JP5317381B2 - Method for producing high purity 2-methoxyestradiol - Google Patents

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.

Description

[Related applications]
This application claims priority to US Provisional Patent Application No. 60 / 150,293, filed Aug. 23, 1999.

[Field of the Invention]
The present invention relates to a method for producing estradiol metabolite 2-methoxyestradiol and purified 2-methoxyestradiol.

[Background of the invention]
2-Methoxyestradiol, 1,3,5 (10) -estraditrien-2,3,17β-triol-2-methyl-ether (2-ME2) is an endogenous metabolite of estradiol, the major ovarian estrogen . The chemical formula of 2-ME2 is C ₁₉ H ₂₆ O ₃ and the compound has a molecular weight of 302.4. 2-ME2 has been found to have low estrogenic activity, but other biological effects.

  US Pat. Nos. 5,504,074, 5,661,143 and 5,892,069 to D'Amato et al. Are mammals characterized by abnormal cell mitosis using 2-ME2. Disclosed is a method for treating a disease. Undesirable cell mitosis is characteristic of numerous diseases including, but not limited to, cancer, atherosclerosis, solid tumor growth, vascular dysfunction, endometriosis, retinopathy, arthropathy, and abnormal wound healing It is. Furthermore, cell mitosis is important in a wide variety of biological functions, including but not limited to normal embryonic development, luteinization, endometrial periodic proliferation, wound healing and inflammation, and immune responses.

  US Pat. No. 5,521,168 to Clark discloses the use of 2-ME2 to lower intraocular pressure. 2-ME2 further inhibits estrogen-induced pituitary tumor angiogenesis in Fischer 344 rats as reported in Banerjee, SK et al., Proc. Amer. Assoc. Cancer Res. 39, March 1998. Inhibits tumor growth.

  Currently, commercial formulations of 2-ME2 are less than 98% pure or contain undesirable steroid contaminants that affect pharmaceutical use. Important contaminants in these formulations are estradiol, 4-hydroxyestradiol, 4-methoxyestradiol, 2-hydroxyestradiol, estrone, and 2-methoxyestrone. The amount of these contaminants found in currently available 2-ME2 formulations is unacceptable for pharmaceutical use.

  Any therapeutic use of 2-ME2 in humans requires 2-ME2 with a high level of purity. In general, the therapeutic agent is required to be substantially pure to avoid the negative side effects of contaminants. In particular, since 2-ME2 has the effect of being neutralized by estradiol and other estrogenic metabolites, it is extremely important to have a 2-ME2 preparation that is substantially free of such contaminants. The effects that can be observed by contamination of estradiol, estrone and 2-hydroxyestradiol are feminization, endometrial proliferation, increased risk of uterus and breast cancer, developmental effects on sexual organs, suppression of leukocyte production, and hematopoietic cells Including estrogenic effects such as 4-hydroxyestradiol, 4-methoxyestradiol and estradiol are known mutagens and carcinogens.

  Therefore, what is needed is a composition of 2-ME2 that is more than 98% pure and substantially free of estradiol or other steroids that have estrogenic or carcinogenic effects.

  What is further needed is a composition containing 2-ME2 having a purity of greater than 99.5%.

  What is further needed is a process for producing 2-ME2 having a purity of greater than 98% and substantially free of estradiol or other steroids having an estrogenic or carcinogenic effect.

  There is also a need for a method that substantially separates 2-ME2 from estradiol, related molecules, and other contaminants to yield 2-ME2 having a purity greater than 99.5%.

[Summary of the Invention]
The present invention provides 2-ME2 having a purity greater than 98%, more preferably greater than 99%, and most preferably greater than 99.5%. The 2-ME2 formulation is preferably less than 0.03% estradiol, 0.02% or less 2-hydroxyestradiol, 0.02% or less 4-hydroxyestradiol, 0.02% or less Contains 4-methoxyestradiol and less than 0.02% estrone. More preferably, the 2-ME2 formulation is 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.

  The present invention also provides a method for producing 2-ME2 having a purity of greater than 98%, more preferably greater than 99%, and most preferably greater than 99.5%. In some embodiments, the method includes a synthesis technique. In other embodiments, the methods include purification techniques for separating 2-ME2 from other compounds. In yet other embodiments, the method encompasses both the synthesis and purification techniques described herein.

