of the chromatogram in FIG. 1
indicating the estradiol impurity.
[0022] FIG. 3 is a chromatogram from the reversed phase HPLC
analysis of 2-methoxyestradiol available from Research Plus
(10699). This graph shows that the Research Plus product contains
about 0.024% estrone and about 0.93% other undesirable
estrogens.
[0023] FIG. 4 is an expanded view of the chromatogram in FIG. 3
indicating the estrone impurity.
[0024] FIG. 5 is a chromatogram from the reversed phase HPLC
analysis of the unpurified 2-methoxyestradiol employed as the
starting material in Example 2 of the present invention.
[0025] FIG. 6 is a chromatogram of the 2-ME2 of the present
invention produced in Example 2. The HPLC was run with a
non-overloaded amount of sample, 75.6 .mu.g (14 .mu.l at 5.4
.mu.l/ml).
[0026] FIG. 7 is an expanded view of the chromatogram in FIG.
6.
[0027] FIG. 8 is a chromatogram of the 2-ME2 of the present
invention produced in Example 2. The HPLC was run with an
overloaded amount of sample, 270 .mu.g (50 .mu.l at 5.4
.mu.l/ml).
[0028] FIG. 9 is an expanded view of the chromatogram in FIG.
8.
[0029] FIG. 10 is a chromatogram of the pooled fractions from
Example 2, assayed using a gradient (20 to 70% acetonitrile over 25
minutes, 1% acetic add, and remainder water). 43.2 .mu.g (8 .mu.l
of the 5.4 .mu.l/ml sample) was injected.
[0030] FIG. 11 is an expanded view of the chromatogram in FIG.
10.
[0031] FIG. 12 depicts a synthetic reaction scheme for the
production of the 2-methoxyestradiol of the present invention,
using estradiol as a starting material and employing bromine, a
crown ether, and a blocking group on the 3- and 17-position
hydroxyloxygen atoms of the estradiol.
[0032] FIG. 13 depicts a synthetic reaction scheme for the
production of the 2-methoxyestradiol of the present invention,
using estradiol as a starting material and employing bromination at
the 2-position of the A ring of unblocked estradiol and a crown
ether.
[0033] FIG. 14 depicts a synthetic reaction scheme for the
production of the 2-methoxyestradiol of the present invention,
using estradiol as a starting material and employing a blocking
group on the 3- and 17-position hydroxyloxygen atoms of estradiol,
nitration, and a Sandmeyer reaction.
[0034] FIG. 15 depicts a synthetic reaction scheme for the
production of the 2-methoxyestradiol of the present invention,
using estrone as a starting material and employing a blocking group
on the 3-position hydroxyloxygen atom, nitration, and a Sandmeyer
reaction.
[0035] FIG. 16 depicts a synthetic reaction scheme for the
production of the 2-methoxyestradiol of the present invention,
using estradiol as a starting material and employing bromination at
the 2-position of the A ring of unblocked estradiol and reaction
with methanol.
DETAILED DESCRIPTION OF THE INVENTION
[0036] The present invention is directed to 2-methoxyestradiol
having a purity of greater than 98.0%, more preferably greater than
99.0%, and most preferably of 99.5% or higher. 2-ME2 can be
obtained through synthesis methods or purification methods
described herein that yield highly pure 2-ME2. The synthesis
methods described herein may also be supplemented with the
purification methods described herein to yield 2-ME2 having even
greater purity.
[0037] Although the terms "2-methoxyestradiol" and 2-ME2 are
specifically used herein, it should be understood that the methods
disclosed herein can be used for synthesis or purification of other
compounds, such as, but not limited to, estradiol and other
structurally related steroids.
[0038] Methods of Synthesis
[0039] The present invention provides methods of synthesizing 2-ME2
to a purity of greater than 98.0%, more preferably greater than
99.0%, and most preferably of 99.5% or higher. The synthetic
methods described herein can also be used, with minor
modifications, to synthesize other 2- and 4-derivatives or
analogues of estradiol, such as, for example, 4-methoxyestradiol
and 4-hydroxyestradiol.