  Purification techniques include the use of liquid-body chromatography (LSC) to separate 2-ME2 from other compounds. The chromatographic medium is preferably silica. The solvent system includes a nonpolar solvent, such as chloroform, and a polar solvent, such as methanol.

  The object of the present invention is therefore to provide 2-ME2 with a purity higher than 98%.

  Another object of the present invention is to provide 2-ME2 that is substantially free of estradiol, related compounds, and other undesirable impurities.

  Yet another object of the present invention is to provide a method for producing substantially pure 2-ME2 by synthetic techniques.

  Another object of the present invention is to provide a method for producing substantially pure 2-ME2 by purification techniques.

  Other features and advantages of the present invention will become apparent from the following description of preferred embodiments thereof.

[Detailed Description of the Invention]
The present invention is directed to 2-methoxyestradiol having a purity greater than 98.0%, more preferably greater than 99.0%, and most preferably 99.5% or higher. 2-ME2 is obtained by the synthesis or purification methods described herein that produce high purity 2-ME2. The synthetic methods described herein can also be supplemented by the purification methods described herein to produce 2-ME2 with even higher purity.

  Although the term “2-methoxyestradiol” and 2-ME2 are specifically used herein, the methods disclosed herein are not limited to those of estradiol and other structurally related steroids. It should be understood that it can be used for the synthesis or purification of such other compounds.

<Synthesis method>
The present invention provides a method for synthesizing 2-ME2 to a purity of greater than 98.0%, more preferably greater than 99.0%, most preferably 99.5% or higher. The synthetic methods described herein are also minor modifications to synthesize other 2- and 4-derivatives or analogs of estradiol, such as 4-methoxyestradiol and 4-hydroxyestradiol, Can be used.

  There are several synthetic approaches that can be employed to prepare 2-ME2 having a purity greater than 98.0%. Alternatively, 2-ME2 can be purified according to the following purification method to have a purity of greater than 98.0%. These different synthetic approaches utilize different starting materials and intermediates, resulting in different yields, side reactions and impurities.

  Two similar approaches are described using estradiol as the starting material as taught by Rao, PN et al., Synthesis, 1977, 168 and Chen, SH et al., Steroids, 1986, 47, 63. Utilized intermediate materials. The first approach is illustrated in FIG. The free hydroxyl group of estradiol is protected with a protecting group. A wide range of protecting groups are used in the present invention. These protecting groups include, but are not limited to, alkyl, aryl, aralkyl groups, and alkyl, aryl, and aralkyl groups that contain one or more heteroatoms. For example, protection can be achieved using an alkyl halide to produce an alkyl ether, such as benzyl bromide. Suitable conditions for hydroxyl protection include reaction of estradiol with an alkyl halide, optionally in the presence of a solvent such as dimethylformamide (DMF), in the presence of NaH and TBAI. The protected estradiol is then reacted with bromine, for example in the presence of acetic acid. Protection of free hydroxyl during bromination yields high yields of 2-brominated intermediate (about 70% versus about 20% without a protecting group) (Cushman, M. et al., J Med. Chem. 1997, 40, 2323).

  Bromine is then replaced with methoxide groups 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 carried out in a solvent such as DMF, optionally in the presence of a promoter. Acceptable accelerators include, but are not limited to, crown ethers such as benzo-15-crown-5.

  For example, removal of the protecting group by catalytic hydrogenation of the alkyl moiety yields 2-ME2. Unfortunately, this synthetic route results in about 1-2% estradiol impurities from the methoxylation step (hydrides suppress reactive copper complexes rather than methoxides). Estradiol can be removed to levels not detectable by chromatography as described below, or is significantly reduced by continuous crystallization in chloroform.

  Another synthetic method utilizing a brominated intermediate and using estradiol as a starting material is illustrated in FIG. In this synthesis reaction, estradiol is ring brominated without first protecting the hydroxyl group. Bromine is then replaced with methoxide in the same manner as described above using a copper catalyst.

  In another approach, estradiol or estrone can be used as a starting material in reaction schemes utilizing nitro / amine intermediates (see Cushmam, 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, free hydroxyl groups are protected. This protection can be accomplished using an alkyl halide to produce an alkyl ether, such as benzyl bromide. Suitable conditions for hydroxyl protection include reaction of the starting material with an alkyl halide, optionally in the presence of a solvent such as dimethylformamide (DMF), in the presence of NaH and TBAI.

  The protected starting material is then nitrated using, for example, nitric acid and acetic acid or with nitric acid and sulfuric acid to produce the corresponding 2-nitro product. The nitro group is then reduced. Selective reduction is accomplished by catalytic hydrogenation, for example hydrogenation in the presence of Pd / C, to produce the corresponding 2-amine. The catalytic reduction is optimally performed for 1 hour. Using Zandmeier conditions (nitrous acid and sodium methoxide), the 2-amino group can be converted to a 2-methoxy substituent. Catalytic hydrogenation removes the protecting group to yield 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.

  Yet another method uses estradiol as a starting material and utilizes a brominated intermediate. In this synthesis reaction, estradiol is ring brominated without first protecting the hydroxyl group. Bromination is accomplished, for example, using bromine and acetic acid in a solvent such as THF. This reaction results in bromination at different sites on the ring, including multiple brominated species. 2-Bromo-estradiol can then be isolated from other brominated intermediates by, for example, chromatography or crystallization, followed by displacement of the bromine with methoxide. Bromine can be replaced with a methoxide group in a manner similar to that described above, using sodium methoxide and methanol, for example, in the presence of a copper catalyst such as CuI. Alternatively, the intermediate is reacted to produce the corresponding methoxide, after which 2-methoxyestradiol is isolated by the methods described above.

<Purification method>
The present invention provides a method for purifying 2-ME2 to a purity of greater than 98.0%, more preferably greater than 99.0%, most preferably 99.5% or higher. The 2-ME2 formulation is preferably less than 0.03% estradiol, 0.02% or less 2-hydroxyestradiol, 0.02% or less 4-hydroxyestradiol, 0.02% or less Contains 4-methoxyestradiol and less than 0.02% estrone. Most preferably, the 2-ME2 formulation is 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.

  The purification method of the present invention involves liquid chromatography on an adsorption / distribution medium, such as silica, using a solvent system comprising polar and nonpolar solvents. The purification methods described herein can be used, for example, for 4-methoxyestradiol, 4-hydroxyestradiol, 2-hydroxyestradiol, estradiol, estrone, 2-methoxyestrone, and 4-methoxyestrone, with minor modifications. It can also be used to purify compounds similar to 2-ME2.

<Sample>
The sample to be purified can be synthesized or obtained from a biological source. The sample may be a 2-ME2 commercial formulation such as that sold by Sigma-Aldrich Chemicals (St. Louis, Missouri), Rearch Plus, Inc. (Bayonne, NJ) or Calbiochem (San Diego, CA). . 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, such as selective crystallization, prior to the methods described herein.

  The sample is preferably dissolved or solvent exchanged in the loading solvent, as further described below. The sample is preferably at a concentration in the range of about 0.01-2 g / ml, preferably about 0.01-1 g / ml, more preferably about 0.05-0.2 g / ml.

<Chromatography medium>
Silica is preferably used as the chromatography medium. About 70-400 mesh silica gel is preferred, most preferably about 70-230 mesh as supplied by Merck and other vendors. The medium can be used as roses in batch chromatography or packed into a column. Pre-packed columns such as those sold by Biotage (Charlottesville, Va.) Can also be used. The medium should be equilibrated in a suitable solvent prior to application of the sample to the medium, as discussed further below.

<Column dimensions>
The chromatographic methods described herein can be accomplished using batch or column chromatography. In batch chromatography, the sample and the chromatographic medium are combined in the vessel for a time sufficient to keep 2-ME2 in the medium. The medium is then preferably washed with a washing solvent. The elution solvent is then applied to the medium. After the loading, washing and elution steps, the solvent is removed from the medium, for example by filtration.

  For column chromatography, a column with appropriate dimensions is packed with a chromatography medium. After the column is equilibrated with a suitable solvent, the sample is loaded by applying the sample to the top or inlet of the column. The ratio of sample volume to column diameter should preferably be about 0.2-3 ml / cm, more preferably about 0.5-1.5 ml / cm for best results.

<Solvent>
A solvent system comprising a polar solvent such as methanol (MeOH) and a nonpolar solvent such as chloroform (CHCl 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 nonpolar 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 ₃ , propanol / CHCl ₃ , isopropanol / CHCl ₃ , and combinations thereof.

  The sample is soluble in polar solvents. A small amount, typically about 10%, of polar solvent is required to make the sample soluble in the loading solvent. As an example, the loading solvent comprises up to about 10% polar solvent and about 90% non-polar solvent.