[0040] There are several synthetic approaches that can be taken to
prepare 2-ME2 having a purity greater than 98.0%. Alternatively,
2-ME2 can be purified according to the following purification
methods to have a purity greater than 98.0%. These different
synthetic approaches utilize different starting materials and
intermediates; consequently, different yields, side reactions and
impurities will be obtained.
[0041] Two similar approaches employ estradiol as a starting
material and utilize a brominated intermediate, as taught by Rao,
P. N. et al., Synthesis, 1977, 168 and Chen, S-H et al., Steroids,
1986, 47, 63. The first approach is illustrated in FIG. 12. The
free hydroxyl groups of estradiol are protected with a blocking
group. A wide range of blocking groups can be used in the present
invention. These blocking groups include, but are not limited to,
alkyl, aryl, aralkyl groups, and alkyl, aryl, and aralkyl group
containing one or more heteroatoms. For example, protection can be
accomplished using an alkyl halide, such as benzyl bromide, to form
an alkyl ether. Appropriate conditions for hydroxyl protection
include reaction of the estradiol and alkyl halide in the presence
of NaH and TBAI, optionally in the presence of a solvent, such as
dimethyl formamide (DMF). The protected estradiol is then reacted
with bromine, for example, in the presence of acetic add.
Protection of the free hydroxyls during bromination gives a higher
yield of the 2-brominated intermediate (about 70% vs. about 20%
without the protecting groups) (see Cushman, M. et al., J. Med.
Chem. 1997, 40, 2323).
[0042] The bromine is then replaced with a methoxide group using a
copper catalyst. For example, the brominated intermediate can be
reacted with NaOMe in the presence of a copper catalyst, such as
CuI. The reaction is preferably conducted in a solvent, such as
DMF, optionally in the presence of a promoter. Acceptable
promoters, include, but are not limited to, crown ethers, such as
benzo-15-crown-5.
[0043] Removal of the protecting groups, for example, by catalytic
hydrogenation of the alkyl moiety, yields 2-ME2. Unfortunately,
this synthetic route yields about 1-2% impurity of estradiol from
the methoxylation step (a hydride quenches the reactive copper
complex rather than a methoxide). The estradiol can be removed to
undetectable levels by chromatography, such as described below, or
significantly reduced by successive crystallization in
chloroform.
[0044] Another synthetic method utilizing a brominated intermediate
and employing estradiol as the starting material is illustrated by
FIG. 13. In this synthetic reaction, the estradiol is ring
brominated without first blocking the hydroxyl groups. The bromine
is then replaced with a methoxide using a copper catalyst in a
manner similar to that described above.
[0045] In another approach, estradiol or estrone can be used as a
starting material in a reaction scheme that utilizes nitro/amine
intermediates (see Cushman, M. et al., J. Med. Chem. 1995, 38,
2041). These synthetic approaches are illustrated in FIG. 14
(estradiol starting material) and FIG. 15 (estrone starting
material). In these approaches, the free hydroxyl groups are
protected. This protection can be accomplished, for example, using
an alkyl halide, such as benzyl bromide, to form an alkyl ether.
Appropriate conditions for hydroxyl protection include reaction of
the starting material and alkyl halide in the presence of NaH and
TBAI, optionally in the presence of a solvent, such as dimethyl
formamide (DMF).
[0046] The protected starting material is then nitrated, for
example, with nitric acid and acetic acid or with nitric add and
sulfuric add, to form the corresponding 2-nitro product. The nitro
group is then reduced. Selective reduction can be accomplished by
catalytic hydrogenation, for example, hydrogenation in the presence
of Pd/C to produce the corresponding 2-amine. The catalytic
reduction is optimally carried out for a period of one hour. Using
Sandmeyer conditions (nitrous acid and sodium methoxide), the
2-amino group can be converted to the 2-methoxy substituent.
Catalytic hydrogenation removes the protecting groups to give 2-ME2
when the starting material is estradiol and 2-methoxyestrone when
the starting material is estrone. Reduction of the 17-keto group of
2-methoxyestrone with sodium borohydride yields 2-ME2.
[0047] Yet another method employs estradiol as the starting
material and utilizes brominated intermediates. In this synthetic
reaction, the estradiol is ring brominated without first blocking
the hydroxyl groups. Bromination is accomplished, for example, with
bromine and acetic acid in a solvent, such as THF. This reaction
results in bromination at different sites on the ring, including
multi-brominated species. The 2-bromo-estradiol can then be
isolated from the other brominated intermediates, for example, by
chromotography or crystallization, followed by replacement of the
bromine with a methoxide. The bromine can be replaced with a
methoxide group, for example, using sodium methoxide and methanol
in the presence of a copper catalyst, such as CuI, in a manner
similar to that described above. Alternatively, the intermediates
can be reacted to form the corresponding methoxides, followed by
isolation of the 2-methoxyestradiol by the methods described
above.
[0048] Methods of Purification
[0049] The present invention provides methods of purifying 2-ME2 to
a purity of greater than 98.0%, more preferably greater than 99.0%
and most preferably of 99.5% or higher. The 2-ME2 preparations
preferably contain less than 0.03% estradiol, 0.02% or less
2-hydroxyestradiol, 0.02% or less 4-hydroxyestradiol, 0.02% or less
4-methoxyestradiol, and less than 0.02% estrone. Most preferably,
the 2-ME2 preparations contain 0.01% or less estradiol, 0.02% or
less 2-hydroxyestradiol, 0.01% or less 4-hydroxyestradiol, 0.01% or
less 4-methoxyestradiol, and 0.01% or less estrone.
[0050] The purification methods of the present invention involve
liquid chromatography on an adsorption/partition medium such as
silica, using a solvent system comprising a polar and a non-polar
solvent. The purification methods described herein can also be
used, with minor modifications, to purify compounds similar to
2-ME2, such as, for example, 4-methoxyestradiol,
4-hydroxyestradiol, 2-hydroxyestradiol, estradiol, estrone,
2-methoxyestrone, and 4-methoxyestrone.
[0051] The Sample
[0052] The sample to be purified can be synthesized, or obtained
from a biological source. The sample may be a commercially
available 2-ME2 preparation, such as those sold by Sigma-Aldrich
Chemicals of St. Louis, Mo., Research Plus, Inc. of Bayonne, N.J.,
or Calbiochem of San Diego, Calif. The sample is preferably at
least about 50% pure, more preferably about 75% pure, even more
preferably about 90% pure, and most preferably about 98% pure. The
sample can be subjected to other purification steps prior to the
methods described herein, such as selective crystallization.
[0053] The sample is preferably dissolved into or solvent-exchanged
into a loading solvent, as further described below. The sample is
preferably at a concentration in the range of about 0.01 to 2 g/ml,
preferably about 0.01 to 1 g/ml, more preferably about 0.05 to 0.2
g/ml.
[0054] Chromatographic Media
[0055] Silica is preferably used as the chromatographic medium.
Silica gel of about 70-400 mesh is preferred, most preferably about
70-230 mesh, such as supplied by Merck and other vendors. The
medium can be used loose, in batch chromatography, or packed into a
column. Pre-packed columns, such as those sold by Biotage of
Charlottesville, Va., can also be used. The medium should be
equilibrated in an appropriate solvent before application of the
sample to the medium, as further discussed below.
[0056] Column Dimensions
[0057] The chromatographic methods described herein can be achieved
using batch or column chromatography. In batch chromatography, the
sample and the chromatographic medium are combined in a container
for a period of time sufficient to allow the 2-ME2 to be retained
by the medium. The medium is then preferably washed with wash
solvent. Elution solvents are then applied to the medium. After the
loading, wash, and elution steps, the solvent is removed from the
medium, such as by filtration.