  After the sample is loaded onto the medium, the medium can be washed with a washing solvent that washes away contaminants from the medium but does not elute 2-ME2. The wash solvent mainly comprises a non-polar solvent with a polar solvent that is sufficient to prevent 2-ME2 from precipitating but not sufficient to elute 2-ME2.

  The sample is eluted with an elution solvent containing sufficient polar solvent to elute 2-ME2 from the medium. The elution solvent can be applied as a step gradient or as a linear gradient, as described below.

<Column conditions>
Wash and elution solvents can be applied to the column in a step gradient or in a linear gradient. In a preferred embodiment, the solvent can be applied using a step gradient that increases the concentration of the polar solvent.

  The column can be operated using gravity or can be operated using a pump that forces liquid through the column. The rate at which the column is operated depends on the volume and size of the column and the silica gel particle size. In general, the column can be operated at a rate of about 0.5-5 ml / min.

  The eluent can be monitored visually or can be monitored using a spectrometer at a wavelength of about 288 nm, the maximum absorbance of 2-ME2, and collected as 2-ME2 eluting from the column.

  The column can optionally be operated under pressure and optionally heated. Normal or reverse phase preparative high performance liquid chromatography (HPLC), or high performance liquid chromatography (FPLC) techniques may be used. Commercially available preparative chromatography equipment such as that sold by Biotage (Charlottesville, Va.) Can also be used. Other known methods for improving column efficiency and / or speed can also be used.

<Sample collection and processing>
The eluent can then be collected as a fraction that is analyzed for 2-ME2 content and purity. These fractions can then be combined to achieve the desired purity of 2-ME2. Fractions can be analyzed for purity using a reverse phase HPLC using a C-18 column (Waters) and a 30: 69: 1 acetonitrile: water: acetic acid isocratic solvent system. Other systems, such as those using a solvent gradient instead of an isocratic solvent system, those using trifluoroacetic acid or formic acid instead of acetic acid, and those using methanol rather than acetonitrile can be used for sample analysis.

  Alternatively or in combination, the eluent can be monitored in real time and sample collection can be initiated when the desired purity of 2-ME2 is eluted from the column.

  The solvent is removed from the pool fraction by use of vacuum and / or other solvent removal methods. Freeze drying and other evaporation methods can be used.

<Preferred embodiment>
In a preferred embodiment, the medium is silica packed into the column. The sample is dissolved in a mixture of CHCl ₃ and MeOH, typically about 90:10 CHCl ₃ : MeOH, with sufficient MeOH to dissolve 2-ME2. The elution conditions are a step gradient from 99: 1 CHCl ₃ : MeOH to 98: 2 CHCl ₃ : MeOH.

  The present invention is further illustrated by the following examples that should not be construed in a direction that would limit the scope of the invention. On the contrary, after reading this specification, various other embodiments, modifications, and equivalents may be taken without departing from the spirit of the invention and / or the scope of the appended claims. It is clearly understood that this can be suggested to the vendor.

<Example 1>
Analyze commercial samples of 2-ME2 by analytical HPLC to determine the purity of all certain contaminants: estradiol, 4-hydroxyestradiol, 4-methoxyestradiol, 2-hydroxyestradiol, estrone and 2-methoxyestrone The amount was measured.

  These analytical HPLC chromatograms are reversed using a C-18 column (Waters) and a solvent gradient (20 → 50% acetonitrile over 30 minutes, 50 → 80% acetonitrile, 1% acetic acid, residual water over 30 minutes). Formed using phase HPLC. The eluent 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, and 4-hydroxyestradiol takes about 15.0 minutes. Elute, 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.

  A chromatogram of a sample from Sigma-Aldrich Chemicals (St. Louis, Missouri) is shown in FIG. The sample has an overall purity of 99.2%, but has an unacceptable amount of about 0.034% contaminated estradiol. FIG. 2 is an enlarged view of the chromatogram of FIG.

  FIG. 3 is a chromatogram of a sample obtained from Rearch Plus, Inc. (Bayonne, NJ) showing that 2-ME2 has a purity of 98.6%. The automatic peak calculator and the enlarged view shown in FIG. 4 show that the formulation contains 0.024% estrone, an unacceptable amount of this contaminant. Other samples tested, including secondary batches obtained from Rearch Plus (97.2% 2-ME2) and samples from Calbiochem (San Diego, California) (91.8% 2-ME2) were 98% Less than 2-ME2 purity was shown.