[0058] For column chromatography, a column having appropriate
dimensions is packed with the chromatography medium. The column,
after equilibration with appropriate solvent, is loaded with sample
by applying the sample to the top, or entrance, of the column. The
ratio of the sample volume to column diameter should preferably be
between about 0.2 to 3 ml/cm, and more preferably between about 0.5
and 1.5 ml/cm for best results.
[0059] Solvents
[0060] A solvent system including a polar solvent, such as methanol
(MeOH), and a non-polar solvent, such as chloroform (CHCl.sub.3),
is used. Other polar solvents that can be used include, but are not
limited to, tetrahydrofuran (THF), ethyl acetate, isopropanol,
ethanol, propanol, and combinations thereof. Other non-polar
solvents that can be used include, but are not limited to, hexane,
dichloromethane, cyclohexane, pentane, and combinations thereof.
More specifically, solvent systems that can be used include
THF/hexane, ethyl acetate/hexane, isopropanol/hexane,
ethanol/CHCl.sub.3, propanol/CHCl.sub.3, isopropanol/CHCl.sub.3,
and combinations thereof.
[0061] The sample is soluble in the polar solvent. Some amount of
the polar solvent, generally about 10%, is needed to render the
sample soluble in the loading solvent. The loading solvent thus
will include up to about 10% polar solvent and about 90% non-polar
solvent.
[0062] After the sample is loaded onto the medium, the medium may
be washed with a wash solvent that will wash contaminants off the
medium but will not elute the 2-ME2. The wash solvent comprises
mostly non-polar solvent, with enough polar solvent to prevent the
2-ME2 from precipitating but not enough polar solvent to elute the
2-ME2.
[0063] The sample is eluted with elution solvent that contains
enough polar solvent to elute the 2-ME2 from the medium. The
elution solvent may be applied as a step gradient or as a linear
gradient, as described below.
[0064] Column Conditions
[0065] The wash and elution solvents can be applied to the column
in a step gradient or in a linear gradient. In a preferred
embodiment, the solvents are applied using a step gradient of
increasing concentration of polar solvent.
[0066] The column can be operated using the force of gravity or can
be operated with a pump that forces liquids through the column. The
rate at which the column is operated will depend upon the volume
and dimensions of the column and the silica gel particle size. In
general, the column can be operated at a rate from about 0.5 to 5
ml/min.
[0067] The eluant can be monitored visually or, monitored with a
spectrophotometer at a wavelength of about 288 nm, which is the
absorbance maximum of 2-ME2, and collected as the 2-ME2 elutes from
the column.
[0068] The column can optionally be operated under pressure and can
optionally be heated. Preparative high performance liquid
chromatography (HPLC), either normal phase or reversed phase, or
fast performance liquid chromatography (FPLC) techniques can be
used. Commercial preparative chromatography apparatus, such as that
sold by Biotage of Charlottesville, Va., can also be used. Other
known methods of improving column efficiency and/or speed can also
be employed.
[0069] Sample Collection and Treatment
[0070] The eluant can be collected as fractions which are then
assayed for 2-ME2 content and purity. These fractions can then be
combined to achieve the desired purity of 2-ME2. The fractions can
be assayed for purity using reverse phase HPLC with a C-18 column
(Waters) and an isocratic solvent system of 30:69:1
acetonitrile:water:acetic acid. Other systems can be used for
sample analysis, such those that use solvent gradients instead of
the isocratic solvent system; those that use trifluoroacetic acid
or formic acid rather than acetic acid; and those that use methanol
rather than acetonitrile.
[0071] Alternatively, or in combination, the eluant can be
monitored in real time and sample collection begun when. 2-ME2 of
desired purity elutes from the column.
[0072] The solvent is removed from the pooled fractions by use of a
vacuum and/or other solvent removal methods. Lyophilization and
other evaporative methods can be used.
[0073] Preferred Embodiment
[0074] In a preferred embodiment the medium is silica, which is
packed into a column. The sample is dissolved in a mixture of
CHCl.sub.3 and MeOH, with enough MeOH to solubilize the 2-ME2,
generally about 90:10 CHCl.sub.3:MeOH. The elution conditions are a
step gradient from 99:1 CHCl.sub.3:MeOH to 98:2
CHCl.sub.3:MeOH.
[0075] This invention is further illustrated by the following
examples, which are not to be construed in any way as imposing
limitations upon the scope thereof. On the contrary, it is to be
clearly understood that resort may be had to various other
embodiments, modifications, and equivalents thereof which, after
reading the description herein, may suggest themselves to those
skilled in the art without departing from the spirit of the present
invention and/or the scope of the appended claims.
EXAMPLE 1
[0076] Commercially available samples of 2-ME2 were assayed by
analytical HPLC to determine their overall purity and the amounts
of certain contaminants, namely estradiol, 4-hydroxyestradiol,
4-methoxyestradiol, 2-hydroxyestradiol, estrone, and
2-methoxyestrone.
[0077] These analytical HPLC chromatograms were generated using
reverse phase HPLC with a C-18 column (Waters) and a solvent
gradient (20 to 50% acetonitrile over 30 minutes, 50 to 80%
acetonitrile over 5 minutes, 1% acetic acid, remainder water). The
eluant was monitored at a wavelength of 288 nm. In this system
2-ME2 elutes at about 21.5 minutes, estradiol elutes at about 20.0
minutes, estrone elutes at about 23.2 minutes, 4-hydroxyestradiol
elutes at about 15.0 minutes, 4-methoxyestradiol elutes at about
20.4 minutes, 2-hydroxyestradiol elutes at about 15.4 minutes, and
2-methoxyestrone elutes at about 24.4 minutes.
[0078] The chromatogram of a sample from Sigma-Aldrich Chemicals of
St. Louis, Mo. is shown in FIG. 1. The sample has an overall purity
of 99.2% but has contaminating estradiol of about 0.034%, an
unacceptable amount. FIG. 2 is an expanded view of the chromatogram
of FIG. 1.
[0079] FIG. 3 is a chromatogram of a sample obtained from Research
Plus, Inc. of Bayonne, N.J. that shows that the 2-ME2 has a purity
of 98.6%. The automatic peak calculator and the expanded view shown
in FIG. 4 show that the preparation contains 0.024% estrone, an
unacceptable amount of this contaminant. Other samples tested
showed 2-ME2 purity less than 98%, including a second batch
obtained from Research Plus (97.2% 2-ME2) and a sample from
CalBiochem of San Diego, Calif. (91.8% 2-ME2).
[0080] Table 1, below, illustrates the purity and contaminants of
these commercially available samples of 2-ME2 and the purified
2-ME2 of the present invention.
1 TABLE 1 Research Plus, Research Plus, Sigma Lot #1 Lot #2
Calbiochem PharmEco purified 2-ME2 99.18 98.61 97.17 91.80 97.80
99.98 estradiol 0.03 n.d. n.d. 1.78 2.2 less than 0.01% estrone
n.d. 0.02 0.43 0.011 n.d. 4-hydroxy- n.d. n.d. n.d. n.d. n.d.
estradiol 4-methoxy- 0.49 0.121 0.18 1.99 n.d. estradiol 2-hydroxy-
n.d. n.d. n.d. 0.06 n.d. estradiol 2-methoxy- n.d. n.d. n.d. 0.20
n.d. estrone *n.d. means none was detected.
EXAMPLE 2
[0081] A 55 cm diameter (60 cm height) glass column was packed with
600 g silica gel (70-230 mesh from Merck) in 90:10 CHCl.sub.3:MeOH.
The column was washed with one liter of CHCl.sub.3 to remove the
MeOH from the column.
[0082] The sample was 3.5 g 2-ME2 in 60 ml 90:10 CHCl.sub.3:MeOH.
The 2-ME2 was obtained from PharmEco Laboratories, Inc. of
Lexington, Mass., and was 97.8% pure as determined by analytical
HPLC (FIG. 5). The peak eluting at 10.917 is estradiol (2.2%).