  Table 1 below shows the purity and contaminants of these commercial samples of 2-ME2 and the purified 2-ME2 of the present invention.

<Example 2>
A glass column 55 cm in diameter (height 60 cm) was packed with 600 g of silica gel (70-230 mesh, Merck) in 90:10 CHCl ₃ : MeOH. The column was washed with 1 liter CHCl ₃ to remove MeOH from the column.

The sample was 3.5 g of 2-ME2 in 60 ml of 90:10 CHCl ₃ : MeOH. 2-ME2 was obtained from PharmEco Labortories, Inc. (Lexington, Mass.) And was 97.8% pure as determined by analytical HPLC (FIG. 5). The peak eluting at 10.1717 is estradiol (2.2%).

  Using reverse phase HPLC using a C-18 column (Waters) and an isocratic gradient of 30: 69: 1 acetonitrile: water: acetic acid to provide a good separation of 2-ME2 and estradiol, starting material, Analytical HPLC of the column fraction and its pooled components was performed. The eluent was monitored at a wavelength of 288 nm.

The sample was poured into the top of the column and placed in the column volume. The column was eluted with 1 liter of 99: 1 CHCl ₃ : MeOH, then 1.5 L of 98: 2 CHCl ₃ : MeOH. Each 50 ml fraction was collected and 15 fractions containing 2-ME2 were analyzed for 2-ME2 purity using the analytical isocratic HPLC system described above. The 9-10 fractions that did not show the amount of estradiol were pooled together and the solvent was evaporated. After drying for 4 hours under vacuum, 3.2 g of yellow / white crystals were collected in 91% yield.

  Using the isocratic technique described above, the purity of the fractions pooled by analytical HPLC was determined to be 99.984%. The HPLC chromatogram is shown in FIGS. FIG. 6 was obtained using 75.6 μg of non-overloaded sample (14 μl at 5.4 μl / ml). FIG. 7 is an enlarged view of the chromatogram of FIG. The automatic peak detector calculated 2-ME2 to be 100.0%, but a small unknown impurity peak eluting before 2-ME2 was seen in the enlarged view. FIG. 8 was obtained using 270 μg of overloaded sample (50 μl at 5.4 μl / ml). FIG. 9 is an enlarged view of the chromatogram of FIG. The automatic peak detector calculated 2-ME2 to be 99.984% pure with a small unknown impurity peak eluting before 2-ME2 and after estradiol and calculated to be 0.016%. The enlarged view shown in FIG. 9 shows this impurity peak more clearly and shows that the 2-ME2 peak is very smooth.

  The pool was also analyzed using a gradient (20 → 70% acetonitrile, 1% acetic acid, residual amount of water over 25 minutes). 43.2 μg (5.4 μl / ml sample of 8 μl) was injected. The chromatogram is shown in FIG. The automatic peak detector calculated 2-ME2 to be 99.825% pure. However, when the artificial peak observed at 29.45 minutes in the blank experiment is excluded from the study, the calculated purity is 99.9%. FIG. 11 is an enlarged view of this chromatogram. The unknown impurity at 13.43 minutes was calculated to be 0.012%. If estradiol is present, it elutes between unknown purity and the 2-ME2 peak. Thus, if estradiol is present, it can be present at 1/3 to 1/4 or less of the amount of unknown peaks. Therefore, the amount of estradiol was estimated to be 0.004% or less. The formulation is 0.02% or less 2-hydroxyestradiol, 0.01% or less 4-hydroxyestradiol, as demonstrated for the lack of any measurable peak at the predicted retention time, 0. Contains 01% or less 4-methoxyestradiol and 0.01% or less estrone.

  The purified sample was also subjected to elemental analysis, and the results are shown in Table 2.

  The above description is for explanation and does not limit the present invention. 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 instead should be determined with reference to the appended claims along with the full scope of equivalents represented by such claims. The disclosures of all papers and references, including patents, patent applications, and publications, are hereby incorporated by reference.

  FIG. 5 is a chromatogram from reverse phase HPLC analysis of 2-methoxyestradiol (45H4033) available from Sigma Chemical Company. This graph shows that the Sigma product contains about 0.034% estradiol.   FIG. 2 is an enlarged view of the chromatogram of FIG. 1 showing estradiol impurities.   FIG. 5 is a chromatogram from reverse phase HPLC analysis of 2-methoxyestradiol (10699) available from Research Plus. This graph shows that Research Plus products contain about 0.024% estrone and about 0.93% other undesirable estrogens.   FIG. 4 is an enlarged view of the chromatogram of FIG. 3 showing estrone impurities.   It is a chromatogram from the reverse phase HPLC analysis of the unpurified 2-methoxyestradiol used as a starting material in Example 2 of this invention.   2 is a chromatogram of 2-ME2 of the present invention produced in Example 2. FIG. HPLC was performed using a non-overloaded sample of 75.6 μg (14 μl at 5.4 μl / ml).   It is an enlarged view of the chromatogram of FIG.   2 is a chromatogram of 2-ME2 of the present invention produced in Example 2. FIG. HPLC was performed using 270 μg of overloaded sample (50 μl at 5.4 μl / ml).   It is an enlarged view of the chromatogram of FIG.   FIG. 4 is a chromatogram of the pool fraction from Example 2 analyzed using a gradient (20 → 70% acetonitrile, 1% acetic acid, and residual water in 25 minutes). 43.2 μg (5.4 μl / ml sample of 8 μl) was injected.   It is an enlarged view of the chromatogram of FIG.   FIG. 2 shows a synthetic reaction diagram for the production of 2-methoxyestradiol of the present invention using estradiol as a starting material, using bromine, crown ether, and protecting groups on the 3- and 17-position hydroxyl oxygen atoms of estradiol.   Figure 2 shows a synthetic reaction diagram for the production of 2-methoxyestradiol of the present invention using estradiol as a starting material, bromination of the 2-position of the A ring of unprotected estradiol, and crown ether.   FIG. 2 shows a synthetic reaction diagram for the production of 2-methoxyestradiol of the present invention using estradiol as a starting material, using protecting groups on the 3- and 17-position hydroxyl oxygen atoms of estradiol, nitration, and Sandmeyer reaction.   FIG. 3 shows a synthetic reaction diagram for the production of 2-methoxyestradiol of the present invention using estrone as a starting material, using a protecting group on the 3-position hydroxyl oxygen atom, nitration, and a Sandmeyer reaction.   FIG. 2 shows a synthetic reaction diagram for the production of 2-methoxyestradiol of the present invention using estradiol as a starting material, 2-position bromination of the unprotected estradiol A-ring, and reaction with methanol.

Claims (24)