[0083] Analytical HPLC of the starting material, the column
fractions, and the pooled product was performed using reverse phase
HPLC with a C-18 column (Waters) and an isocratic gradient of
30:69:1 acetonitrile:water:acetic acid, which provides good
separation of 2-ME2 and estradiol. The eluant was monitored at a
wavelength of 288 nm.
[0084] The sample was applied to the top of the column and allowed
to enter the bed volume. The column was eluted with one liter of
99:1 CHCl.sub.3:MeOH and then 1.5 L of 98:2 CHCl.sub.3:MeOH.
Fractions of 50 ml each were collected and 15 fractions containing
2-ME2 were assayed for 2-ME2 purity using the analytical isocratic
HPLC system described above. Nine to ten fractions that showed no
amount of estradiol were pooled together and solvent was
evaporated. After drying under vacuum for 4 hours, 3.2 g of
yellow/white crystals were collected, for a 91% yield.
[0085] Purity of the pooled fractions was determined by analytical
HPLC to be 99.984%, using the isocratic technique described above.
The HPLC chromatograms are shown in FIGS. 6 through 9. FIG. 6 was
generated with a non-overloaded amount of sample, 75.6 .mu.g (14
.mu.l at 5.4 .mu.l/ml). FIG. 7 is an expanded view of the
chromatogram of FIG. 6. The automatic peak finder calculated the
2-ME2 to be 100.0%, although a small, unknown impurity peak is seen
in the expanded view, eluted prior to the 2-ME2. FIG. 8 was
generated with an overloaded amount of sample, 270 .mu.g (50 .mu.l
at 5.4 l/ml). FIG. 9 is an expanded view of the chromatogram of
FIG. 8. The automatic peak finder calculated the 2-ME2 to be
99.984% pure, with a small, unknown, impurity that eluted prior to
the 2-ME2, and after estradiol, that was calculated to be 0.016%.
The expanded view shown in FIG. 9 shows this impurity peak more
clearly and shows that the 2-ME2 peak is very clean.
[0086] The pool was also assayed using a gradient (20 to 70%
acetonitrile over 25 minutes, 1% acetic acid, and remainder water).
43.2 .mu.g (8 .mu.l of the 5.4 .mu.l/ml sample) was injected. The
chromatogram is shown in FIG. 10. The automatic peak finder
calculated the 2-ME2 to have a purity of 99.825%. However, when an
artifact peak, present in a blank run, at 29.45 minutes is removed
from consideration, the calculated purity is 99.9%. FIG. 11 is an
expanded view of this chromatogram. The unknown impurity at 13.43
minutes was calculated to be 0.012%. If estradiol were present, it
would elute between the unknown purity and the 2-ME2 peak. If
estradiol is present, therefore, it can be present at no more than
1/3 to 1/4 of the amount of the unknown peak. Accordingly, the
estradiol amount was estimated to be no more than 0.004%. The
preparation contained 0.02% or less 2-hydroxy-estradiol, 0.01% or
less 4-hydroxy-estradiol, 0.01% or less 4-methoxy-estradiol, and
0.01% or less estrone, as demonstrated by the lack of any
measurable peaks at the expected retention times.
[0087] The purified sample was also subjected to elemental analysis
and the results are shown in Table 2.
2TABLE 2 Elemental Analysis Element Theoretical Found Carbon 75.46
75.21 Hydrogen 8.67 8.65 Oxygen 15.87 16.13 (obtained by
difference) Chlorine 0.00 0.0
[0088] The above description is intended to be illustrative and not
restrictive. Many embodiments will be apparent to those of skill in
the art upon reading the above description. The scope of the
invention should, therefore, be determined not with reference to
the above description, but should instead be determined with
reference to the appended claims, along with the full scope of
equivalents to which such claims are entitled. The disclosures of
all articles and references, including patents, patent applications
and publications, are incorporated herein by reference.
* * * * *