Look containing 2-methoxyestradiol with greater than 99.5% purity, and, compositions containing estrone estradiol and less than 0.02% to less than 0.03%.
The composition of claim 1 containing estrone estradiol and less than 0.01% to less than 0.01%.
Furthermore composition of claim 1 containing 2-hydroxy estradiol of less than 0.02%.
Furthermore composition of claim 1 containing 4-hydroxy estradiol of less than 0.02%.
Furthermore composition of claim 1 containing 4-methoxy estradiol of less than 0.02%.
0.01% or less estradiol, 0.02% or less 2-hydroxyestradiol, 0.01% or less 4-hydroxyestradiol, 0.01% or less 4-methoxyestradiol, and 2. A composition according to claim 1 containing 0.01% or less estrone.
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.
8. The composition of claim 7 , comprising less than 0.01% estradiol and less than 0.01% estrone.
0.01% or less estradiol, 0.02% or less 2-hydroxyestradiol, 0.01% or less 4-hydroxyestradiol, 0.01% or less 4-methoxyestradiol, and The composition of claim 7 containing 0.01% or less estrone.
The composition of claim 7, wherein the 2-methoxyestradiol has a purity of greater than 99.0%.
11. The composition of claim 10, comprising less than 0.01% estradiol and less than 0.01% estrone.
0.01% or less estradiol, 0.02% or less 2-hydroxyestradiol, 0.01% or less 4-hydroxyestradiol, 0.01% or less 4-methoxyestradiol, and 11. A composition according to claim 10 containing 0.01% or less estrone.
A process for purifying 2-methoxyestradiol to produce 2-methoxyestradiol having a purity greater than 98% and containing less than 0.03% estradiol and less than 0.02% estrone, comprising:
Adding a solution comprising 2-methoxyestradiol to the chromatography medium;
Eluting 2-methoxyestradiol from the medium using a solvent system comprising a polar solvent and a nonpolar solvent;
Including methods.
The medium is silica and the 2-methoxyestradiol is 99: 1 CHCl _3.
: From MeOH 98: 2 in CHCl _3: claim 1 which is eluted with a step gradient of MeOH
3. The method according to 3 .
A process for producing 2-methoxyestradiol having a purity of greater than 98% and containing less than 0.03% estradiol and less than 0.02% estrone,
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 a 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;
Purifying 2-methoxyestradiol using liquid chromatography on an adsorption / distribution medium using a solvent system comprising polar and nonpolar solvents;
Including methods.
A process for producing 2-methoxyestradiol having a purity of greater than 98% and containing less than 0.03% estradiol and less than 0.02% estrone,
Ring bromination of estradiol by reacting estradiol with bromine in the presence of acetic acid to produce a ring brominated intermediate;
Reacting the ring brominated intermediate with sodium methoxide in the presence of a copper catalyst to produce 2-methoxyestradiol;
Purifying 2-methoxyestradiol using liquid chromatography on an adsorption / distribution medium using a solvent system comprising polar and nonpolar solvents;
Including methods.
A process for producing 2-methoxyestradiol having a purity of greater than 98% and containing less than 0.03% estradiol and less than 0.02% estrone,
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 a 2-amino derivative of estradiol under Zandmeier conditions to produce 3-, 17-hydroxyl protected 2-methoxyestradiol;
Removing the protecting groups on the 3- and 17-hydroxyl groups to produce 2-methoxyestradiol;
Including methods.
A process for producing 2-methoxyestradiol having a purity of greater than 98% and containing less than 0.03% estradiol and less than 0.02% estrone,
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 a 2-amino derivative of estrone under Zandmeier conditions to produce 3-hydroxyl-protected 2-methoxyestrone;
Removing the protecting group on the 3-hydroxyl group to produce 2-methoxyestrone;
Reducing the 17-keto group of 2-methoxyestrone to produce 2-methoxyestradiol;
Including methods.
A process for producing 2-methoxyestradiol having a purity of greater than 98% and containing less than 0.03% estradiol and less than 0.02% estrone,
Brominating estradiol in the presence of acetic acid to produce a mixture of ring brominated estradiol;
Isolating 2-bromoestradiol from a mixture of estradiol;
Reacting 2-bromoestradiol with sodium methoxide in the presence of a copper catalyst to produce 2-methoxyestradiol;
Including methods.
2-methoxyestradiol having a purity greater than 98% and containing less than 0.03% estradiol and less than 0.02% estrone,
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 a 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;
Purifying 2-methoxyestradiol using liquid chromatography on an adsorption / distribution medium using a solvent system comprising polar and nonpolar solvents;
2-methoxyestradiol produced by a process comprising:
2-methoxyestradiol having a purity greater than 98% and containing less than 0.03% estradiol and less than 0.02% estrone,
Ring bromination of estradiol by reacting estradiol with bromine in the presence of acetic acid to produce a ring brominated intermediate;
Reacting the ring brominated intermediate with sodium methoxide in the presence of a copper catalyst to produce 2-methoxyestradiol;
Purifying 2-methoxyestradiol using liquid chromatography on an adsorption / distribution medium using a solvent system comprising polar and nonpolar solvents;
2-methoxyestradiol produced by a process comprising:
2-methoxyestradiol having a purity greater than 98% and containing less than 0.03% estradiol and less than 0.02% estrone,
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 a 2-amino derivative of estradiol under Zandmeier conditions to produce 3-, 17-hydroxyl protected 2-methoxyestradiol;
Removing the protecting groups on the 3- and 17-hydroxyl groups to produce 2-methoxyestradiol;
2-methoxyestradiol produced by a process comprising:
2-methoxyestradiol having a purity greater than 98% and containing less than 0.03% estradiol and less than 0.02% estrone,
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 a 2-amino derivative of estrone under Zandmeier conditions to produce 3-hydroxyl-protected 2-methoxyestrone;
Removing the protecting group on the 3-hydroxyl group to produce 2-methoxyestrone;
Reducing the 17-keto group of 2-methoxyestrone to produce 2-methoxyestradiol;
2-methoxyestradiol produced by a process comprising:
2-methoxyestradiol having a purity greater than 98% and containing less than 0.03% estradiol and less than 0.02% estrone,
Brominating estradiol in the presence of acetic acid to produce a mixture of ring brominated estradiol;
Isolating 2-bromoestradiol from a mixture of estradiol;
Reacting 2-bromoestradiol with sodium methoxide in the presence of a copper catalyst to produce 2-methoxyestradiol;
2-methoxyestradiol produced by a process comprising:

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